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Interleukin-1β-Induced Extracellular Matrix Degradation and Glycosaminoglycan Release Is Inhibited by Curcumin in an Explant Model of Cartilage Inflammation
Annals of the New York Academy of Sciences
Abstract
Osteoarthritis (OA) is a degenerative and inflammatory disease of synovial joints that is characterized by the loss of articular cartilage, for which there is increasing interest in natural remedies. Curcumin (diferuloylmethane) is the main polyphenol in the spice turmeric, derived from rhizomes of the plant Curcuma longa. Curcumin has potent chemopreventive properties and has been shown to inhibit nuclear factor kappaB-mediated inflammatory signaling in many cell types, including chondrocytes. In this study, normal articular cartilage was harvested from metacarpophalangeal and metatarsophalangeal joints of eight horses, euthanized for reasons other than research purposes, to establish an explant model mimicking the inflammatory events that occur in OA. Initially, cartilage explants (N= 8) were stimulated with increasing concentrations of the proinflammatory cytokine IL-1beta to select effective doses for inducing cartilage degeneration in the explant model. Separate cartilage explants were then cotreated with IL-1beta at either 10 ng/mL (n= 3) or 25 ng/mL (n= 3) and curcumin (0.1 micromol/L, 0.5 micromol/L, 1 micromol/L, 10 micromol/L, and 100 micromol/L). After 5 days, the percentage of glycosaminoglycan (GAG) release from the explants was assessed using a dimethylmethylene blue colorimetric assay. Curcumin (100 micromol/L) significantly reduced IL-1beta-stimulated GAG release in the explants by an average of 20% at 10 ng/mL and 27% at 25 ng/mL back to unstimulated control levels (P < 0.001). Our results suggest that this explant model effectively simulates the proinflammatory cytokine-mediated release of articular cartilage components seen in OA. Furthermore, the evidence suggests that the inflammatory cartilage explant model is useful for studying the effects of curcumin on inflammatory pathways and gene expression in IL-1beta-stimulated chondrocytes.
... Second, inflammation induced by pro-catabolic cytokines IL-1, IL-6, and tumor necrosis factor-α (TNFα) has been suggested to result in rapid GAG depletion over 5-25-days (Li et al., 2015(Li et al., , 2013 in a dosedependent manner (Clutterbuck et al., 2009) and synergize with injuries in 6-8-day-long explant cultures (Sui et al., 2009). Furthermore, pro-inflammatory factors may decrease aggrecan biosynthesis and promote cell death (Li et al., 2015). ...
... Our findings are generally aligned with current literature. However, the time scale of GAG loss was slower here (day 12, 1 ng/ml IL-1α, accumulated GAG loss 30%) than in other studies; Li et al. (2015) found 80% GAG loss by day 12 (1 ng/ml IL-1α, immature bovine) and Clutterbuck et al. (2009) found 20% GAG loss by day 5 in vitro (0.5 ng/ml IL-1β, equine). This may be explained by animal (species)-wise variation and differences in culture media. ...
... This may be explained by animal (species)-wise variation and differences in culture media. GAG loss has been found to be cytokine concentration-dependent (Clutterbuck et al., 2009;McNulty et al., 2013) and chondral injuries aggravate inflammation-modulated degradation (Li et al., 2013;Sui et al., 2009), for which our results give further support. We also noted early rapid GAG loss post-injury (Mohanraj et al., 2014;Rolauffs et al., 2010) and localized GAG loss near lesions (Orozco et al., 2018). ...
Injurious overloading and inflammation perturbate homeostasis of articular cartilage, leading to abnormal tissue-level loading during post-traumatic osteoarthritis. Our objective was to gain time- and cartilage depth-dependent insights into the early-stage disease progression with an in vitro model incorporating for the first time the coaction of (1) mechanical injury, (2) pro-inflammatory interleukin-1 challenge, and (3) cyclic loading mimicking walking and considered beneficial for cartilage health. Cartilage plugs (n=406) were harvested from the patellofemoral grooves of young calves (N=6) and subjected to injurious compression (50% strain, rate 100%/s; INJ), interleukin-1α-challenge (1 ng/ml; IL), and cyclic loading (intermittent 1h loading periods, 15% strain, 1 Hz; CL). Plugs were assigned to six groups (control, INJ, IL, INJ-IL, IL-CL, INJ-IL-CL). Bulk and localized glycosaminoglycan (GAG) content (DMMB assay, digital densitometry), aggrecan biosynthesis (³⁵S-sulfate incorporation), and chondrocyte viability (fluorescence microscopy) were assessed on days 3–12. The INJ, IL, and INJ-IL groups exhibited rapid early (days 2–4) GAG loss in contrast to CL groups. On day 3, deep cartilage of INJ-IL-CL group had higher GAG content than INJ group (p<0.05). On day 12, INJ-IL-CL group showed more accumulated GAG loss (normalized with control) than INJ-IL group (average fold changes 1.97 [95%CI: 1.23–2.70]; 1.66 [1.42–1.89]; p=0.007). Aggrecan biosynthesis increased in CL groups on day 12 compared to day 0. Despite promoting aggrecan biosynthesis, this cyclic loading protocol seems to be beneficial early-on to deep cartilage, but later becoming incapable of restricting further degradation triggered by marked but non-destructive injury and cytokine transport.
... These studies are important as they serve to illuminate mechanism of anti-arthritic action on the target tissue. Two studies have reported effects of curcumin on equine [7] and human [8] cartilage explants. Conditioning of equine cartilage explants with curcumin (100 μmol/L) for 5 days significantly reduced interleukin-(IL-)1β-induced release of glycosaminoglycan (GAG; a measure of cartilage breakdown) [7]. ...
... Two studies have reported effects of curcumin on equine [7] and human [8] cartilage explants. Conditioning of equine cartilage explants with curcumin (100 μmol/L) for 5 days significantly reduced interleukin-(IL-)1β-induced release of glycosaminoglycan (GAG; a measure of cartilage breakdown) [7]. This inhibition of GAG release was not observed in human cartilage explants, perhaps due to a lower curcumin exposure rate (5-20 μmol/L), but these authors did report an inhibition of IL-1-induced nitric oxide (NO), prostaglandin E 2 (PGE 2 ), IL-6, IL-8 and matrix metalloproteinase 3 [8]. ...
... We did not characterize the post-hepatic species in our biological extract for the current study, and future research should attempt to identify the major phytochemical parents and downstream metabolic endproducts in TUR sim to further define those with inhibitory effects on PGE 2 . The slight inhibitory effect of TUR sim (15 μg/mL) on LPS-induced cartilage breakdown (as measured by increased release of GAG into tissue culture media) demonstrated in the current study is less marked than that reported for curcumin in IL-1-stimulated equine cartilage explants [7], perhaps due (at least in part) to the very high concentration of curcumin employed in the latter study (100 μM, equivalent to 36.8 μg/mL). Assuming a curcumin concentration of approximately 3% in turmeric powder [18], a curcumin concentration of 36.8 μg/ mL would be represented in a turmeric concentration of about 1227 μg/mLmarkedly higher than the concentration of TUR sim in the current study. ...
Background:
Turmeric is commonly used as a dietary treatment for inflammation, but few studies have evaluated the direct effect of turmeric on cartilage. The purpose of this study was to characterize cartilage explants' inflammatory responses to lipopolysaccharide in the presence of a simulated biological extract of turmeric.
Methods:
Turmeric was incubated in simulated gastric and intestinal fluid, followed by inclusion of liver microsomes and NADPH. The resulting extract (TURsim) was used to condition cartilage explants in the presence or absence of lipopolysaccharide. Explants were cultured for 96 h (h); the first 24 h in basal tissue culture media and the remaining 72 h in basal tissue culture media containing TURsim (0, 3, 9 or 15 μg/mL). Lipopolysaccharide (0 or 5 μg/mL) was added for the final 48 H. media samples were collected immediately prior to lipopolysaccharide exposure (0 h) and then at 24 and 48 h after, and analyzed for prostaglandin E2 (PGE2), glycosaminoglycan (GAG), and nitric oxide (NO). Explants were stained with calcein-AM for an estimate of live cells. Data were analyzed using a 2-way repeated measures (GAG, PGE2, NO) or 1-way ANOVA without repeated measures (viability). Significance accepted at p < 0.05.
Results:
TURsim significantly reduced PGE2, NO and GAG, and calcein fluorescence was reduced.
Conclusions:
These data contribute to the growing body of evidence for the utility of turmeric as an intervention for cartilage inflammation.
... The present study aimed to analyze whether PEMF stimulation (75 Hz, 1.5 mT), applied to bovine cartilage explants, derived from two different joints of the same animals, was able to counteract the catabolic effect of a high dose of IL1β. In comparison to 2D monolayer chondrocyte cultures, the cartilaginous 3D explants cultured with IL1β are a well-accepted method to measure and evaluate the effect and mechanism of action of a therapy, by simultaneously evaluating chondrocytes and cartilage matrix and by closely mimicking the clinical situation of OA joint, in which a high dose of IL1β is found in the early stages of OA [30] and its concentration can be further increased by surgical intervention [2]. ...
... The in vitro effects were investigated on bovine cartilage explants harvested from two different joints. To simulate the OA microenvironment, a high IL1β concentration (50 ng/ ml) was administered in explant cultures, as set in an our previous in vitro study [30], for a longer period of 21 days and the changes in the structure and cellularity of CTR and IL1β-treated cartilage explants, either exposed or not to PEMFs, were analyzed. The concentration of IL-1β found in the synovial fluid of patients affected by OA is between 0.068 and 0.33 pg/ml [35]. ...
... The addition of a high dose of IL1β to the culture medium induced a significant reduction, not only in structural parameters, but also ECM components and TGFβ1 synthesis, a decline observed at all experimental times until 21 days. These results are in agreement with those of previous in vitro studies, that observed GAGs, PGs and Coll II loss with the addition of IL1β in the culture medium of human, bovine and horse cartilage explants [30,42,43]. Although the joint inflammatory microenvironment of OA consists of different inflammatory mediators, such as matrix metalloproteinases (MMPs), the disintegrin and metalloproteinases with thrombospondin motifs (ADAMTs) and other pro-inflammatory cytokines, TNF-α and Interleukin-6 (IL-6), data suggested that IL1β might have the strongest effect. ...
Background
Osteoarthritis (OA) is the final result of progressive alterations to articular cartilage structure, composition and cellularity, followed by an increase in the concentration of pro-inflammatory cytokines in joint synovial fluid. Even though the effect of pulsed electromagnetic field (PEMF) stimulation in counteracting OA progression and inflammation is of increasing interest, because of its anabolic and anti-inflammatory properties, the present study aimed to improve the knowledge on cartilage extracellular matrix (ECM) and chondrocyte changes related to the exposure of PEMF, from a histological and histomorphometric point of view.
Methods
An in vitro OA model was realized, culturing bovine cartilage explants with a high dose of interleukin 1β (IL1β, 50 ng/ml) at different experimental times (24 h, and 7 and 21 days). The effects of PEMFs (75 Hz, 1.5 mT) were evaluated in cartilage explants treated with IL1β or not (control), in terms of cartilage structure, cellularity and proteoglycans, glycosaminoglycans, collagen II and transforming growth factor β1 synthesis by using histology, histomorphometry and immunohistochemistry.
Results
Making a comparison with control cartilage, IL1β-treated explants showed a decrease in cartilage matrix, structure and cellularity parameters. PEMFs were able to counteract the progression of OA acting on both cartilage cellularity and ECM in cartilage previously treated with IL1β. Normal distribution (Kolmogroc-Smirnov test) and homoscedasticity (Levene test) of data were verified, then, the non-parametric Kruskal Wallis test followed by Mann-Whiteny U test for pairwise comparisons were performed. The p-value was adjusted according to the Dunn-Sidak correction.
Conclusions
These results, obtained by culturing and treating cartilage explants from two different joints, confirmed that PEMF stimulation can be used as adjuvant therapy to preserve cartilage from detrimental effects of high inflammatory cytokine levels during OA.
... Osteoarthritis (OA) can be idiopathic in nature or initiated by trauma, joint malformation or excessive load on joints (Clutterbuck et al., 2009). Recent evidence has shown that OA also has an inflammatory component (Berenbaum, 2013;Konttinen et al., 2012), where matrix fragments can signal through toll-like receptors. ...
... MMP-13 is induced in chondrocytes by inflammatory cytokines in osteoarthritis, and modulated by several intracellular factors including NF-κB and p38-MAPK (Mengshol et al., 2000;Vincenti and Brinckerhoff, 2002). FGF-2 has been demonstrated to have a chondroprotective effect (Chia et al., 2009), and we recently showed that sulphated alginates decrease the expression of MMP-13 and ADAMTS-5 in chondrocytes by mediating FGF signalling (Öztürk et al., 2016). In the present study, sulphated alginate suppressed the expression of MMP-13 in non-stimulated chondrocytes but not following IL-1β stimulation, indicating different signalling mechanisms during intrinsic expression compared to inflammatory induction. ...
Loss of articular cartilage from ageing, injury or degenerative disease is commonly associated with inflammation, causing pain and accelerating degradation of the cartilage matrix. Sulphated glycosaminoglycans (GAGs) are involved in the regulation of immune responses in vivo, and analogous polysaccharides are currently being evaluated for tissue engineering matrices to form a biomimetic environment promoting tissue growth while suppressing inflammatory and catabolic activities. Here, we characterise physical properties of sulphated alginate (S-Alg) gels for use in cartilage engineering scaffolds, and study their anti-inflammatory effects on encapsulated chondrocytes stimulated with IL-1β. Sulphation resulted in decreased storage modulus and increased swelling of alginate gels, whereas mixing highly sulphated alginate with unmodified alginate resulted in improved mechanical properties compared to gels from pure S-Alg. S-Alg gels showed extensive anti-inflammatory and anti-catabolic effects on encapsulated chondrocytes induced by IL-1β. Cytokine-stimulated gene expression of pro-inflammatory markers IL-6, IL-8, COX-2 and aggrecanase ADAMTS-5 were significantly lower in the sulphated gels compared to unmodified alginate gels. Moreover, sulphation of the microenvironment suppressed the protein expression of COX-2 and NF-κB as well as the activation of NF-κB and p38-MAPK. The sulphated alginate matrices were found to interact with IL-1β, and proposed to inhibit inflammatory induction by sequestering cytokines from their receptors. This study shows promising potential for sulphated alginates in biomimetic tissue engineering scaffolds, by reducing cytokine-mediated inflammation and providing a protective microenvironment for encapsulated cells.
... Some studies suggest that the immunomodulatory activity of curcumin may involve direct targeting (activation) of TLRs (such as TLR4: a receptor of LPS) by pathogen-associated molecular patterns (PAMPs). PAMPS are microbial products that are recognized by cell surface receptors such as TLRs and cytosolic receptors (Buhrmann et al., 2011;Clutterbuck, Mobasheri, Shakibaei, Allaway, & Harris, 2009;Csaki, Mobasheri, & Shakibaei, 2009;Guimarães et al., 2012;Mathy, Sanchez, Priem, & Henrotin, 2007 Other mechanisms underlying curcumin modulation of immune responses are attributed to the regulation of various transcription factors such as nuclear factor (NF-κB), activator protein 1 (AP-1), signal transducer, and activator of transcription (STAT) and also their downstream signaling pathways (Gonzales & Orlando, 2008;Han, Keum, Seo, & Surh, 2002;Jiang, Wu, Wu, Yu, & Li, 2016;Kim et al., 2005;Soetikno et al., 2011;Shishodia, Sethi, & Aggarwal, 2005;Shishodia, Singh, & Chaturvedi, 2007). ...
... Curcumin blocks IL-1-mediated recruitment of IL-1 receptor-associated kinase (IRAK) to the IL-1RI in murine Thymoma EL-4 cells and thereby inhibits NF-κB activation (Jurrmann et al., 2005). Curcumin has been found to reduce serum levels of various inflammatory mediators such as cytokines, chemokines, and surface receptors including IL-1β, IL-6, soluble CD40 ligand, IL-8, macrophage inflammatory protein-1 (MIP-1), macrophage chemotactic protein-1 (MCP-1), TNF-α, adhesion molecules, C-Reactive Protein (CRP), CXCR-4, PG-E2, and soluble vascular cell adhesion molecule-1, as well as the erythrocyte sedimentation rate (ESR) (Banerjee, Tripathi, Srivastava, Puri, & Shukla, 2003;Clutterbuck et al., 2009;Davis et al., 2007;Jagetia & Aggarwal, 2007;Mathy-Hartert et al., 2009;Rahimnia et al., 2015). The effects of curcumin on the production of key cytokines and chemokines is shown in Table 1. ...
Curcumin is a dietary polyphenol from turmeric with numerous pharmacological activities. Novel animal and human studies indicate that curcumin can affect different immune cells, such as various T lymphocyte subsets, macrophages, dendritic cells, B lymphocytes and natural killer cells, which results in decreasing severity of various diseases with immunological etiology. The present review provides a comprehensive overview of the effects of curcumin on different immune cells and immune system-related diseases. This article is protected by copyright. All rights reserved.
... In the context of OA, curcumin exerts its action through various mechanisms, as evidenced by multiple studies over the years [307][308][309]. The storm of secretion of inflammatory mediators in OA causes joint destruction, and hence, this represents an important therapeutic target. ...
Osteoarthritis (OA) stands as a prevalent and progressively debilitating clinical condition globally, impacting joint structures and leading to their gradual deterioration through inflammatory mechanisms. While both non-modifiable and modifiable factors contribute to its onset, numerous aspects of OA pathophysiology remain elusive despite considerable research strides. Presently, diagnosis heavily relies on clinician expertise and meticulous differential diagnosis to exclude other joint-affecting conditions. Therapeutic approaches for OA predominantly focus on patient education for self-management alongside tailored exercise regimens, often complemented by various pharmacological interventions primarily targeting pain alleviation. However, pharmacological treatments typically exhibit short-term efficacy and local and/or systemic side effects, with prosthetic surgery being the ultimate resolution in severe cases. Thus, exploring the potential integration or substitution of conventional drug therapies with natural compounds and extracts emerges as a promising frontier in enhancing OA management. These alternatives offer improved safety profiles and possess the potential to target specific dysregulated pathways implicated in OA pathogenesis, thereby presenting a holistic approach to address the condition’s complexities.
... Together these results suggest minimal-to-no detrimental effect of G2HP BPL molecules on chondrocyte viability and function.We also examined the penetration of G2 and G2HP BPL molecules across an IL-1β-challenged cartilage explant as a function of time. Exposure to IL-1β is widely used to mimic the proteoglycan loss associated with early stages of OA.39 The IL-1β-conditioned cartilage explants exhibited higher uptake of G2 and G2HP BPL molecules compared to healthy cartilage, with approximately 20% higher uptake occurring after 6 h (Figure S20a). ...
Joint diseases, such as osteoarthritis, often require delivery of drugs to chondrocytes residing within the cartilage. However, intra‐articular delivery of drugs to cartilage remains a challenge due to their rapid clearance within the joint. This problem is further exacerbated by the dense and negatively charged cartilage extracellular matrix (ECM). Cationic nanocarriers that form reversible electrostatic interactions with the anionic ECM can be an effective approach to overcome the electrostatic barrier presented by cartilage tissue. For an effective therapeutic outcome, the nanocarriers need to penetrate, accumulate, and be retained within the cartilage tissue. Nanocarriers that adhere quickly to cartilage tissue after intra‐articular administration, transport through cartilage, and remain within its full thickness are crucial to the therapeutic outcome. To this end, we used ring‐opening polymerization to synthesize branched poly(l‐lysine) (BPL) cationic nanocarriers with varying numbers of poly(lysine) branches, surface charge, and functional groups, while maintaining similar hydrodynamic diameters. Our results show that the multivalent BPL molecules, including those that are highly branched (i.e., generation two), can readily adhere and transport through the full thickness of cartilage, healthy and degenerated, with prolonged intra‐cartilage retention. Intra‐articular injection of the BPL molecules in mouse knee joint explants and rat knee joints showed their localization and retention. In summary, this study describes an approach to design nanocarriers with varying charge and abundant functional groups while maintaining similar hydrodynamic diameters to aid the delivery of macromolecules to negatively charged tissues.
... Curcumin, the bioactive component of turmeric, has demonstrated potent anti-inflammatory, antioxidant, and chondroprotective properties [61]. Studies have shown that curcumin can inhibit the expression of proinflammatory cytokines, including TNF-α, IL-1β, and interleukin-6, as well as MMPs in chondrocytes [62][63][64]. Furthermore, curcumin suppresses the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), a key transcription factor involved in the regulation of inflammation and cartilage destruction [63]. ...
Background:
Chondrocytes are the primary cells responsible for maintaining cartilage integrity and function. Their role in cartilage homeostasis and response to inflammation is crucial for understanding the progression and potential therapeutic interventions for various cartilage-related disorders. Developing an accessible and cost-effective model to generate viable chondrocytes and to assess their response to different bioactive compounds can significantly advance our knowledge of cartilage biology and contribute to the discovery of novel therapeutic approaches.
Objective:
We developed a novel, streamlined protocol for generating chondrocytes from bone marrow-derived mesenchymal stem cells (BMSCs) in a 3D culture system that offers significant implications for the study of cartilage biology and the discovery of potential therapeutic interventions for cartilage-related and associated disorders.
Methods:
We developed a streamlined protocol for generating chondrocytes from BMSCs in a 3D culture system using an "in-tube" culture approach. This simple pellet-based 3D culture system allows for cell aggregation and spheroid formation, facilitating cell-cell and cell-extracellular matrix interactions that better mimic the in vivo cellular environment compared with 2D monolayer cultures. A proinflammatory chondrocyte model was created by treating the chondrocytes with lipopolysaccharide and was subsequently used to evaluate the anti-inflammatory effects of vitamin D, curcumin, and resveratrol.
Results:
The established protocol successfully generated a large quantity of viable chondrocytes, characterized by alcian blue and toluidine blue staining, and demonstrated versatility in assessing the anti-inflammatory effects of various bioactive compounds. The chondrocytes exhibited reduced inflammation, as evidenced by the decreased tumor necrosis factor-α levels, in response to vitamin D, curcumin, and resveratrol treatment.
Conclusions:
Our novel protocol offers an accessible and cost-effective approach for generating chondrocytes from BMSCs and for evaluating potential therapeutic leads in the context of inflammatory chondrocyte-related diseases. Although our approach has several advantages, further investigation is required to address its limitations, such as the potential differences between chondrocytes generated using our protocol and those derived from other established methods, and to refine the model for broader applicability and clinical translation.
... Curcumin has strong anti-inflammatory and antioxidant activity and has been reported to have chondroprotective activity using various in vitro and in vivo models [127][128][129][130]. Curcumin suppressed the expression of MMP-13 and increased the expression of type II collagen by inhibiting the NFκB pathway in rat primary chondrocytes [85]. Curcumin treatment reduced MMP-3 levels and inhibited aggrecan degradation in the equine cartilage explant model [81,82]. In another study, curcumin was found to suppress IL-1β induced MMP-3 expression in human cartilage explant [83]. ...
Osteoarthritis (OA) is the most common degenerative joint disease characterized by enzymatic degradation of the cartilage extracellular matrix (ECM) causing joint pain and disability. There is no disease-modifying drug available for the treatment of OA. An ideal drug is expected to stop cartilage ECM degradation and restore the degenerated ECM. The ECM primarily contains type II collagen and aggrecan but also has minor quantities of other collagen fibers and proteoglycans. In OA joints, the components of the cartilage ECM are degraded by matrix-degrading proteases and hydrolases which are produced by chondrocytes and synoviocytes. Matrix metalloproteinase-13 (MMP-13) and a disintegrin and metalloproteinase with thrombospondin motifs 4 and 5 (ADAMTS5) are the major collagenase and aggrecanase, respectively, which are highly expressed in OA cartilage and promote cartilage ECM degradation. Current studies using various in vitro and in vivo approaches show that natural compounds inhibit the expression and activity of MMP-13, ADAMTS4, and ADAMTS5 and increase the expression of ECM components. In this review, we have summarized recent advancements in OA research with a focus on natural compounds as potential therapeutics for the treatment of OA with emphasis on the prevention of cartilage ECM degradation and improvement of joint health.
... The signs of disease progression include narrowing of the joint space, cartilage lesions and surface fibrillation, loss of PG and collagen contents and increases in the fluid content in cartilage, formation of osteophytes, possible ligamentous and meniscal lesions, cement line advancement, subchondral sclerosis and cysts, chondrocyte clustering, increased subchondral bone plate thickness, fibrosis, and thickening and increased vascularity of synovium [3, 8, [248], and genome editing [249]. Experimental research has discovered that in biomechanical terms, cartilage stiffness decreases after a mechanical injury [250] and that in biochemical terms, the introduction of exogenous pro-inflammatory cytokines leads to PG loss in a dose-dependent manner [251]. However, there is no overall consensus in the OA scientific community about the pathogenesis of PTOA. ...
Osteoarthritis is a debilitating musculoskeletal whole-joint disease affecting the quality of life of over 340 million people globally. One of the most common disease sites is the knee joint. The disease is characterized by degradation of the articular cartilage covering the ends of bones. Degraded cartilage exhibits detrimental changes in its structure and composition, such as loss of proteoglycans (PG). The changes compromise the functioning of the cartilage. Ultimately, articular cartilage degradation leads to pain, joint space narrowing, joint stiffness, and a restricted range of motion, rendering the disease as a leading cause of disability. The disease phenotype initiated by traumatic injuries, i.e. post-traumatic osteoarthritis (PTOA), has been suggested to be triggered by two intertwined mechanisms: (1) biomechanics-related degradation resulting from impact damage, injuries, joint overloading, as well as mechanical shearing and wearing of cartilage, and (2) inflammation-related changes in cellular behavior and subsequent biochemical degradation.
These two fundamental mechanisms of cartilage degradation and their dynamics are not fully understood. As a result, the current clinical treatment options are limited, diagnosis is delayed to the late phases of the disease where extensive and irreparable degradation has already occurred, and there are no known ways to restore cartilage back to its healthy state. The endeavors to treat symptoms, but not the causes, are also expensive. Therefore, the best and most cost-effective cure would be prevention. In order to achieve this goal, there is a need for a novel computational framework capable of predicting the progression of PTOA.
Computational models incorporating biomechanical cartilage degradation mechanisms have recently been a focus of interest. However, there is a myriad of evidence underlining the importance of biochemical mechanisms. Thus, biomechanics-only models are likely to be incomplete in providing a comprehensive picture of the disease progression, and should be enriched with biochemical factors. When validated with experiments where both mechanisms are examined at several time points, such comprehensive models would represent promising tools for predicting PTOA progression and designing personalized intervention strategies.
The first aim of this thesis was to develop tissue-level finite element models to predict PG matrix damage of injured knee cartilage explants incorporating both biomechanical and biochemical degradation mechanisms based on previous in vitro findings. With respect to biomechanical degradation, several mechanical strain measures and one stress measure were investigated; for biochemical degradation, the net catabolic effect of inflammation was modeled with the diffusion of pro-inflammatory messenger proteins into cartilage (interleukin-1 cytokines) followed by cell-level perturbations in the biosynthesis levels of enzymatic proteins (aggrecanases) and PGs (aggrecan). These two approaches were combined, i.e. excessive levels of maximum shear strain were used in tandem with assessments of cytokine diffusion. The second aim was to study in more detail the biomechanical and biochemical degradation mechanisms by conducting a new set of in vitro experiments in a bovine cartilage PTOA model. We investigated these mechanisms at several early time points up until 12 days of explant culture incorporating a combination of injurious loading, cytokine challenge, and a cyclic loading protocol that was considered healthy and mimicking daily activities. The experiments provided important information about the early-stage disease progression in terms of changes in tissue glycosaminoglycan content (GAG, a building block of PGs), aggrecan biosynthesis, and cell viability. The final aim was to generate, for the first time, a subject-specific knee joint-level computational model with both biomechanical (chondral injury, gait information) and biochemical (synovial fluid cytokine concentrations) aspects in a patient who had undergone an anterior cruciate ligament reconstruction surgery. These results were compared to quantitative magnetic resonance imaging (MRI) findings at the 3-year follow-up.
The results of this thesis confirm that inflammation plays a crucial role alongside biomechanical factors in early PTOA progression. Thus, including both degradation mechanisms into adaptive models of cartilage allows a more comprehensive prediction of disease progression than is possible with biomechanics-only models. Specifically, injury-related PG loss and cell death localized near to lesions, cytokine-induced PG loss occurred also near to explant edges, and the combination of injurious loading and the presence of excess cytokine levels caused more PG matrix damage than either of these conditions alone. All of these findings were captured by the tissue-level computational models with elevated maximum shear strain as a biomechanical damage biomarker. Shear strain-driven PG loss was also prominent near a lesion in the joint-level model, corresponding with MRI findings which detected a localized substantial increase of T1rho relaxation time. Relaxation times were also increased in areas away from the lesions, possibly affected by the presence of pro-inflammatory cytokines as suggested in the new model.
Interestingly, in the new experiments, the chosen cyclic loading regime (15% strain amplitude, 1 Hz frequency) was beneficial for GAG retention in inflamed cartilage within the first four days of loading, but became deleterious after 12 days despite the increasing aggrecan biosynthesis rate. The early-stage (1--4 days) protecting effect of cyclic loading is suggested to be prominent in the lower transitional and deep cartilage zones.
In conclusion, the novel biomechanical and inflammation models of cartilage degradation presented in this thesis could be used to predict PTOA progression. In the future, such deterministic, physics-based models could help clinicians to assess either patient-specific or even population-specific risks of PTOA progression based on non-invasively imaged cartilage geometries, gait patterns if available, and synovial fluid biomarker profiles. The effects of different interventions could also be evaluated with the predictive model. Moreover, the new experimental findings provide a foundation for further in vivo studies where anti-catabolic drug treatment could be combined with early well-timed rehabilitation, but physical rehabilitation would not be continued if there were signs of chronic inflammation. The effectiveness of disease-modifying drug interventions could also be investigated with the proposed modeling platform. However, rigorous validation with large patient populations will be needed before this enters clinical use.
... Several transcriptional factors such as NF-κB, AP-1, Fos, and Jun play a significant role in inflammatory processes and arthritis [38,40,49]. Recent studies have shown that curcumin inhibits the NF-κB mediated inflammatory pathway in chondrocytes [56]. Also, curcumin was an effective inhibitor of transcription factors like NF-κB and AP-1, while activator of certain signal transducer (STAT) proteins, peroxisome proliferator-activated receptor-γ (PPARγ), and β-catenin, thus simultaneously regulating the expression of many genes [38,40,49]. ...
The Chemistry inside Spices and Herbs: Research and Development brings comprehensive information about the chemistry of spices and herbs with a focus on recent research in this field. The book is an extensive 2-part collection of 20 chapters contributed by experts in phytochemistry with the aim to give the reader deep knowledge about phytochemical constituents in herbal plants and their benefits. The contents include reviews on the biochemistry and biotechnology of spices and herbs, herbal medicines, biologically active compounds and their role in therapeutics among other topics. Chapters which highlight natural drugs and their role in different diseases and special plants of clinical significance are also included. Part II continues from the previous part with chapters on the treatment of skin diseases and oral problems. This part focuses on clinically important herbs such as turmeric, fenugreek, ashwagandha (Indian winter cherry), basil, Terminalia chebula (black myrobalan). In terms of phytochemicals, this part presents chapters that cover resveratrol, piperine and circumin.
... Curcumin also exerts potent anti-apoptotic and anti-catabolic effects on IL-1β-stimulated cultures of articular chondrocyte. 32 Further study by Huang and his colleagues (2013) showed that curcumin dramatically mitigate the progression and severity of collagen-induced arthritis in mice and inhibits the production of the B-cellactivating factor that belongs to the TNF family. 33 Curcumin also inhibits COX-2, LOX, and inducible NOS enzymes, which are important in the inflammatory process, thereby acting as an anti-inflammatory agent. ...
Background: Osteoarthritis (OA) is one of the most prevalent chronic degenerative arthritis diseases and a major cause of pain and physical disability among elderly patients. It can affect any joint in the body but most commonly, hip and knee joints. The etiology of the disease is multifactorial, OA affected by a range of mechanical and biochemical factors. Various studies provided compelling evidence that low-grade inflammation and synovitis are playing a pivotal role in its pathogenesis along with oxidative stress. Unfortunately, there is no cure for the disease; thus, most current treatments are prescribed for alleviating symptoms only. Curcumin, a natural polyphenolic compound, has been used for centuries in ayurvedic medicine that gained an increasing surge of interest to explore its potential properties. Many in vitro and in vivo studies reported powerful anti-inflammatory and antioxidant capacity for treating various pathological conditions, including OA, curcumin has shown chondroprotective potential on osteoarthritis disease. Aim of the Study: This study was designed to evaluate the anti-inflammatory effect of curcumin as an additive therapy to a non-steroidal anti-inflammatory drug, meloxicam, in the management of knee osteoarthritis. Patients and Method: This prospective open-labeled randomized controlled trial was conducted among patients with mild to moderate knee OA. Sixty-two patients were enrolled in this study; only 42 patients completed the study. Patients were assigned randomly into two groups; group (A) 21 patients treated with meloxicam alone (15 mg/day), group (B) 21 patients treated with a combination of meloxicam (15 mg/day), and curcumin (1600 mg/day) for 12 weeks. Inflammatory biomarkers (IL-1β, IL-6, and TNF-α) serum levels were evaluated at the time of enrolment and after 12 weeks of treatment. Results: Results gained from this study showed that treatment of knee OA patients with a combination of meloxicam and curcumin has a better effect on overall pain and physical function in addition to a remarkable decrease in serum pro-inflammatory biomarkers (IL-1β, IL-6, TNF-α) level (-39%,-24%,-30%) respectively after 12 weeks of treatment in respect to baseline levels. However, this reduction was significant only for IL-6. While those patients treated with meloxicam alone demonstrated no significant reduction. Conclusion: Curcumin represents a safe and effective anti-inflammatory product that exhibits a synergistic effect when used in combination with meloxicam, resulting in pain and physical activity improvement, which its anti-inflammatory effect may reflect.
... Curcumin, a polyphenol derived from the turmeric spice, has demonstrated anti-inflammatory and anti-oxidative properties in ex vivo studies in numerous species including humans and horses (Derochette et al., 2013;Gupta et al., 2013;Siard et al., 2016). In an in vitro equine articular cartilage explant model simulating OA, curcumin resulted in a decrease in glycosaminoglycan release and degeneration of the extracellular matrix (Clutterbuck et al., 2009). Furthermore, turmeric has demonstrated analgesic and anti-inflammatory effects in animal models and in clinical trials (Eke-Okoro et al., 2018). ...
Background
Herbal supplements containing curcumin and other ingredients are used for pain management in horses with osteoarthritis (OA).
Objectives
To test the effects of a herbal supplement containing curcumin and other ingredients in horses with lameness due to naturally occurring OA.
Study design
Two‐period randomised crossover design.
Methods
Ten Thoroughbreds with naturally occurring chronic OA were randomly assigned to the treatment (BLP; Buteless® Performance) or control (CTR, no supplement) groups and fed daily for 30 days. On Days −1 (before treatment), 15 and 30, lameness examination, range of motion, pain on palpation and force platform data were collected. Plasma curcumin concentration and its metabolites were measured on Days 1 and 14. Gastroscopy, a complete blood count and a serum biochemistry panel were performed on each horse before treatment Day −1 and Day 31. Gastric lesions (ulcers) were scored in real time by a masked investigator.
Results
Mean peak vertical force (PVF), measured by the force platform, significantly increased in the lame limb of the BLP‐treated horses on Days 15 (0.40 ± 0.13 N/kg, ( p = 0.0025) and 30 (0.63 ± 0.14 N/kg, p < 0.0001) compared to the CTR group. In addition, mean normalised PVF was higher in the BLP group on Day 15 ( p = 0.0438) and on day 30 ( p = 0.0003) when compared to CTR horses for the same days. The PVF significantly improved (≥5%; range, 5.2–33%) in six of nine individual BLP‐treated horses and did not improve (<5%; range, 0–3.4%) in three of nine BLP‐treated horses. Also, PVF improved (≥5%; range, 7.6–15.4%) in three of nine horses in the CRT group. Squamous gastric lesion scores significantly decreased in both groups by Day 31. Plasma curcumin‐O‐sulphate concentrations (1.2–3.3 ng/ml) were present in 9/10 BLP‐treated horses by Day 14.
Main limitations
Small sample size and absence of a positive treatment (non‐steroidal anti‐inflammatory drug) control.
Conclusions
The BLP supplement containing curcumin achieved plasma concentrations and improved weight bearing in some treated horses with naturally occurring OA.
... An in vitro study reported that curcumin antagonizes glycosaminoglycans release in cartilage explants (Clutterbuck, Mobasheri, Shakibaei, Allaway, & Harris, 2009). Another in vitro trial has demonstrated that curcumin inhibited the production of catabolic and inflammatory mediators by chondrocytes (Mathy-Hartert et al., 2009). ...
Turmeric (Curcuma longa) and its constituent, curcumin, have been used for their therapeutic properties for a long time. Most of the medicinal impacts of turmeric and curcumin might be attributed to their anti‐inflammatory, antinociceptive, and antioxidant effects. In the present review, the preventive and therapeutic potentials of turmeric and its active constituent, curcumin, on inflammatory disorders and pain as well as patents related to their analgesic and anti‐inflammatory effects, have been summarized to highlight their value on human health. A literature review was accomplished in Google Scholar, PubMed, Scopus, Google Patent, Patentscope, and US Patent. Several documents and patents disclosed the significance of turmeric and curcumin to apply in several therapeutic, medicinal, and pharmaceutical fields. These phytocompounds could be applied as a supplementary therapy in phytotherapy, inflammatory disorders such as arthritis, inflammatory bowel diseases, osteoarthritis, psoriasis, dermatitis, and different types of pain including neuropathic pain. However, because of inadequate clinical trials, further high‐quality studies are needed to firmly establish the clinical efficacy of the plant. Consistent with the human tendency to the usage of phytocompounds rather than synthetic drugs, particular consideration must be dedicated to bond the worth of turmeric and curcumin from basic sciences to clinical applications.
... Co-cultures were treated with or without rEq IL-1β (10 ng/mL; R&D Systems) for 24 h, washed with PBS, and medium was replenished without IL-1β. Intraarticular injection of IL-1β is used to model OA in horses by inducing synovitis and MMP activity [25], and GAG loss at a dose of 10 ng/mL [26]. ...
Objective
To gain insight into Treg interactions with synovial tissues in early OA, an equine tri-culture model of OA was used to test the hypothesis that Tregs, in the absence of T Helper 17 cells, are sufficient to resolve inflammation elicited by IL-1β.
Methods
To model normal and OA joints, synoviocytes were co-cultured with chondrocytes in a transwell system and +/- stimulated with IL-1β. Tregs were activated and enriched, then added to co-cultures, creating tri-cultures. At culture end, synoviocytes and chondrocytes were analyzed for gene expression, Treg Foxp3 expression was reexamined by flow cytometry, and conditioned media were evaluated by ELISA.
Results
Tregs increased IL-10 and IL-4 in tri-culture media and increased TIMP1 gene expression in synoviocytes and chondrocytes. Tregs increased IL-6 in conditioned media and Il6 gene expression in synoviocytes, which was additive with IL-1β. In chondrocytes, addition of Tregs decreased Col2b gene expression while Acan gene expression was decreased by IL-1β and addition of Tregs. IL-17A was detected in tri-cultures. CCL2 and CCL5 were increased in tri-cultures.
Conclusions
In a tri-culture model of OA, addition of Tregs resulted in conditions conducive to chondroprotection including increased concentration of IL-10 and IL-4 in conditioned media and increased gene expression of TIMP1 in both chondrocytes and synoviocytes. However, there was increased concentration of the catabolic cytokine IL-6, and decreased gene expression of Col2b and Acan in IL-1β-stimulated chondrocytes. These results suggest that blocking IL-6 could enhance Treg function in mitigating OA progression.
... Furthermore, lower aggrecan loss in IL-1β-stimulated articular cartilage explants has been reported to occur at a curcumin concentration no lower than 100 μM [49]. Additionally, it was reported that probucol would also bring side effects, such as ventricular arrhythmia, torsades de pointes, and syncope [50]. ...
Osteoarthritis (OA) is a chronic joint disease characterized by cholesterol accumulation in chondrocytes, cartilage degeneration, as well as extracellular matrix (ECM) destruction, and joint dysfunction. Curcumin, a chemical that can reduce cholesterol levels in OA patients, also can inhibit the progression of OA. However, a high concentration of curcumin may also trigger apoptosis in normal chondrocytes. Besides curcumin, probucol that is found can also effectively decrease the cholesterol level in OA patients. Considering that high cholesterol is a risk factor of OA, it is speculated that the combination treatment of curcumin and probucol may be effective in the prevention of OA. To investigate the possible effects of such two chemicals on OA pathophysiology, chondrocyte apoptosis and autophagy behavior under inflammatory cytokine stress were studied, and specifically, the PI3K-Akt-mTOR signaling pathway was studied. Methods. Cell proliferation, colony formation, and EdU assay were performed to identify the cytotoxicity of curcumin and probucol on chondrocytes. Transwell assay was conducted to evaluate chondrocyte migration under TNF-α inflammation stress. Immunofluorescence, JC-1, flow cytometry, RT-PCR, and western blot were used to investigate the signal variations related to autophagy and apoptosis in chondrocytes and cartilage. A histological study was carried out on OA cartilage. Glycosaminoglycan (GAG) release was determined to evaluate the ECM degradation under stress. Results. Compared with a single intervention with curcumin or probucol, a combined treatment of these two chemicals is more effective in terms of protecting chondrocytes from stress injury induced by inflammatory cytokines. The promoted protection may be attributed to the inhibition of apoptosis and the blockage of the autophagy-related PI3K/Akt/mTOR pathway. Such results were also verified in vitro by immunofluorescence staining of OA chondrocytes and in vivo by immunohistochemistry staining of cartilage. Besides, in vivo studies also showed that when applied in combination, curcumin and probucol could block the PI3K-AKT-mTOR signaling pathway; promote COL-II expression; suppress P62, MMP-3, and MMP-13 expression; and inhibit TNF-α-stimulated cartilage degradation. Moreover, the combined medication could help reduce the release of ECM GAGs in OA cartilage and alleviate the severity of OA. Conclusion. A combined treatment of curcumin and probucol could be used to protect chondrocytes from inflammatory cytokine stress via inhibition of the autophagy-related PI3K/Akt/mTOR pathway both in vitro and in vivo, which might be of potential pharmaceutical value for OA prevention and therapy.
1. Introduction
OA is a chronic inflammatory disease closely related to cartilage degeneration. Researchers have found that a high level of total cholesterol is related to the OA process. Specifically, in a prospective cohort study, total cholesterol and triglycerides are verified to be associated with new bone marrow lesion formation in asymptomatic middle-aged women [1] and result in cartilage defect and OA eventually. Another possible explanation could be lipid embolism caused by serum cholesterol, which may cause osteonecrosis leading to OA.
Hypertension, obesity, abnormal blood lipids, and high cholesterol, such conditions known as “metabolic syndrome” [2] are common among OA patients. The interrelationship between high cholesterol levels and increased risk of OA has been studied extensively in recent years [3, 4], and previous reports have shown that inhibition of de novo cholesterol synthesis may provide better OA remiment outcome [5, 6]. In this context, OA should be considered as a syndrome rather than merely a joint disease.
Autophagy is an important self-maintenance mechanism by which a cell protects itself when facing harmful stress [7]. Active autophagy is related to cholesterol effluent, and it can delay disease progress to a certain alleviated extent. Specifically, nitro-oleic acid, a ligand of CD36, reduces cholesterol accumulation by modulating fluidized LDL uptake and cholesterol efflux in RAW264.7 macrophages, and FGF21 induces autophagy-mediated cholesterol efflux to inhibit atherogenesis via the upregulation of RACK1 [8].
However, autophagy activity tends to drop in several cells and tissues with age. In OA chondrocytes, autophagy markers decrease significantly [9], accompanying with dysfunctional autophagy, enhanced apoptosis, and less migration [10]. Therefore, chemicals that can regulate autophagy in chondrocytes and stabilize the cholesterol level may be of potential medication value for OA prevention and therapy.
Curcumin, a diferuloylmethane, is extracted from the root of Curcuma longa [11]. In China and India, Curcuma longa has been used as a medicinal herb with a long history. Recent research indicated that curcumin can function to reduce cholesterol levels [12]. Additionally, previous authors have verified that curcumin can promote autophagy and reduce apoptosis in several cells [13]. Moreover, probucol, another cholesterol regulator, can also activate autophagy and inhibit apoptosis in nerve cells by blocking PI3K/Akt/mTOR signals [14]. Since autophagy takes an important role in chondrocyte physiology, and the PI3K/Akt/mTOR pathway is essential in regulating autophagy in OA patients [15], we speculated that curcumin and probucol may be of potential value for OA prevention. In the present study, these two chemicals were applied together to investigate their effects on chondrocytes in vitro and on cartilage in vivo.
2. Materials and Methods
2.1. Animals
Healthy male Sprague Dawley rats from the Animal Experimental Center of Wuhan University (Wuhan, China) were involved in this study. The rats were fed under specific pathogen-free conditions at a constant room temperature (24°C) and relative humidity (45%–55%). All rats had free access to sterile food and water and lived under a light/dark cycle of 12 h. The present study was approved by the Laboratory Animal Welfare & Ethics Committee, Renmin Hospital of Wuhan University. Efforts were made to minimize animal suffering in the study.
2.2. Reagents
The reagents included DMEM/F12 high glucose (Hyclone, Utah, USA), penicillin (Hyclone, Utah, USA), streptomycin (Hyclone, Utah, USA), curcumin (Bellancom, Beijing, China), trypsin (Google Biotechnology, Wuhan, China), collagenase-II, bovine serum albumin (BSA), probucol (Sigma-Aldrich, St. Louis, MO, USA), KeyFluor488 Click-iT EdU kits, DAPI, (KeyGEN BioTECH, Nanjing, China), AnnexV-PI kits (BD, USA), Counting Kit-8 (CCK-8) reagents, goat serum (Beyotime Institute of Biotechnology, Shanghai, China), TNF-α (Peprotech, Inc., Suzhou, China), Caspase-3, Bcl-2, Lc3, Bax, PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, GADPH, Berclin-1, COL-II, P62, FITC, Cy3, MMP-3, JC-1 assay kits (Abcam, USA), TRIzol reagents (Invitrogen, Thermo Fisher Scientific, Inc. USA), a RevertAid First Strand cDNA Synthesis kit (Fermentas; Thermo Fisher Scientific, Inc. USA), MMP detection kits (Solarbio Science & Technology, Beijing, China), and chemiluminescent luminol reagent (Santa Cruz Biotechnology, Texas, USA).
2.3. Chondrocyte Culture and Identification
Briefly, cartilage was extracted from the knee joints of 35 male Sprague-Dawley rats (4 weeks, weighing ). Cartilage samples were minced into thin slices (1 mm³) and digested with 3 ml of 0.25% trypsin for 40 min followed by further treatments with type II collagenase for another 6 h. Chondrocytes were then been centrifuged and collected. Subsequently, the isolated chondrocytes were cultured in 5 ml of DMEM/F12 with 20% fetal bovine serum and incubated at 37°C in 5% CO2.
2.4. CCK-8 Assay
To determine the appropriate study concentration of probucol and curcumin for further investigation in the subsequent experiments, cell viability was detected by the CCK-8 test. The chondrocytes were first cultured in a 96-well plate, and CCK-8 reagents were added, which was incubated at 37°C for another 2 h. The chondrocyte viability was detected by OD 450 nm with an automatic microplate reader. All studies were conducted in triplicate.
2.5. Cell Groups
Based on the above CCK-8 results, cells were randomly divided into five groups (): control, TNF-ɑ, TNF-ɑ + curcumin (50 μM), TNF-ɑ + probucol (100 μM), and TNF-ɑ + probucol (50 μM) + curcumin (25 μM). After excluding other cytokines or growth factors, TNF-α aqueous solution (20 ng/ml) was mixed with normal chondrocytes to mimic the inflammatory cytokine environment in OA [16]. 36 h later, curcumin, probucol, or both of them were added, and the chondrocytes were further incubated for another 24 h.
2.6. Flow Cytometry of Annexin V-FITC-PI Staining
The apoptosis rates of chondrocytes were measured with an AnnixV-PI apoptosis detection kit. In short, the chondrocytes were held at 25°C for 15 min and treated with PI solution (5 μl) and FITC-labeled annexin V (5 μl) for 10 min in the dark. The apoptosis rates were evaluated with a flow cytometer (BD Biosciences, USA).
2.7. Colony Formation Assay
The chondrocytes were placed on a six-well plate and mixed with curcumin and probucol at predescribed concentrations. After that, the cells were incubated for another two weeks without curcumin and probucol. Subsequently, the colonies were fixed with methanol, stained with Wright-Giemsa solution, and counted for their numbers [17].
2.8. Transwell Migration Assay
Transwell assays were used to evaluate cell migration. First, the transwell chambers were washed with serum-free medium, and chondrocytes were cultured in DMEM medium with 10% FBS as the chemical attractant. After incubation for 48 h, cells attached to the membrane were discarded, and those entering the lower membrane were fixed with methanol and stained with 0.2% crystal violet. Under a microscope (×200), the cells invaded by the matrix gel in 5 random fields of view were photographed.
2.9. JC-1 for Mitochondrial Membrane Permeability (MMP) Assessment
An MMP detection kit was used to evaluate the MMP in chondrocytes. After the chondrocytes were washed with PBS, 800 μl of JC-1 working fluid was mixed with the chondrocytes and stained at 37°C for 25 minutes. Subsequently, 2 ml of medium containing serum was added to the working fluid after staining. The red-green fluorescence ratio was measured by a FACS Caliber flow cytometer (Becton, Dickinson, and Company) and an Olympus fluorescence microscope (Olympus Corporation, Japan).
2.10. EdU Incorporation Assay
Chondrocyte proliferation was assessed by a keyFluor488 Click-iT-EdU kit. First, the chondrocytes were placed in a six-well plate, and 100 μl of EdU was added into the plate, followed by incubation at 37°C for 2 h. Second, the cells were fixed with 4% paraformaldehyde at room temperature, washed with BSA containing 3% glycine, and incubated with 0.5% TritonX-100 and 1× click-it reaction solution in the dark at room temperature. Last, Hoechst 33342 was added to the six-well plate, and the whole plate was placed in a dark environment for 20 minutes and then washed three times with PBS. The stained cells were observed with a fluorescence microscope.
2.11. Reverse Transcription Quantitative Polymerase Chain Reaction (RT-PCR)
TRIzol reagents were used to isolate the total RNA from chondrocytes. To determine the expression levels of inflammation-related genes, first-strand complementary cDNA chains were synthesized using the RevertAid First Strand cDNA Synthesis kit (Fermentas; Thermo Fisher Scientific, Inc.). Quantitative PCR was performed for 40 cycles in a StepOnePlus device (Applied Biosystems; Thermo Fisher Scientific, Inc.), and each cycle contained temperature at 95°C for 10 secs, followed by 5 seconds at 95°C and 20 seconds at 60°C. The additional primers were as follows: COL2, 5-CTTAGGACAGAGAGAGAAGG-3; Rev, 5-ACTCTGGGTGGCAGAGTTTC-3; MMP-3, 5-TTTGGCCGTCTCTTCCATCC-3; Rev, 5-GGAGGCCCAGAGTGTGAATG-3; MMP-13, 5-GG AGCATGGCGACTTCTAC-3; Rev, 5-GAGTGCTCCAGGGTCCTT; GADPH, 5-CTCAACTACATGGTCTACATGTTCCA-3; and Rev, 5-CTTCCCATTCTCAGCCTTGACT-3. GADPH was used as an internal reference. Moreover, the 2-ΔΔCq method was employed to calculate the relative levels of mRNA expression.
2.12. Western Blot
To extract the total proteins from the chondrocytes, organophosphorus inhibitors, protease inhibitors, and RIPA lysates were mixed at a ratio of 1 : 1 : 50. The proteins were separated by electrophoresis and transferred to polyvinylidene fluoride membranes, which were sealed for one hour. After that, a primary antibody was added to the membranes, which were then washed three times with TBST and incubated with horseradish peroxidase-labeled anti-rabbit goat IgG for 1 hour. Subsequently, the membranes were washed with TBST again, and the protein bands were observed with chemiluminescent luminol reagent (Santa Cruz Biotechnology, Inc.) and an Image Lab quantitative analysis system (Bio-Rad Laboratories Inc.). The relative protein levels were compared by normalizing to GADPH. The primary antibodies were as follows: Bcl-2, Bax, Beclin-1, LC3, mTOR, PI3K, Akt, p-Akt, p-PI3K, p-mTOR, and GAPDH.
2.13. OA Animal Model In Vivo Study
SD rats (8 weeks old, weighing 250-280 g) were randomly divided into five groups, which are denoted as control (), OA (), OA+50 mg/kg curcumin (), OA+100 mg/kg probucol (), and OA+75 mg/kg curcumin-probucol (). The specific dosages were determined according to the earlier literature [18]. A rat OA model was created by excising the medial meniscus and the anterior cruciate ligament of the rats’ right knee. Four weeks later, the groups with medications were treated with curcumin and probucol intramuscular injections once every three days for a total of 8 weeks, while the OA and the control groups were injected with normal saline. All rats were sacrificed after 3 months.
2.14. Immunofluorescence and Immunohistochemistry
After washed with PBS, the cartilage tissues and chondrocytes were fixed with paraformaldehyde for 12 h at 4°C and then dehydrated in 30% sucrose solution. Next, the tissues were sliced into pieces of 10 μM and incubated with P62 and COL-II at room temperature for 1 h. Subsequently, the section slices were then immunostained with FITC or Cy3-labeled secondary antibodies for 1 h, and DAPI was applied to counterstain the nuclei for 5 min. The sections were then incubated overnight with the primary antibodies for MMP-3 or MMP-13 at 4°C, and they were then incubated with biotinylated secondary antibodies. All sections were observed under an Olympus fluorescence microscope mentioned above. The proportions of stain-positive cells in the samples were analyzed by Image Pro Plus 6.0 (Media Cybernetics, Inc., USA).
2.15. Glycosaminoglycan Release Assay
Papain-digested cartilage explants and their defrosted supernatants were examined in 96-well plates using the dimethyl methylene blue (DMMB) method [16]. Briefly, each sample was diluted in distilled water to a total volume of 40 μl per well in triplicate. Shark chondroitin sulfate (Sigma-Aldrich) was used as a standard (0-70 ng). DMMB solution (200 μl) was added to each well, and the whole plate was immediately transferred to a Multiskan Ascent Scanner (Thermo Labsystems, Basingstoke, UK) with Ascent Software (version 2.6, Thermo Labsystems, Finland). Total GAG release was observed from a spectrophotometric reading of the digested cartilage and its supernatants at 540 nm. For each well, the percentage of GAG release was calculated by dividing the GAG readings from the supernatants by the total GAG release.
2.16. Statistical Analysis
For each group, the data are expressed as . Intragroup differences were assessed with Student’s -test and one-way analysis of variance by SPSS 16.0 (SPSS, Inc., USA) followed by a Bonferroni posthoc correction for multiple testing with GraphPad Prism (version 7.04; GraphPad Software, Inc., USA). Specifically, differences with were considered statistically significant.
3. Results
3.1. Effects of Probucol and Curcumin on Chondrocyte Proliferation
CCK-8 was used to detect chondrocyte activity. The most appropriate concentrations of probucol and curcumin to counteract inflammatory cytokine stress were found to be 100 μM and 50 μM, respectively (Figures 1(a) and 1(b)). It is noteworthy that both these substances could promote chondrocyte proliferation in a dose-dependent manner. Here, we chose these substances at optimal concentrations of 12.5%, 25%, 50%, and 100% to the most appropriate concentration for combinations [10]. Considering the possible reported side effects of such substances [19], a combination of curcumin 25 μM + probucol 50 μM was used in this study, and the results suggest that such a combination can promote chondrocyte proliferation (Figures 1(c)). Colony formation assays further confirmed that they play a promotive role in chondrocyte proliferation (Figures 1(d) and 1(e)), and such effect is in a synergistic way by the two chemicals. In the EdU assays with TNF-α treatment, the chondrocytes showed a low proliferation ratio. However, after treating with 50 μM curcumin or 100 μM probucol, the proliferation ratio got increased; and with the combined treatment, such increasement became more significant (Figures 1(f) and 1(g)).
(a)
... Stimulation of osteochondral slices with biological stimuli showed that chondrocytes in slice cultures responded adequately to external stimulation with catabolic molecules. TNF-α is known to be a proinflammatory cytokine in the joint and to induce cartilage matrix degradation both in vivo and in vitro [25,26], but other explant models either took significantly longer for a similarly strong reduction in matrix expression and degradation of proteoglycans or required additional catabolic stimuli such as oncostatin-M or interleukin-1β [11,27,28]. Here, the short perfusion distance between the medium and cells could be an advantage for pharmacological testing, since a shorter diffusion distance results in a higher and faster penetration by the corresponding factors than in thicker explant cultures. ...
For in vitro modeling of human joints, osteochondral explants represent an acceptable compromise between conventional cell culture and animal models. However, the scarcity of native human joint tissue poses a challenge for experiments requiring high numbers of samples and makes the method rather unsuitable for toxicity analyses and dosing studies. To scale their application, we developed a novel method that allows the preparation of up to 100 explant cultures from a single human sample with a simple setup. Explants were cultured for 21 days, stimulated with TNF-α or TGF-β3, and analyzed for cell viability, gene expression and histological changes. Tissue cell viability remained stable at >90% for three weeks. Proteoglycan levels and gene expression of COL2A1, ACAN and COMP were maintained for 14 days before decreasing. TNF-α and TGF-β3 caused dose-dependent changes in cartilage marker gene expression as early as 7 days. Histologically, cultures under TNF-α stimulation showed a 32% reduction in proteoglycans, detachment of collagen fibers and cell swelling after 7 days. In conclusion, thin osteochondral slice cultures behaved analogously to conventional punch explants despite cell stress exerted during fabrication. In pharmacological testing, both the shorter diffusion distance and the lack of need for serum in the culture suggest a positive effect on sensitivity. The ease of fabrication and the scalability of the sample number make this manufacturing method a promising platform for large-scale preclinical testing in joint research.
... Epigallocatechin-3-gallate inhibits expression of ADAMTS-1, -4 and -5 in chondrocytes, the activity of proteolytic enzymes in joint tissues, including synoviocytes and tendons, TNF-α-induced production of MMP-1 and MMP-3, IL-1β-induced expression of MMP-1, -3, and -13 in human tendon fibroblasts [114][115][116][117]. In vitro studies have confirmed an increase in the anticatabolic potential and activity of tissue inhibitors of MMP (TIMP) -1 and -2 under the action of epigallocatechin-3-gallate [95,118]. Curcumin inhibits the release of proteoglycans IL-1β, the production of MMP-1, MMP-9, and MMP-13 in tenocytes [12,27]. ...
Osteoarthritis (OA) is a degenerative joint disease and an important cause of incapacitation. There is a lack of drugs and effective treatments that stop or slow the OA progression. Modern pharmacological treatments, such as analgesics, have analgesic effects but do not affect the course of OA. Long-term use of these drugs can lead to serious side effects. Given the OA nature, it is likely that lifelong treatment will be required to stop or slow its progression. Therefore, there is an urgent need for disease-modifying OA treatments that are also safe for clinical use over long periods. Phytonutraceuticals are herbal products that provide a therapeutic effect, including disease prevention, which not only have favorable safety characteristics but may have an alleviating effect on the OA and its symptoms. An estimated 47% of OA patients use alternative drugs, including phytonutraceuticals. The review studies the efficacy and action mechanism of widely used phytonutraceuticals, analyzes the available experimental and clinical data on the effect of some phytonutraceuticals (phytoflavonoids, polyphenols, and bioflavonoids) on OA, and examines the known molecular effect and the possibility of their use for chondroprotection.
... Furthermore, many clinical trials revealed that curcumin could reduce the pain and improve the functionality of the osteoarthritic joint [92]. The anti -inflammatory properties of curcumin act through reduction in the synthesis of some inflammatory mediators [93] also, stimulate inhibition of IL-1β-that make induction of extracellular matrix degradation [94] and chondrocyte apoptosis [95]. ...
... Several studies have reported that the treatment of human articular chondrocytes with curcumin inhibited IL-1b and TNFa-induced NFkB activation, resulting in downregulation of COX-2 (57). Similarly, curcumin promoted inhibition of IL-1b-induced IL-6 and MMP-3 production by human chondrocytes (58) and extracellular matrix degradation (59). Moreover, curcumin supplementation decreased mRNA expressions of pro-inflammatory mediators, including IL-1b, TNF-a and MMP-3 in a post-traumatic OA mouse model Blots were repeated at least three times. ...
Osteoarthritis (OA) is a chronic and debilitating disease of the knee joint. OA of the knee is initiated by physical damage and accumulated oxidative stress, followed by an exaggerated inflammation leading to cartilage damage. Currently, no effective and safe therapeutic option capable of restoring articular cartilage tissue and joint architecture is available. We here report a novel and highly bioavailable formulation of curcumin, labeled as Next Generation Ultrasol Curcumin (NGUC), which was 64.7 times more bioavailable than natural 95% curcumin extract as demonstrated in rat bioavailability studies. We further investigated the protective effect of NGUC against monosodium iodoacetate (MIA)‐induced knee OA in rats. Analysis of X-ray and histopathological images revealed that NGUC supplementation restored joint architecture and reduced swelling of joints induced by MIA. NGUC treatment caused a significant reduction in the levels of inflammatory mediators such as TNF-α, IL-1β, IL-6, COMP, and CRP, and expressions of MMP-3, 5-LOX, COX-2, and NFκB in synovial tissue of rats with MIA-induced OA. NGUC also decreased serum MDA level and increased the levels of antioxidant enzymes SOD, CAT, and GPX. Thus, our results indicate that a novel formulation of curcumin with enhanced bioavailability effectively ameliorates the pathophysiology of OA.
... The major advantage of such a model is that the cell response can be determined in their natural environment and they are relatively simple and easy to produce. Most commonly used explant-based models thus far were of bovine origin and applied a super-physiological perturbing factor of either a fierce inflammatory cytokines treatment [10][11][12] or cartilage loading [13][14][15]. Next to inflammation and mechanical loading, recapitulation of endochondral ossification and thereby hypertrophy is also thought to be one of the major mechanisms driving the processes in OA [16]. ...
IntroductionLikely due to ignored heterogeneity in disease pathophysiology, osteoarthritis (OA) has become the most common disabling joint disease, without effective disease-modifying treatment causing a large social and economic burden. In this study we set out to explore responses of aged human osteochondral explants upon different OA-related perturbing triggers (inflammation, hypertrophy and mechanical stress) for future tailored biomimetic human models.Methods
Human osteochondral explants were treated with IL-1β (10 ng/ml) or triiodothyronine (T3; 10 nM) or received 65% strains of mechanical stress (65% MS). Changes in chondrocyte signalling were determined by expression levels of nine genes involved in catabolism, anabolism and hypertrophy. Breakdown of cartilage was measured by sulphated glycosaminoglycans (sGAGs) release, scoring histological changes (Mankin score) and mechanical properties of cartilage.ResultsAll three perturbations (IL-1β, T3 and 65% MS) resulted in upregulation of the catabolic genes MMP13 and EPAS1. IL-1β abolished COL2A1 and ACAN gene expression and increased cartilage degeneration, reflected by increased Mankin scores and sGAGs released. Treatment with T3 resulted in a high and significant upregulation of the hypertrophic markers COL1A1, COL10A1 and ALPL. However, 65% MS increased sGAG release and detrimentally altered mechanical properties of cartilage.Conclusion
We present consistent and specific output on three different triggers of OA. Perturbation with the pro-inflammatory IL-1β mainly induced catabolic chondrocyte signalling and cartilage breakdown, while T3 initiated expression of hypertrophic and mineralization markers. Mechanical stress at a strain of 65% induced catabolic chondrocyte signalling and changed cartilage matrix integrity. The major strength of our ex vivo models was that they considered aged, preserved, human cartilage of a heterogeneous OA patient population. As a result, the explants may reflect a reliable biomimetic model prone to OA onset allowing for development of different treatment modalities.
... In cartilage explant models, curcumin reduced IL-1-induced MMP-3, GAG, and PGE2 release. 26,48 These studies further aid to the promise of using resveratrol and curcumin as anti-AGE and anti-IL-1 therapeutics; however, our study is the first to report the ability of either of these drugs to suppress both AGE crosslinking and IL-1-induced catabolic activity in a live tissue matrix model. To study nonenzymatic glycation, past work has either crosslinked the collagen network of dead cartilage explants using high sugar concentrations to study changes in tissue mechanical properties or separately evaluated the downstream cellular response of AGE accumulation using chondrocyte culture. ...
Objective
Advanced glycation end-product (AGE) accumulation is implicated in osteoarthritis (OA) pathogenesis in aging and diabetic populations. Here, we develop a representative nonenzymatic glycation-induced OA cartilage explant culture model and investigate the effectiveness of resveratrol, curcumin, and eugenol in inhibiting AGEs and the structural and biological hallmarks of cartilage degeneration.
Design
Bovine cartilage explants were treated with AGE–bovine serum albumin, threose, and ribose to determine the optimal conditions that induce physiological levels of AGEs while maintaining chondrocyte viability. AGE crosslinks, tissue stiffness, cell viability, metabolism and senescence, nitrite release and loss of glycosaminoglycans were assessed. Explants were cotreated with resveratrol, curcumin, or eugenol to evaluate their anti-AGE properties. Blind docking analysis was conducted to estimate binding energies of drugs with collagen II.
Results
Treatment with 100 mM ribose significantly increased AGE crosslink formation and tissue stiffness, resulting in reduced chondrocyte metabolism and enhanced senescence. Blind docking analysis revealed stronger binding energies of both resveratrol and curcumin than ribose, with glycation sites along a human collagen II fragment, indicating their increased likelihood of competitively inhibiting ribose activity. Resveratrol and curcumin, but not eugenol, successfully inhibited AGE crosslink formation and its associated downstream biological response.
Conclusions
We establish a cartilage explant model of OA that recapitulates several aspects of aged human cartilage. We find that resveratrol and curcumin are effective anti-AGE therapeutics with the potential to decelerate age-related and diabetes-induced OA. This in vitro nonenzymatic glycation-induced model provides a tool for screening OA drugs, to simultaneously evaluate AGE-induced biological and mechanical changes.
... TNF-α is a pro-in ammatory cytokine and well known for the induction of cartilage matrix degradation -both in-vivo and in-vitro (26) (27). However, Pretzel et al. required 14 days to achieve minimal proteoglycan reduction with TNF-α mono treatment in a 3 mm diameter porcine explant model; other models required the addition of other potent degrading proteins such as Oncostatin-M or Interleukin-1β to reach similar results (14), (28), (29). In the F-cultivated group, addition of TNF-α to the slices showed a dose dependent decrease in expression cartilage ECM relevant genes COL2A1, ACAN and COMP and an active reduction of proteoglycans in the tissue by 32% in only 7 days. ...
Background: Current advances in musculoskeletal research yielded numerous new insights and therapy options for diseases of the joints but preclinical in-vitro testing is currently limited to self-manufactured 2D and 3D cell cultures only remotely mimicking the in-vivo situation in regards of tissue composition and cell configuration. While in-vivo animal models are a lot more lifelike, they are inherently connected with ethical concerns, translation to the human situation, logistic efforts and high costs. Here we explored the use of native human life slice cultures from explanted tibial plateaus upon their feasibility to serve as a highly lifelike and easy-to-handle model of the human joint which can be obtained in masses from few tissue samples.
Methods: Osteochondral slices (1cm x 1cm x 500µm) were prepared with a special microtome from 23 human tibial plateaus and subsequently cultivated in hanging inserts over the course of 7 or 21 days. Short-term cultivated slices were stimulated with either 800 pM/1.2 nM TNF-α or 800 pM TGF-β3. During cultivation, viability of tissue cells was assessed via laser microscopy / resazurin assay. After cultivation, gene expression of cartilage ECM Marker proteins was quantified with RT-PCR. TNF-α stimulated slices and their controls were stained with Safranin-O and analyzed via histomorphometry to quantify tissue proteoglycan content.
Results: Tissue cell viability remained >90% over the three weeks. Laser scanning microscopy revealed highly conserved spatial alignment of cells inside the cartilage. Incubation of the slice cultures with TNF-α showed a significant, dose dependent decrease in mRNA expression of cartilage proteins collagen II, cartilage oligomeric matrix protein and aggrecan while incubation with TGF-β3 caused a significant increase of early bone formation proteins collagen I and also cartilage oligomeric matrix protein. Histologically, TNF-α incubation caused a 32% reduction of proteoglycans, detachment of collagen fibers and cell swelling.
Conclusion: In summary, native osteochondral slice cultures provide a stable and manipulable, physiological model of human joint bone and cartilage biology. The setup is easy to produce and handle, scalable, and contributes to 3R principles in biomedical research, suggesting itself as a platform for preclinical testing especially regarding high-throughput applications.
... Furthermore, curcumin was shown to suppress pro-inflammatory signaling pathways in human and rat articular chondrocytes by targeting IL-1β-induced transcription factor nuclear factor kappa B (NF-κB) activation and the suppression of NF-κB-regulated gene end-products such as cyclooxygenase−2 (COX−2) and MMP−9 [37,50]. Moreover, in IL-1β-stimulated cartilage explants, curcumin was able to block the IL−1β-induced loss of glycosaminoglycans (GAGs) [51]. Contrary to these findings, a comparative study investigating the effect of glucosamine (GlcN), curcumin, and diacerein in human chondrocytes found that curcumin was not as effective as GlcN and diacerein in stimulating expression of cartilage-specific genes such as aggrecan (AGC) and COL2A1, and it was even toxic at high concentrations [52]. ...
It is estimated that by 2023, approximately 20% of the population of Western Europe and North America will suffer from a degenerative joint disease commonly known as osteoarthritis (OA). During the development of OA, pro-inflammatory cytokines are one of the major causes that drive the production of inflammatory mediators and thus of matrix-degrading enzymes. OA is a challenging disease for doctors due to the limitation of the joint cartilage's capacity to repair itself. Though new treatment approaches, in particular with mesenchymal stem cells (MSCs) that integrate the tissue engineering (TE) of cartilage tissue, are promising, they are not only expensive but more often do not lead to the regeneration of joint cartilage. Therefore, there is an increasing need for novel, safe, and more effective alternatives to promote cartilage joint regeneration and TE. Indeed, naturally occurring phytochemical compounds (herbal remedies) have a great anti-inflammatory, anti-oxidant, and anabolic potential, and they have received much attention for the development of new therapeutic strategies for the treatment of inflammatory diseases, including the prevention of age-related OA and cartilage TE. This paper summarizes recent research on herbal remedies and their chondroinductive and chondroprotective effects on cartilage and progenitor cells, and it also emphasizes the possibilities that exist in this research area, especially with regard to the nutritional support of cartilage regeneration and TE, which may not benefit from non-steroidal anti-inflammatory drugs (NSAIDs).
... [9,10] Curcuminoids stimulated matrix synthesis by restoring glycosaminoglycan (GAG) synthesis and type II collagen. [11] There are increasing clinical studies investigating the therapeutic efficacy of curcuminoids in treating patients with KOA. However, the limitations of these clinical studies is relatively small patient sample size. ...
Background:
Knee osteoarthritis (KOA) is 1 of the commonest cause of disability with joint pain in adults and a burden on healthcare resources. The limitations of current KOA treatment necessitate further researches to discover the more efficacious and safety treatments. There are increasing clinical studies investigating the potential protective effects of Curcuminoids in the alleviation of symptoms in patients suffering from KOA. However, the convincing evidence indicating the efficacy of curcuminoids for patients suffering from KOA remains unclear.
Methods:
Several databases including PubMed, Web of Science, Cochrane Library, Embase, Chinese Biomedical Literature Database, Chinese National Knowledge Infrastructure, and Wanfang Database will be searched. And the language was not limited. We will include all Randomized controlled trials that use curcuminoids to treat patients with KOA, regardless of blinding. If the pre-crossover data can be analyzed to avoid carryover effects, the crossover randomized trials also are included. Meanwhile, We will exclude non-randomized controlled trials, qualitative studies, uncontrolled clinical trials and laboratory studies. The primary end point include Western Ontario and McMaster Universities Osteoarthritis Index, visual analog scale scores and Lequesne's pain functional index. The secondary end points are total effective rate and adverse effects. The Review Manager Version 5.3 will be used to perform the data synthesis and subgroup analysis.
Discussion:
There are evidences that supports the potential protective effects of Curcuminoids in the alleviation of symptoms in patients suffering from KOA. This systematic review and meta-analysis would provide convincing evidence indicating that curcuminoids relieve the symptoms of patients suffering from KOA.
Registration:
Open Science Framework (OSF) registries (https://osf.io/fz29b) with the registration DOI: 10.17605/OSF.IO/FZ29B.
... MMP-1 and -13 were known as collagenases, MMP-2 and -9 were gelatinases, and MMP-3 was stromelysin that degrade aggrecan's core protein. A previous study demonstrated that when MMPs were upregulated, PGs, glycosaminoglycans (GAGs), and COL2 were also upregulated (Clutterbuck et al., 2009;Jackson et al., 2006). Upregulated MMPs led to degrade PG and GAGs, therefore, in the osteoarthritis status, the levels of PG and GAGs become to be increased (Jackson et al., 2006). ...
A complex of curcumin and lecithin developed to improve the solubility of curcumin, enhanced its chondroprotective effect via an anti-inflammatory mechanism. In macrophage, proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, prostaglandin E2 (PGE2), and nitric oxide (NO) were quantified. In addition, the activity of nuclear factor (NF)-κB was examined. With chondrocytes, inflammatory mediators were assessed by measuring the secretion levels of IL-6, IL-8, and PGE2, also the mRNA expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Metalloproteinases (MMPs), tissue inhibitor of metalloprotease (TIMP)-1, type II collagen (COL2), proteoglycan (PG), and hyaluronic acid (HA) were measured with respect to the articulation surface. The complex promoted the anti-inflammatory effect by the inhibition of inflammatory mediators. In addition, mRNA expression levels ameliorated. Furthermore, it was effective in decreasing extracellular secretion of polypeptides, also corresponding intracellular MMPs and TIMP-1. In conclusion, the complex may be developed as a functional supplement to maintain articulation health.
... Osteoarthritis (OA) is a complex systemic disease in which the whole joint, including the synovium, articular cartilage, subchondral bone, tendons, and muscles, is affected [1]. OA can be idiopathic or initiated by aging, trauma, malformations, or inflammatory disease [2,3]. As it affects up to 10% of males and 18% of females greater than 48 yr of age, OA is becoming a serious health problem, and currently established therapies for OA insufficiently address the clinical need [1]. ...
Osteoarthritis is an inflammatory disease in which all joint-related elements, articular cartilage in particular, are affected. The poor regeneration capacity of this tissue together with the lack of pharmacological treatment has led to the development of regenerative medicine methodologies including microfracture and autologous chondrocyte implantation (ACI). The effectiveness of ACI has been shown in vitro and in vivo , but the use of other cell types, including bone marrow and adipose-derived mesenchymal stem cells, is necessary because of the poor proliferation rate of isolated articular chondrocytes. In this investigation, we assessed the chondrogenic ability of human dental pulp stem cells (hDPSCs) to regenerate cartilage in vitro and in vivo . hDPSCs and primary isolated rabbit chondrocytes were cultured in chondrogenic culture medium and found to express collagen II and aggrecan. Both cell types were cultured in 3% alginate hydrogels and implanted in a rabbit model of cartilage damage. Three months after surgery, significant cartilage regeneration was observed, particularly in the animals implanted with hDPSCs. Although the results presented here are preliminary, they suggest that hDPSCs may be useful for regeneration of articular cartilage.
... The present study further demonstrated that NF-κB signaling was involved in the regulation of type II collagen and MMP-13 expression levels in IL-1β-stimulated chondrocytes by using an inhibitor of NF-κB activation. Clutterbuck et al (29,30) reported that treatment with curcumin at concentrations >100 µM for 48 h or 5 days led to the death of chondrocytes, and that the release of glycosaminoglycan by tissues cultured in vitro was suppressed by curcumin at high concentrations, which subsequently inhibited the proliferation of chondrocytes. However, no significant effects of 50 µM curcumin treatment on survival and migration of chondrocytes were identified in the present study (data not shown), thereby indicating the safety of the curcumin concentration employed. ...
Curcumin possesses strong anti-inflammatory, anti-rheumatoid and anti-oxidative activities, and has the potential to inhibit nuclear factor‑κB (NF‑κB) signaling. Cartilage damage in osteoarthritis (OA) is largely mediated by interleukin-1β (IL-1β) via activation of various transcription factors, including NF‑κB and activator protein‑1. The aim of the present study was to determine whether IL‑1β induces matrix metalloproteinase-13 (MMP-13) expression and inhibits type II collagen expression, as well as to examine whether cell proliferation may be inhibited by curcumin through the inhibition of NF‑κB signaling. The effects of curcumin were investigated in rat articular chondrocyte cell cultures treated with IL‑1β in the presence or absence of curcumin or the NF‑κB inhibitor pyrrolidine dithiocarbamate. Western blotting and reverse transcription‑quantitative polymerase chain reaction were conducted to evaluate protein and mRNA expression levels of type II collagen, MMP‑13, NF‑κB inhibitor α (IκBα), phosphorylated‑IκBα and NF‑κB subunit p65/RelA. Western blotting and immunofluorescence were performed to examine the effects of curcumin on the expression, phosphorylation and nuclear translocation of NF‑κB‑associated proteins. The effects of curcumin on cell proliferation were evaluated by Cell Counting Kit‑8 (CCK‑8). Curcumin was demonstrated to inhibit the IL‑1β‑induced activation of NF‑κB by suppressing IκBα phosphorylation and p65/RelA nuclear translocation. These events were associated with the downregulation of MMP‑13 expression and the upregulation of type II collagen expression, both of which are considered to be NF‑κB targets. CCK‑8 assays revealed that co‑treatment with curcumin resulted in increased proliferation in IL‑1β‑treated chondrocytes. These findings implicated curcumin as a naturally occurring anti‑inflammatory agent for the treatment of OA via inhibition of NF‑κB signaling.
... 21 In vitro studies have also shown that curcuminoids inhibit IL-1b-induced glycosaminoglycan (GAG) release from animal and human OA cartilage explants. 22,23 Randomized clinical trials (RCTs) in animals have suggested that Curcuma extracts (comprised of mixtures of Indian and Javanese turmeric with essential oils) may have beneficial effects in treatment of OA 24 and results of a recent large observational study including 820 adults with painful OA showed evidence of benefits with curcuminoids in real-life clinical practice. 25 Several clinical trials investigating the effect of curcuminoids in OA have also been published. ...
Aim:
To critically appraise and evaluate the evidence for effectiveness of curcuminoids in the treatment of osteoarthritis (OA) in adults.
Methods:
We conducted electronic searches in Medline, Embase, AMED, Cinahl and the Cochrane library. We included randomized controlled trials (RCTs) that investigated the effectiveness of orally-administered curcuminoids in OA in adults, and assessed risk of bias using the Cochrane risk of bias criteria. We used a random-effect model for meta-analysis.
Results:
We included seven studies with a total of 797 participants with primarily knee OA. All studies were conducted in Asia. The overall risk of bias was moderate. Compared with placebo, curcuminoids significantly reduced knee pain (visual analogue scale): (standardized mean difference: -3.45; 95% CI: -5.52 to -1.38; I(2) = 95% P = 0.001), and improved quality of life (Lequesne pain-function index): (mean difference: -2.69; 95% CI: -3.48 to -1.90; I(2) = 0% P < 0.00001). There were significantly fewer effects on pain relief, knee stiffness and physical function with curcuminoids compared with ibuprofen. Significant improvements in Western Ontario and McMaster Universities Arthritis Index total scores, with significant reductions in the use of rescue medication were also observed with curcuminoids. No serious adverse events were reported.
Conclusions:
Curcuminoids may have some beneficial effects on knee pain and quality of life in patients with knee OA. However, they are less effective at relieving pain compared with ibuprofen. Curcuminoids appear safe on the short-term, and may reduce the need for rescue medication. Published RCTs vary in reporting quality, are characterized by small sample sizes, and have all been conducted in Asia. Further clinical trials are therefore warranted.
... Evidence from several recent in vitro studies suggests that curcumin may exert a chondroprotective effect through actions such as antiinflammatory, anti-oxidative stress, and anti-catabolic activity that are critical for mitigating OA disease pathogenesis and symptoms. For example, curcumin has been shown to mitigate the inflammatory process by decreasing synthesis of inflammatory mediators such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, IL-8, prostaglandin E2 (PGE 2 ) , and cyclooxygenase-2 (COX-2) [6][7][8], inhibit IL-1β-induced extracellular matrix degradation [9] and chondrocyte apoptosis [10,11], and mitigate the overproduction of reactive oxygen and nitrogen species [12,13]. Moreover, curcumin, by inhibiting the activator protein 1 (AP-1) pathway [14] and nuclear factor kappa B (NF-kB) activation [14][15][16], suppresses the gene expression of a number of matrix metalloproteinases (MMPs), which play critical roles in the breakdown of the cartilage extracellular matrix [7,[14][15][16][17]. ...
Background:
Curcumin has been shown to have chondroprotective potential in vitro. However, its effect on disease and symptom modification in osteoarthritis (OA) is largely unknown. This study aimed to determine whether curcumin could slow progression of OA and relieve OA-related pain in a mouse model of destabilization of the medial meniscus (DMM).
Methods:
Expression of selected cartilage degradative-associated genes was evaluated in human primary chondrocytes treated with curcumin and curcumin nanoparticles and assayed by real-time PCR. The mice subjected to DMM surgery were orally administered curcumin or topically administered curcumin nanoparticles for 8 weeks. Cartilage integrity was evaluated by Safranin O staining and Osteoarthritis Research Society International (OARSI) score, and by immunohistochemical staining of cleaved aggrecan and type II collagen, and levels of matrix metalloproteinase (MMP)-13 and ADAMTS5. Synovitis and subchondral bone thickness were scored based on histologic images. OA-associated pain and symptoms were evaluated by von Frey assay, and locomotor behavior including distance traveled and rearing.
Results:
Both curcumin and nanoparticles encapsulating curcumin suppressed mRNA expression of pro-inflammatory mediators IL-1β and TNF-α, MMPs 1, 3, and 13, and aggrecanase ADAMTS5, and upregulated the chondroprotective transcriptional regulator CITED2, in primary cultured chondrocytes in the absence or presence of IL-1β. Oral administration of curcumin significantly reduced OA disease progression, but showed no significant effect on OA pain relief. Curcumin was detected in the infrapatellar fat pad (IPFP) following topical administration of curcumin nanoparticles on the skin of the injured mouse knee. Compared to vehicle-treated controls, topical treatment led to: (1) reduced proteoglycan loss and cartilage erosion and lower OARSI scores, (2) reduced synovitis and subchondral plate thickness, (3) reduced immunochemical staining of type II collagen and aggrecan cleavage epitopes and numbers of chondrocytes positive for MMP-13 and ADAMTS5 in the articular cartilage, and (4) reduced expression of adipokines and pro-inflammatory mediators in the IPFP. In contrast to oral curcumin, topical application of curcumin nanoparticles relieved OA-related pain as indicated by reduced tactile hypersensitivity and improved locomotor behavior.
Conclusion:
This study provides the first evidence that curcumin significantly slows OA disease progression and exerts a palliative effect in an OA mouse model.
... Tannic acid (TA) extracted from oak trees also has beneficial biological activities in cancer and diabetes [19][20][21]. Previous findings relating to the role of polyphenols in arthritis mostly elucidate the mechanisms of inhibiting inflammatory cytokines or MMPs [15,[22][23][24][25][26][27][28]. ...
Arthritic diseases, such as osteoarthritis and rheumatoid arthritis, inflict an enormous health care burden on society. Osteoarthritis, a degenerative joint disease with high prevalence among older people, and rheumatoid arthritis, an autoimmune inflammatory disease, both lead to irreversible structural and functional damage to articular cartilage. The aim of this study was to investigate the effect of polyphenols such as catechin, quercetin, epigallocatechin gallate, and tannic acid, on crosslinking type II collagen and the roles of these agents in managing in vivo articular cartilage degradation. The thermal, enzymatic, and physical stability of bovine articular cartilage explants following polyphenolic treatment were assessed for efficiency. Epigallocatechin gallate and tannic acid-treated explants showed >12 °C increase over native cartilage in thermal stability, thereby confirming cartilage crosslinking. Polyphenol-treated cartilage also showed a significant reduction in th
Nutritional epigenomics is exceptionally important because it describes the complex interactions among food compounds and epigenome modifications. Phytonutrients or bioactive compounds, which are secondary metabolites of plants, can protect against osteoarthritis by suppressing the expression of inflammatory and catabolic mediators, modulating epigenetic changes in DNA methylation, and the histone or chromatin remodelling of key inflammatory genes and noncoding RNAs. The combination of natural epigenetic modulators is crucial because of their additive and synergistic effects, safety and therapeutic efficacy, and lower adverse effects than conventional pharmacology in the treatment of osteoarthritis. In this review, we have summarized the chondroprotective properties of bioactive compounds used for the management, treatment, or prevention of osteoarthritis in both human and animal studies. However, further research is needed into bioactive compounds used as epigenetic modulators in osteoarthritis, in order to determine their potential value for future clinical applications in osteoarthritic patients as well as their relation with the genomic and nutritional environment, in order to personalize food and nutrition together with disease prevention.
Nutritional epigenomics is exceptionally important because describes the complex interactions among food compounds and epigenome modifications. A healthy diet can improve the quality of life and, alleviate the progression and symptomatology of many complex diseases such as osteoarthritis (OA). Phytonutrients or bioactive compounds, which are secondary metabolites of plants, can protect against OA by suppressing the expression of inflammatory and catabolic mediators, and modulating epigenetic changes in DNA methylation, histone or chromatin remodelling of key inflammatory genes and noncoding RNAs. The combination of natural epigenetic modulators is crucial because their additive and synergistic effects, safety and therapeutic efficacy, and lower adverse effects than conventional pharmacology in the treatment of OA. In this review, we have summarised the chondroprotective properties of bioactive compounds and nutraceuticals for the management, treatment, or prevention of OA in both human and animal [1]studies. Some of them have been considered as natural epigenetic modulators that can modify the activity of various epigenetic factors and, alter the expression of genes related to inflammation and cartilage destruction. However, this complex pathology with inflammatory mediators has been little studied at the nutriepigenomic level. Further research is needed towards bioactive compounds as epigenetic modulators in OA, likewise, determine their potential value for future clinical applications in OA patients.
No studies have evaluated the effect of culture in serum-free media (SF) vs. media supplemented with equine serum (ES) on co-culture of synovial membrane and cartilage tissue explants. The study objective was to evaluate the effects of equine serum supplementation on induced production of inflammatory and catabolic mediators from articular cartilage and synovial explants while in co-culture. Articular cartilage and synovial membrane explants were harvested from femoropatellar joints of 5 adult horses. Cartilage and synovial explants were harvested from the stifle of 5 horses, placed in co-culture, stimulated with IL-1β (10 ng/ml) and maintained in culture for 3, 6 and 9 days in 10% ES or SF. At each time point, media was harvested for analysis of cellular viability (Lactate dehydrogenase) and elution of glycosaminoglycans (Dimethylene Blue Binding Assay). Tissue explants were harvested for histopathologic and gene expression analyses. No differences in cell viability were observed between SF and ES groups. SF culture produced an upregulation of TNF-α in synovial membrane and ADAMTS-4 and 5 in articular cartilage at 9 days of culture. ES produced an upregulation of aggrecan expression in cartilage at 9 days of culture. No differences in tissue viability were found between culture media, but SF media produced a higher glycosaminoglycan concentration in media at 3 days of culture. The addition of 10% ES produced a slight chondroprotective effect in an inflamed co-culture system. This effect should be considered when designing studies evaluating treatment of serum or plasma-based orthobiologic studies in vitro.
Background: Curcuminoids (CURs) are the principal ingredients of Curcuma longa L. [Zingiberaceae] (CL)—an herbal plant used in east Asia to alleviate pain and inflammation. Thus far, the therapeutic effects of CURs for knee osteoarthritis (OA) uncovered by multiple reviews remained uncertain due to broadly involving trials with different agents-combined or CURs-free interventions. Therefore, we formed stringent selection criteria and assessment methods to summarize current evidence on the effectiveness of CURs alone in the treatment of knee OA.
Methods: A series of databases were searched for randomized controlled trials (RCTs) evaluating the efficacy of CURs for knee OA. Clinical outcomes were evaluated using meta-analysis and the minimum clinically important difference (MCID) for both statistical and clinical significance.
Results: Fourteen studies with 1633 patients were included. Overall, CURs were significantly more effective than controls in the improvements of VAS for pain (WMD: −1.46, 95% CI: −2.13 to −0.8), WOMAC total score (WMD: −7.06, 95% CI: −12.27 to −1.84), WOMAC pain score (WMD: −1.42, 95% CI: −2.41 to −0.43), WOMAC function score (WMD: −5.04, 95% CI: −7.65 to −2.43), but not in WOMAC stiffness score (WMD: −0.34, 95% CI: −0.79 to 0.1). Moreover, the effect sizes of VAS for pain and WOMAC total score exceeded their MCID, while WOMAC subscale scores did not. Additionally, CURs did not significantly increase the incidence of adverse events (AEs) compared with placebo (RR: 1.22, 95% CI: 0.89 to 1.67), and significantly lowered the incidence of AEs when compared with NSAIDs (RR: 0.71, 95% CI: 0.59 to 0.9).
Conclusions: CURs alone can be expected to achieve considerable analgesic and functional promotion effects for patients with symptomatic knee OA in short term, without inducing an increase of adverse events. However, considering the low quality and substantial heterogeneity of present studies, a cautious and conservative recommendation for broader clinical use of CURs should still be made. Further high-quality studies are necessary to investigate the impact of different dosages, optimization techniques and administration approaches on long-term safety and efficacy of CURs, so as to strengthen clinical decision making for patients with symptomatic knee OA.
The Chemistry inside Spices & Herbs: Research and Development brings comprehensive information about the chemistry of spices and herbs with a focus on recent research in this field. The book is an extensive 2-part collection of 20 chapters contributed by experts in phytochemistry with the aim to give the reader deep knowledge about phytochemical constituents in herbal plants and their benefits. The contents include reviews on the biochemistry and biotechnology of spices and herbs, herbal medicines, biologically active compounds and their role in therapeutics among other topics. Chapters which highlight natural drugs and their role in different diseases and special plants of clinical significance are also included.
Part II continues from the previous part with chapters on the treatment of skin diseases and oral problems. This part focuses on clinically important herbs such as turmeric, fenugreek, ashwagandha (Indian winter cherry), basil, Terminalia chebula (black myrobalan). In terms of phytochemicals, this part presents chapters that cover resveratrol, piperine and circumin.
Audience: This book is an ideal resource for scholars (in life sciences, phytomedicine and natural product chemistry) and general readers who want to understand the importance of herbs, spices and traditional medicine in pharmaceutical and clinical research.
The aim of this systematic review was to evaluate the efficacy and safety of all types of Curcuma longa extract versus placebo for knee osteoarthritis (OA) treatment. The research was conducted by using the databases of PubMed, Embase, Scopus, and Cochrane Library through April 2021. Randomized controlled trials (RCTs) that compared the effect of Curcuma longa extract with placebo for patients with knee OA were considered eligible. The pooled results were expressed as mean differences or relative risks with 95% confidence intervals. A total of 10 RCTs with 783 patients were eligible for this meta-analysis. The pooled analysis showed that Curcuma longa extract was associated with significantly better pain relief and functional improvement compared with placebo for knee OA. Moreover, the smallest effect sizes of VAS for pain and WOMAC total score exceeded the minimum clinically important differences (MCIDs). Current evidence indicates that, compared with placebo, Curcuma longa extract has more benefit in pain relief and functional improvement for symptomatic knee OA. However, considering the potential heterogeneity in the included studies, more future high-quality RCTs with large sample sizes are necessary to confirm the benefits of Curcuma longa extract on knee OA.
Nitric oxide (NO) is an important inflammatory mediator involved in the development and progression of osteoarthritis (OA). Increased production of NO in the affected joints promote cartilage damage. As NO synthesis is catalysed by the inducible NO synthase (iNOS) enzyme, iNOS inhibition serves as an attractive therapeutic target to prevent NO release. Despite a number of direct and indirect iNOS inhibitor molecules demonstrating chondro-protective effect, none have reached the clinic. Its limited bioavailability and adverse side effects served as a deterrent for pursuing clinical trials in OA patients. With the advent of nanotechnology, interest in targeting NO for preventing cartilage degeneration has revived. In this article, we discuss the limitations of the existing molecules and provide an insight on recent nanotechnology-based strategies that have been explored for the diagnosis and inhibition of NO in OA. These approaches hold promise in reviving the hitherto under explored potential of targeting NO to address OA.
Supplements containing turmeric (Curcuma longa) and devil’s claw (Harpagophytum species) are commonly fed to horses to decrease inflammation and pain, but because these supplements contain spices and plant irritants, warning labels such as 'these supplements might cause gastric irritation' are required. The purpose of this study was to determine whether supplements containing turmeric and devil’s claw cause or worsen gastric ulcers in stall‐confined horses, as suggested in required cautions that appear on the labels of these animal products. Twelve clinically healthy Thoroughbred horses with naturally occurring equine gastric ulcer syndrome (EGUS) scores >0 were included in a noncrossover study design. Horses were stratified by EGUS score and assigned to either the treatment (supplements containing turmeric and devil’s claw) or control (same supplements without active ingredients) group and fed the supplements for 28 days. Gastroscopy was performed on Days 0, 14 and 28. The EGUS score, nonglandular ulcer number (NGN) and severity (NGS) scores, and glandular number (GN) and severity (GS) scores were recorded during each gastroscopy by a masked investigator (F.M.A.). In addition, bodyweight, gastric juice pH, packed cell volume (PCV), total protein (TP) and blood biochemical results were measured on Day 0 and Day 28. Mean EGUS and NGS scores were significantly lower in both treatment and control groups by Days 14 and 28, when compared to Day 0. NGN score was not different among groups. Bodyweight, gastric juice pH and blood parameters did not change during the study. In conclusion, supplements containing turmeric and devil’s claw did not cause or worsen gastric ulcers or alter health parameters after 28 days of feeding.
This chapter reviews the structure and function of articular cartilage and the pathogenesis of Osteoarthritis (OA) before exploring the challenges associated with cartilage repair and regeneration. Surgical techniques for cartilage repair are critically reviewed and special emphasis is placed upon arthroscopic lavage and debridement, microfracture, bone marrow stimulation, and autologous osteochondral allografting. Biomimetic models of cartilage relevant to cartilage regeneration and tissue engineering research are reviewed along with microenvironmental approaches that preserve the chondrocyte phenotype using a combination of biomimetic 3-dimensional cell culture systems and growth factor supplementation. Future refinement of tissue-engineering approaches for cartilage repair will benefit significantly from advances in cell-based repair strategies. The authors provide their own perspectives by highlighting and prioritizing areas for future investigation.
Objective:
A combination of curcumagalactomannosides (CGM) (400 mg) with glucosamine hydrochloride (GLN) (500 mg) was evaluated against a standard dietary supplement combination chondroitin sulfate (CHN) (415 mg)/GLN (500 mg) for their effectiveness in alleviating the pain and symptoms among osteoarthritic subjects.
Design:
Randomized, double-blinded and active-controlled study.
Settings/Location:
The study was conducted in a hospital-based research center in Vadodara, Gujarat, India.
Subjects:
Eighty subjects (38 males and 42 females), with confirmed osteoarthritis (OA) (Class I-III), were randomized into two parallel groups designated as Group I (CGM-GLN) and Group II (CHN-GLN).
Interventions:
All the study subjects were supplemented with their corresponding intervention capsules (ether CGM along with GLN or CHN along with GLN), as a single oral dose twice a day, once in the morning 10-15 min before breakfast and again in the evening before dinner, for 84 days.
Outcome measures:
A validated treadmill uphill walking protocol was used for the study, and the efficiency of supplementation was evaluated using visual analogue scale (VAS) score, Karnofsky Performance Scale (KPS) score, and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) questionnaire at the baseline, 28th, and 84th day following the treatment. Mechanism of action of CGM-GLN combination was analyzed by measuring the levels of serum inflammatory markers interleukin 1 beta (IL-1β), interleukin 6 (IL-6), and soluble vascular cell adhesion molecule-1 (sVCAM) at the baseline and 84th day.
Results:
CGM-GLN was found to offer significant beneficial effects to pain, stiffness, and physical function of OA subjects compared with CHN-GLN, which was evident from the improvement in walking performance, VAS score, KPS score, and WOMAC score. The efficiency of CGM-GLN was almost double compared with the CHN-GLN by the end of the study (84th day). A significant reduction of inflammatory serum marker levels was observed among CGM-GLN subjects compared with CHN-GLN subjects. Compared with the baseline, CGM-GLN produced 54.52%, 59.08%, and 22.03% reduction in IL-1β, IL-6, and sVCAM levels, respectively. Whereas CHN-GLN group of subjects expressed only 23.17%, 21.38%, and 6.82% reduction in IL-1β, IL-6, and sVCAM levels, respectively.
Conclusions:
In conclusion, the present study demonstrated the potential benefits of CGM-GLN supplements in alleviating the symptoms and function of OA subjects compared with the standard CHN-GLN treatment. The augmented efficacy of CGM-GLN combination could be attributed to the enhanced anti-inflammatory effect of CGM.
Temporomandibular joint osteoarthritis (TMJ OA) is a complex multifactorial disease that can be induced by inflammation and oxidative stress. Curcumin has been reported to have anti-inflammatory and antioxidant properties. Herein, the anti-inflammatory and antioxidant mechanisms of curcumin in TMJ OA were investigated. Curcumin treatment inhibited the expression of the inflammation mediators IL-6, iNOS, and COX-2 and of the matrix-degrading proteinases MMP-1, MMP-3, MMP-9, MMP-13, ADAMTS-4, and ADAMTS-5 and upregulated the mRNA levels of the cartilage anabolic factors COL2A1 and ACAN after IL-1β treatment. Curcumin treatment also decreased oxidative stress injury following IL-1β stimulation. Pathway analysis demonstrated that the ROS/Nrf2/HO-1-SOD2-NQO-1-GCLC signaling axis is a key axis through which curcumin activates the Nrf2/ARE pathway in TMJ inflammatory chondrocytes. Curcumin-induced anti-inflammatory and cartilage protective effects were significantly abrogated by specific Nrf2 siRNA. In vivo results demonstrated that curcumin treatment protected TMJ articular cartilage from progressive degradation. Our experimental results indicate that curcumin inhibits inflammation, oxidative stress, and the matrix degradation of TMJ inflammatory chondrocytes through the Nrf2/ARE signaling pathway, thereby exerting cartilage protective effects. This study provides insight into potential therapeutic approaches for TMJ OA.
Introduction:
Estrogen receptor α (ERα) plays important roles in the etiology of osteoarthritis (OA), in which cartilage degradation and cellular inflammation are involved. MiR-203 is reported to direct target ERα, but its roles in chondrocytes remain uncovered.
Methods:
In this study, ELISA showed that the level of estrogen hormone in the serum of postmenopausal OA patients was significantly lower than the one in patients without OA. RT-PCR revealed that the expression level of miR-203 was significantly up-regulated in the OA patients. Furthermore, western blotting demonstrated the lower expression levels of aggrecan, Col2A1, and ERα in the isolated articular cartilage tissues of OA patients. To decipher the association between ERα and miR-203 in the pathogenesis of OA, IL-1β stimulated cultured chondrocyte cell model was established to measure the cell viability, cellular inflammation, cell injury, as well as cartilage degradation with miR-203 inhibitor and ERα.
Results:
The results showed that IL-1β stimulation induced the expression of miR-203, which promoted cellular inflammation and cell injury, and caused down-regulation of aggrecan and Col2A1. Luciferase assay indicated the direct binding between miR-203 and ERα, and ERα-specific SiRNA inversed the protective role of miR-203 inhibitor in the progression of OA in the cell system.
Conclusions:
MiR-203 is critical in the onset and progression of OA, at least in part, caused by estrogen deficiency and ERα instability in OA patients, providing a novel therapeutic target for the treatment of OA.
Curcumin has been found to be very efficacious against many different types of diseases. However, the major disadvantage associated with the use of curcumin is its low systemic bioavailability. In the present study the protective effects of curcumin-loaded poly lactic-co-glycolic acid nanoparticles (nanocurcumin) against mono-iodoacetate-induced osteoarthritis in rats was investigated. Mono-iodoacetate was injected into right knee joints to induce osteoarthritis. In experimental groups, 14 days after injection of mono-iodoacetate, curcumin (200 mg kg⁻¹) and nanocurcumin (200 mg kg⁻¹) were gavaged, respectively, for two weeks. Then the rats were sacrificed and the right knee joints were removed and fixated in 10% formalin for histological assessments. Cellularity and matrix staining were significantly increased in articular cartilage of curcumin-treated animals compared to mono-iodoacetate group (p < 0.01). These effects were significantly (p < 0.01) more in nanocurcumin-treated animals. These results suggested that administration of nanocurcumin prevented the structural changes of articular cartilage in mono-iodoacetate model of osteoarthritis in rats.
Objective: Curcumin (diferuloylmethane) is a phytochemical with potent anti-inflammatory and anti-oxidant properties, and has therapeutic potential for the treatment of a range of inflammatory diseases, including osteoarthritis (OA). The aim of this study was to determine whether non-toxic concentrations of curcumin can reduce interleukin-1beta (IL-1β)-stimulated inflammation and catabolism in an explant model of cartilage inflammation. Methods: Articular cartilage explants and primary chondrocytes were obtained from equine metacarpophalangeal joints. Curcumin was added to monolayer cultured primary chondrocytes and cartilage explants in concentrations ranging from 3μM-100μM. Prostaglandin E2 (PGE2) and matrix metalloproteinase (MMP)-3 release into the secretome of IL-1β-stimulated explants was measured using a competitive ELISA and western blotting respectively. Proteoglycan (PG) release in the secretome was measured using the 1,9-dimethylmethylene blue (DMMB) assay. Cytotoxicity was assessed with a live/dead assay in monolayer cultures after 24 hours, 48 hours and five days, and in explants after five days. Results: Curcumin induced chondrocyte death in primary cultures (50μM p
This chapter reports on a systematic review of the literature of crenobalneotherapy in the management of knee OA. Crenobalneotherapy is defined as the spectrum of techniques based on mineral or tap water and its derivatives, as used in a medical context. We searched Medline using the following keywords: "spa therapy", "mud", "radon", "balneotherapy", and "hydrotherapy" in combination with "OA", "arthrosis", and "gonarthrosis". We also reviewed the reference lists of articles retrieved by the Medline search. All studies that compared crenobalneotherapy to any other intervention or to no intervention were selected, and a checklist was used to assess their internal validity, external validity and the quality of the statistical analysis. We analyzed separately some components of crenobalneotherapy and comparators and four types of outcome criteria (pain, function, stiffness and quality of life). We calculated standardized response mean. There is middle level evidence that "multiple mineral interventions" that combine two or more components of crenobalneotherapy are superior to no treatment, high level evidence that its combination with home exercises is superior to home exercises alone and low level evidence that it is superior to short wave. There is high level but conflicting evidence that water exercise is superior to no treatment. There is a high level of evidence that water exercise is similar to land based exercise (but the studies noted that it is better tolerated). There is middle level evidence that massage is superior to no treatment. There is low level and conflicting evidence that bathing in mineral water is superior to or similar to bathing in tap water and that mineral mud and bathing in mineral water is superior to hot water. The only study evaluating heat (heat sleeve vs regular sleeve) found no differences but was a pilot study with insufficient sample size. Crenobalneotherapy seems to improve, pain, function, stiffness and quality of life in lower limb OA. As a whole treatment, its efficacy has a high level of evidence but efficacy of each component has middle level (massage) and sometimes high but conflicting level of evidence (exercise in water). There is low level evidence that chemical composition of water has a clinical relevant effect. More studies with higher methodology quality and sufficient sample size are needed in these fields.
Background: Curcumin is proved to possess anti-inflammatory, antioxidant and anti-apoptotic roles. Objective: To summarize and discuss the mechanisms and effects of curcumin used for the treatment of osteoarthritis Methods: PubMed, Embase, Elseveir databases were retrieved for relevant articles published from 1973 to 2014, with the key words of “osteoarthritis, curcumin, chondrocyte, articular cartilage” in English. After the quality of the included studies was evaluated, valid data were extracted and analyzed. Results and Conclusion: Curcumin prevents the occurrence and development osteoarthritis by inhibiting oxidative enzyme and scavenging free radicals. Curcumin increases production of collagen type 2 through suppressing the reduction of cartilage matrix by matrix metalloproteinases. Anti-inflammatory reaction of curcumin can be achieved by inhibiting cytosolic phospholipase A2, cyclooxygenase-2, and 5-lipoxygenase. Curcumin inhibits interleukin-1β-induced apoptosis of chondrocytes and mitochondrial swelling. Results from clinical trials suggest that curcumin or its derivatives can reduce joint pain and improve the function of knee joints in patients with osteoarthritis. High-concentration curcumin in vitro have been demonstrated toxic effects. © 2015, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.
Spices, defined by the US Food and Drug Administration as aromatic vegetable substances, in the whole, broken, or ground form, whose significant function in food is seasoning rather than nutrition, are an integral part of any Indian cuisine and are added to impart tastes, flavor, and color to the curries. In addition to their use in cooking, spices are also known to possess medicinal benefits and have been used in the various traditional and folk systems of medicine to treat ailments. Scientific studies carried out in the past have shown conclusively that most of the commonly used Indian spices and some of their phytochemicals possess antioxidant, free radical scavenging, anti-inflammatory, antibacterial, and antiviral effects; modulate detoxification enzymes, stimulate the immune system, reduce inflammation, and modulate steroid metabolism; and have antimutagenic, gastroprotective, and anticarcinogenic effects. Studies carried out during the past two decades have shown that commonly used Indian spices, such as fenugreek (. Trigonella foenum-graecum L., family Fabaceae), coriander (. Coriandrum sativum L., family Apiaceae), ginger (. Zingiber officinale Roscoe, family Zingiberaceae), turmeric (. Curcuma longa L., family Zingiberaceae), and their phytochemicals 6-shogaol (present in ginger), curcumin (present in turmeric), eugenol (present in clove Syzygium aromaticum L., family Myrtaceae), and thymoquinone (present in black cumin Nigella sativa L., family Ranunculaceae), possess beneficial effects in amelioration of rheumatoid arthritis in preclinical studies. Of these, ginger, turmeric and the oil of Nigella sativa have been shown also to possess beneficial effects in humans. For the first time, this chapter summarizes the findings from both preclinical and clinical studies with these spices and phytochemicals in rheumatoid arthritis, and also emphasizes the mechanisms of action responsible for the observed effects of each of the spices/phytochemicals.
The rhizome of Curcuma longa Linn [family: Zingiberaceae], commonly known as "turmeric," is one of the most regularly used spices, especially in the Indian subcontinent, and is popularly known as the "curry spice." In addition to its dietary use, turmeric also has a long history of medicinal uses in the various traditional and folk systems of medicine and is used to treat burns, wounds, gastrointestinal ailments, heart conditions, arthritis, and inflammatory conditions. Studies carried out in the past have validated many of the ethnomedicinal observations; and observations indicate that the validated properties of turmeric are mediated predominately by the principle bioactive polyphenol curcumin. Preclinical and clinical studies have also shown curcumin to possess myriad benefits, such as turmeric used as an aid in arthritic diseases. The current review compiles validated observations and addresses the mechanism responsible for the beneficial effects of turmeric.
The transcription factor NF-κB is a pivotal regulator of inflammatory responses. While the activation of NF-κB in the arthritic
joint has been associated with rheumatoid arthritis (RA), its significance is poorly understood. Here, we examine the role
of NF-κB in animal models of RA. We demonstrate that in vitro, NF-κB controlled expression of numerous inflammatory molecules in synoviocytes and protected cells against tumor necrosis
factor α (TNFα) and Fas ligand (FasL) cytotoxicity. Similar to that observed in human RA, NF-κB was found to be activated
in the synovium of rats with streptococcal cell wall (SCW)-induced arthritis. In vivo suppression of NF-κB by either proteasomal inhibitors or intraarticular adenoviral gene transfer of super-repressor IκBα
profoundly enhanced apoptosis in the synovium of rats with SCW- and pristane-induced arthritis. This indicated that the activation
of NF-κB protected the cells in the synovium against apoptosis and thus provided the potential link between inflammation and
hyperplasia. Intraarticular administration of NF-kB decoys prevented the recurrence of SCW arthritis in treated joints. Unexpectedly,
the severity of arthritis also was inhibited significantly in the contralateral, untreated joints, indicating beneficial systemic
effects of local suppression of NF-κB. These results establish a mechanism regulating apoptosis in the arthritic joint and
indicate the feasibility of therapeutic approaches to RA based on the specific suppression of NF-κB.
When activated, NF-κB, a ubiquitous transcription factor, binds DNA as a heterodimeric complex composed of members of the
Rel/NF-κB family of polypeptides. Because of its intimate involvement in host defense against disease, this transcription
factor is an important target for therapeutic intervention. In the present report we demonstrate that curcumin (diferuloylmethane),
a known anti-inflammatory and anticarcinogenic agent, is a potent inhibitor of NF-κB activation. Treatment of human myeloid
ML-1a cells with tumor necrosis factor (TNF) rapidly activated NF-κB, which consists of p50 and p65 subunits, and this activation
was inhibited by curcumin. AP-1 binding factors were also found to be down-modulated by curcumin, whereas the Sp1 binding
factor was unaffected.
Besides TNF, curcumin also blocked phorbol ester- and hydrogen peroxide-mediated activation of NF-κB. The TNF-dependent phosphorylation
and degradation of IκBα was not observed in curcumin-treated cells; the translocation of p65 subunit to the nucleus was inhibited
at the same time. The mechanism of action of curcumin was found to be different from that of protein tyrosine phosphatase
inhibitors. Our results indicate that curcumin inhibits NF-κB activation pathway at a step before IκBα phosphorylation but
after the convergence of various stimuli.
We demonstrate the direct involvement of increased collagenase activity in the cleavage of type II collagen in osteoarthritic human femoral condylar cartilage by developing and using antibodies reactive to carboxy-terminal (COL2-3/4C(short)) and amino-terminal (COL2-1/4N1) neoepitopes generated by cleavage of native human type II collagen by collagenase matrix metalloproteinase (MMP)-1 (collagenase-1), MMP-8 (collagenase-2), and MMP-13 (collagenase-3). A secondary cleavage followed the initial cleavage produced by these recombinant collagenases. This generated neoepitope COL2-1/4N2. There was significantly more COL2-3/4C(short) neoepitope in osteoarthritis (OA) compared to adult nonarthritic cartilages as determined by immunoassay of cartilage extracts. A synthetic preferential inhibitor of MMP-13 significantly reduced the unstimulated release in culture of neoepitope COL2-3/4C(short) from human osteoarthritic cartilage explants. These data suggest that collagenase(s) produced by chondrocytes is (are) involved in the cleavage and denaturation of type II collagen in articular cartilage, that this is increased in OA, and that MMP-13 may play a significant role in this process.
To compare selected immunohistological features of inflammation in synovial tissue from patients with early and late osteoarthritis (OA).
Synovial tissue samples were obtained from 10 patients with knee pain, normal radiographs, and arthroscopic manifestations of OA (early OA), and from 15 patients with OA undergoing knee joint arthroplasty (late OA). Conventional immunohistochemical techniques were used to measure microscopic manifestations of inflammation. The inflammatory cell infiltrate, blood vessel formation, and angiogenic factors, NF-kappaB activation, expression of tumour necrosis factor alpha (TNFalpha) and interleukin 1beta (IL1beta), and the presence of cyclo-oxygenase (COX)-1 and COX-2 were quantified. Fibroblast-like synoviocytes (FLS) were isolated from early and late OA tissue samples to compare in vitro production of prostaglandin E2 (PGE2)
Synovial tissue from patients with early OA demonstrated significantly greater CD4+ (p = 0.017) and CD68+ (p<0.001) cell infiltration, blood vessel formation (p = 0.01), vascular endothelial growth factor (p = 0.001), and intercellular adhesion molecule-1 expression (p<0.001). Numbers of cells producing TNFalpha and IL1beta were also significantly greater in early OA (p<0.001). Manifestations of inflammation in early OA were associated with increased expression of the NF-kappaB1 (p<0.001) and RelA (p = 0.015) subunits, and with increased COX-2 expression (p = 0.04). Cytokine-induced PGE2 production by cultured FLS was similar in both groups.
Increased mononuclear cell infiltration and overexpression of mediators of inflammation were seen in early OA, compared with late OA. Isolated FLS were functionally similar in both groups, consistent with microenvironmental differences in the synovial tissue during different phases of OA. These observations may have important therapeutic implications for some patients during the early evolution of OA.
Curcumin (diferuloylmethane) is an orange-yellow component of turmeric (Curcuma longa), a spice often found in curry powder. Traditionally known for its an antiinflammatory effects, curcumin has been shown in the last two decades to be a potent immunomodulatory agent that can modulate the activation of T cells, B cells, macrophages, neutrophils, natural killer cells, and dendritic cells. Curcumin can also downregulate the expression of various proinflammatory cytokines including TNF, IL-1, IL-2, IL-6, IL-8, IL-12, and chemokines, most likely through inactivation of the transcription factor NF-kappaB. Interestingly, however, curcumin at low doses can also enhance antibody responses. This suggests that curcumin's reported beneficial effects in arthritis, allergy, asthma, atherosclerosis, heart disease, Alzheimer's disease, diabetes, and cancer might be due in part to its ability to modulate the immune system. Together, these findings warrant further consideration of curcumin as a therapy for immune disorders.
Objective
To evaluate potential stimulatory or matrixsparing effects of insulin-like growth factor 1 (IGF-1 ), alone or in combination with a corticosteroid, in an interleukin 1 (IL-1)-induced model of cartilage degradation.
Samples
Cartilage from the weightbearing surfaces of trochlea and condyles of clinically normal 2-year-old male horses.
Procedure
Triamcinolone acetonide and IGF-1 effects were evaluated by assessing: matrix responses by sulfated glycosaminoglycan (GAG) assay and [ ³⁵ S]sulfated GAG synthesis; collagen content by hydroxyproline assay; and mitogenic response by [ ³ H]thymidine incorporation into DNA and fluorometric assay of total DNA concentration.
Results
Conditioning of cartilage expiants with 10 ng of human recombinant IL-1α increased degradation and decreased synthesis of matrix proteoglycans (PG), without affecting matrix collagen content. Human recombinant IGF-1 decreased PG loss and reversed the reduction of PG synthesis in cartilage expiants conditioned with IL-1. Given alone, steroids decreased PG concentration and synthetic rate in normal cartilage. However, the previously diminished PG content, attributable to IL-1 conditioning, was not further exacerbated by steroid administration in IL-1-conditioned expiants. Combined treatment of normal cartilage expiants with IGF-1 and steroids resulted in PG preservation and increase in collagen content. Similar PG and collagen effects were not evident when treating IL-1-conditioned cartilage with IGF-1/steroid combinations. Decrease in chondrocyte proliferation was associated with steroid administration. Exposure to IGF and steroids prevented the decrease in mitogenesis that could lead to cellular loss, particularly in IL-1-conditioned expiants.
Conclusion
Combination IGF-1 and steroid treatment of normal cartilage cultures indicated substantial ability to override the anabolic suppression associated with steroids alone. Potentially, administration of corticosteroids, followed by IGF-1, may act to decrease propagation of detrimental mediator release while allowing appreciation of the chondroenhancing effects of IGF-1. These beneficial effects were considerably reduced in IL-1-induced cartilage damage. ( Am J Vet Res 1997;58:524–530)
Interleukin 1β (IL-1β) is a pleiotropic pro-inflammatory cytokine that plays a key role in mediating cartilage degradation in osteoarticular disorders such as osteoarthritis (OA) and rheumatoid arthritis (RA). At the cellular level, IL-1β activates matrix degrading enzymes, down-regulates expression of matrix components and induces chondrocyte apoptosis. Curcumin (diferuloylmethane) is an anti-inflammatory phytochemical agent that has recently been shown to antagonize the pro-inflammatory effects of cytokines in chondrocytes and other cells. To test the hypothesis that curcumin also protects chondrocytes from morphological alterations induced by IL-1β, we investigated its in vitro effects on apoptotic signalling proteins and key cartilage-specific matrix components in IL-1β-stimulated chondrocytes.
Human articular chondrocytes were pre-treated with 10 ng/ml IL-1β alone for 30 min before being co-treated with IL-1β and 50 μM curcumin for 5, 15 or 30 min, respectively. The ultrastructural morphology of chondrocytes was investigated by transmission electron microscopy. The production of collagen type II, the adhesion and signal transduction receptor β1-integrin, the apoptosis marker activated caspase-3 was analysed by immunohistochemistry, immunoelectron microscopy and Western blotting.
Transmission electron microscopy of chondrocytes stimulated with IL-1β revealed early degenerative changes which were relieved by curcumin co-treatment. The suppression of collagen type II and β1-integrin synthesis by IL-1β was inhibited by curcumin. Additionally, curcumin antagonized IL-1β-induced caspase-3 activation in a time-dependent manner.
This study clearly demonstrates that curcumin exerts anti-apoptotic and anti-catabolic effects on IL-1β-stimulated articular chondrocytes. Therefore curcumin may have novel therapeutic potential as an adjunct nutraceutical chondroprotective agent for treating OA and related osteoarticular disorders.
Honors thesis (Biological Sciences)--Cornell University, May 2005. Includes bibliographical references (leaves 16-19).
Prostaglandin E2 (PGE2) and stromelysin are produced by equine chondrocytes and synovial cells in vitro in response to recombinant human (rh) interleukin-1 (IL-1) alpha and beta, and equine mononuclear cell supernatants (MCS) containing IL-1. However, culture conditions are important. PGE2 concentrations increase in proportion to the concentration of fetal calf serum (FCS) in the culture medium, whereas stromelysin concentrations are inversely proportional to the concentration of FCS. Equine MCS, containing a lower concentration of IL-1 than the concentration of rhIL-1 used in these experiments, stimulated production of much higher levels of PGE2 than rhIL-1. In addition, equine MCS induced the production of broadly similar levels of PGE2 by both chondrocytes and synovial cells, whereas rhIL-1 was more active on equine synovial cells than equine chondrocytes. Although equine MCS induced both stromelysin and PGE2 production by equine articular cells, on the whole rhIL-1 failed to induce stromelysin production. This supports previous observations of species restrictions in the activity of human IL-1 on equine cells. Therefore, experiments using mammalian cells and heterologous IL-1 should be interpreted with caution.
Articular cartilage covers the ends of long bones in synovial joints, providing smooth articulation and cushioning of the underlying bone during joint movement. The tissue can be viewed as a viscoelastic, composite material composed of collagen type II (and smaller amounts of other collagens) entrapping compressed (underhydrated) proteoglycan aggregates which generate a high osmotic/swelling pressure. This abundant extracellular matrix (ECM) is synthesized and turned over by relatively few cells, the chondrocytes. These cells produce a compartmentalized ECM, the components of which are heterogeneous and vary with anatomical location. They also undergo changes with age and altered functional requirements. Articular cartilage contains no separating basement membranes, nerves, lymphatics, or blood vessels. Access to nutrients and elimination of waste products occur via diffusion through the extracellular matrix. The turnover of collagen is much slower than that of proteoglycans. Products of the metabolic turnover of the matrix macromolecules are released continuously into the synovial cavity and ultimately reach the blood circulation where they can be measured as "markers" of metabolic changes.
Horse articular cartilage glycosaminoglycans (GAGs) were measured in synovial fluids from 48 joints affected with osteoarthritis (OA), 22 normal joints, four joints with osteochondritis, three joints with traumatic arthritis and seven joints infected with bacteria. Serum and urine from individual horses were also examined for the presence of GAGs. High levels of GAGs were found in synovial fluids (SF) from horses with OA. In each case, the level was higher in the synovial fluid than in the serum or urine from the same horse. Horses with OA showed high GAG levels in SF, serum and urine compared to horses with normal and infected joints. High levels were also found in horses with osteochondritis and traumatic arthritis. Levels of synovial fluid GAG reflect cartilage destruction in arthritis and may be useful for monitoring disease progression in the equine species.
The turnover of proteoglycans was investigated in articular cartilage in explant culture by analysing the components released into the culture medium. The effect of IL-1 alpha on the release of fragments derived from different proteoglycan domains and hyaluronan (HA) was determined over 4 days in culture. The effect of IGF-1 (100 ng/ml) on matrix degradation of proteoglycan and its ability to inhibit the effects of IL-1 (10 ng/ml) was also assessed. The rate of release of G1 and G2 globular domains of proteoglycans into the culture medium was determined by radioimmunoassay. In unstimulated control cartilage there was a greater release of proteoglycan G2 domain than of G1 domain suggesting that cleavage occurred between them and that some G1 was preferentially retained bound in the matrix. Compared with control cartilage IL-1 stimulated the release of all proteoglycan components and hyaluronan. IL-1 had a greater effect on the release of G1 than on G2 domain, but also resulted in some net loss of these proteins (approximately 45% as detected in the immunoassays). In explants treated with both IL-1 and IGF-1 there was much less release of proteoglycan fragments and evidence for less extensive degradation. IGF-1 was particularly affective in preventing any increase in HA release and also preventing the apparent loss of G1 and G2 domains. It also partially inhibited the release of G1 and G2 domains and the sulphated glycosaminoglycan fragments. IGF-1 was therefore an effective antagonist of IL-1 action on cartilage. It is not known at what level it blocks the chondrocyte response to IL-1, but it clearly results in the suppression of matrix degradative activity.
A model system of explanted cartilage has been used in vitro to determine whether insulin-like growth factor 1 (IGF 1), which promotes matrix formation is effective in the presence of cytokines such as interleukin 1 (IL1) and tumour necrosis factor (TNF), which induce net matrix depletion. IGF 1 induced a dose-dependent 2.5-fold stimulation of proteoglycan synthesis, with a half-maximal dose of 25 ng/ml. A similar relative increase occurred in response to IGF 1 (10-100 ng/ml) in cartilage cultured also with IL1 or TNF (5-500 pM). There was no detectable qualitative change in the average molecular size or charge of the aggregating proteoglycan synthesized by explants exposed to IGF 1 alone or with IL1 or TNF. The increased production of prostaglandin E2, which is initiated when IL1 or TNF bind to the chondrocytes, was the same in the presence or absence of IGF 1. The time taken for 50% of pre-labelled proteoglycan to be released from the explants (t1/2) increased in the presence of IGF 1 (100 ng/ml) from 21 to 32 days in control cultures and from 8 to 26 days in cartilage cultured with IL1 (50 pM). It is concluded that IGF 1 enhances the synthesis of aggregating proteoglycan in cartilage exposed to cytokines and can directly decrease both the basal and the cytokine-stimulated degradation of proteoglycan in cartilage.
A rapid spectrophotometric procedure is described for the estimation of sulfated glycosaminoglycans in cartilage cultures. Papain digestion of tissue or culture medium provides glycosaminoglycans in solution for assay; an aliquot of the digest is mixed with the dye 1,9-dimethylmethylene blue. The assay is based on the metachromatic shift in absorption maximum which occurs when the dye is complexed with sulfated glycosaminoglycans. The reagent is stable, and the method is substantially free from interference, is sensitive to less than 1 microgram (4 micrograms/ml) of chondroitin sulfate, and provides a simple alternative to the traditional methods for glycosaminoglycan determinations.
L-Arginine-derived nitric oxide (NO) and its derivatives, such as peroxynitrite and nitrogen dioxide, play a role in inflammation and also possibly in the multistage process of carcinogenesis. We investigated the effect of various non-steroidal anti-inflammatory agents and related compounds on the induction of NO synthase (NOS) in RAW 264.7 macrophages activated with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Low concentrations of curcumin, a potent anti-tumour agent having anti-inflammatory and anti-oxidant properties, inhibited NO production, as measured by the amount of nitrite released into the culture medium in 24 h (IC50 = 6 microM). NOS activity in soluble extracts of macrophages activated for 6-24 h in the presence of curcumin (10 microM) was significantly lower than that of macrophages activated without curcumin. Northern-blot and immunoblotting analyses demonstrated that significantly reduced levels of the mRNA and 130-kDa protein of inducible NOS were expressed in macrophages activated with curcumin, compared to those without curcumin. Inhibition of NOS induction was maximal when curcumin was added together with LPS and IFN-gamma and decreased progressively as the interval between curcumin and LPS/IFN-gamma was increased to 18 h.
When activated, NF-kappa B, a ubiquitous transcription factor, binds DNA as a heterodimeric complex composed of members of the Rel/NF-kappa B family of polypeptides. Because of its intimate involvement in host defense against disease, this transcription factor is an important target for therapeutic intervention. In the present report we demonstrate that curcumin (diferuloylmethane), a known anti-inflammatory and anticarcinogenic agent, is a potent inhibitor of NF-kappa B activation. Treatment of human myeloid ML-1a cells with tumor necrosis factor (TNF) rapidly activated NF-kappa B, which consists of p50 and p65 subunits, and this activation was inhibited by curcumin. AP-1 binding factors were also found to be down-modulated by curcumin, whereas the Sp1 binding factor was unaffected. Besides TNF, curcumin also blocked phorbol ester- and hydrogen peroxide-mediated activation of NF-kappa B. The TNF-dependent phosphorylation and degradation of I kappa B alpha was not observed in curcumin-treated cells; the translocation of p65 subunit to the nucleus was inhibited at the same time. The mechanism of action of curcumin was found to be different from that of protein tyrosine phosphatase inhibitors. Our results indicate that curcumin inhibits NF-kappa B activation pathway at a step before I kappa B alpha phosphorylation but after the convergence of various stimuli.
To evaluate potential stimulatory or matrix-sparing effects of insulin-like growth factor 1 (IGF-1), alone or in combination with a corticosteroid, in an interleukin 1 (IL-1)-induced model of cartilage degradation.
Cartilage from the weightbearing surfaces of trochlea and condyles of clinically normal 2-year-old male horses.
Triamcinolone acetonide and IGF-1 effects were evaluated by assessing: matrix responses by sulfated glycosaminoglycan (GAG) assay and [35S]sulfated GAG synthesis; collagen content by hydroxyproline assay; and mitogenic response by [3H]thymidine incorporation into DNA and fluorometric assay of total DNA concentration.
Conditioning of cartilage explants with 10 ng of human recombinant IL-1 alpha increased degradation and decreased synthesis of matrix proteoglycans (PG), without affecting matrix collagen content. Human recombinant IGF-1 decreased PG loss and reversed the reduction of PG synthesis in cartilage explants conditioned with IL-1. Given alone, steroids decreased PG concentration and synthetic rate in normal cartilage. However, the previously diminished PG content, attributable to IL-1 conditioning, was not further exacerbated by steroid administration in IL-1-conditioned explants. Combined treatment of normal cartilage explants with IGF-1 and steroids resulted in PG preservation and increase in collagen content. Similar PG and collagen effects were not evident when treating IL-1-conditioned cartilage with IGF-1/steroid combinations. Decrease in chondrocyte proliferation was associated with steroid administration. Exposure to IGF and steroids prevented the decrease in mitogenesis that could lead to cellular loss, particularly in IL-1-conditioned explants.
Combination IGF-1 and steroid treatment of normal cartilage cultures indicated substantial ability to override the anabolic suppression associated with steroids alone. Potentially, administration of corticosteroids, followed by IGF-1, may act to decrease propagation of detrimental mediator release while allowing appreciation of the chondroenhancing effects of IGF-1. These beneficial effects were considerably reduced in IL-1-induced cartilage damage.
Proteoglycan degradation was induced in young equine articular cartilage explants cultured for eight days in the presence of 50 ng/ml recombinant human interleukin-1 beta. Degradation was initiated after 6 hours of exposure to the cytokine. This was accompanied by an induction of nitric oxide synthesis and a decrease in the incorporation of [36S]sulphate into the glycosaminoglycan chains of proteoglycans. The addition of 1mM N-iminoethyl-L-ornithine (an inhibitor of nitric oxide synthase) to the explant cultures in the presence of rhIL-1 beta suppressed the synthesis of NO and restored proteoglycan synthesis to control levels. However, treatment of explants with LNIO did not overcome proteoglycan degradation. These results indicate that although IL1 beta regulates both proteoglycan synthesis and degradation in equine cartilage explants, only the inhibition of proteoglycan synthesis is mediated by nitric oxide.
To investigate osteoarthritic cartilage in comparison to normal cartilage in humans for the presence of the most relevant cytokines/growth factors known to be important for degradation and formation of new cartilage.
Cartilage from knee or hip joints was obtained from 10 patients with osteoarthritis (OA) and from 7 age matched control patients with intact cartilage. Additionally, normal cartilage from 2 young patients (12 and 17 years old) was obtained after knee traumas. Immunohistological staining of cartilage sections was performed using antibodies for the following cytokines/growth factors: tumor necrosis factor alpha (TNF-alpha), interleukin 1alpha (IL-1alpha), IL-1beta, interferon-gamma, IL-6, IL-4, IL-10, transforming growth factor beta1 (TGF-beta1), insulin-like growth factor I (IGF-I), IGF-II, platelet derived growth factor AA (PDGF-AA), and PDGF-BB.
Immunohistochemical stainings were positive for all cytokines in OA cartilage, while only a faint or no staining was found in healthy cartilage. Activated chondrocytes expressing most of the cytokines were located in the middle and partly in the lower layer of cartilage, with the exception of IGF-I, which was expressed exclusively in the upper cartilage layer close to the surface. More chondrocytes stained positive for TNF-alpha than for IL-1, and expression of the degrading cytokine TNF-alpha was inversely correlated to the expression of the regulatory cytokines IL-4, IL-10, and TGF-beta.
The most relevant cytokines known to be involved in cartilage metabolism are produced by chondrocytes themselves. They are upregulated in OA cartilage, suggesting that they serve some regulatory function and could be a target for future treatment.
The specific causative agent of the pathological process of osteoarthritis (OA) has not yet been identified, however, episodic inflammation at the clinical stage is now a well documented phenomenon and believed to be involved in the disease progression. Interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) are the predominant proinflammatory cytokines synthesized during the OA process. Other cytokines having proinflammatory properties or catabolic factors could also contribute to this pathological condition, and those having antiinflammatory properties may be able to counteract the negative effects of the former on the disease process. In this chapter, we will review cytokine interactions and their modulatory effects on joint articular tissue metabolism, including their stimulatory and/or inhibitory actions, and their potential relevance to OA. We will also briefly survey the major biological factors, in relation to cytokines, that look promising for future therapeutic approaches.
Oncostatin M (OSM), a member of the IL-6 superfamily of cytokines, is elevated in patients with rheumatoid arthritis and, in synergy with IL-1, promotes cartilage degeneration by matrix metalloproteinases (MMPs). We have previously shown that OSM induces MMP and tissue inhibitor of metalloproteinase-3 (TIMP-3) gene expression in chondrocytes by protein tyrosine kinase-dependent mechanisms. In the present study, we investigated signaling pathways regulating the induction of MMP and TIMP-3 genes by OSM. We demonstrate that OSM rapidly stimulated phosphorylation of Janus kinase (JAK) 1, JAK2, JAK3, and STAT1 as well as extracellular signal-regulated kinase (ERK) 1/2, p38, and c-Jun N-terminal kinase 1/2 mitogen-activated protein kinases in primary bovine and human chondrocytes. A JAK3-specific inhibitor blocked OSM-stimulated STAT1 tyrosine phosphorylation, DNA-binding activity of STAT1 as well as collagenase-1 (MMP-1), stromelysin-1 (MMP-3), collagenase-3 (MMP-13), and TIMP-3 RNA expression. In contrast, a JAK2-specific inhibitor, AG490, had no impact on these events. OSM-induced ERK1/2 activation was also not affected by these inhibitors. Similarly, curcumin (diferuloylmethane), an anti-inflammatory agent, suppressed OSM-stimulated STAT1 phosphorylation, DNA-binding activity of STAT1, and c-Jun N-terminal kinase activation without affecting JAK1, JAK2, JAK3, ERK1/2, and p38 phosphorylation. Curcumin also inhibited OSM-induced MMP-1, MMP-3, MMP-13, and TIMP-3 gene expression. Thus, OSM induces MMP and TIMP-3 genes in chondrocytes by activating JAK/STAT and mitogen-activated protein kinase signaling cascades, and interference with these pathways may be a useful approach to block the catabolic actions of OSM.
To evaluate the effects of equine recombinant interleukin-1alpha (rEqIL-1alpha) and recombinant interleukin-1beta (rEqIL-1beta) on proteoglycan metabolism and prostaglandin E2 (PGE2) synthesis by equine articular chondrocytes in explant culture.
Near full-thickness articular cartilage explants (approx 50 mg) harvested from stifle joints of a 3-year-old and a 5-year-old horse.
Expression constructs containing cDNA sequences encoding EqIL-1alpha and EqIL-1beta were generated, prokaryotically expressed, and the recombinant protein purified. Near full-thickness articular cartilage explants (approx 50 mg) harvested from stifle joints of a 3-year-old and a 5-year-old horse were separately randomized to receive rEqIL-1alpha or rEqIL-1beta treatments 10 to 500 ng/ml). Proteoglycan release was evaluated by 1,9-dimethylmethylene blue spectrophotometric analysis of explant media glycosaminoglycan (GAG) concentration and release of 35S-sulfate-labeled GAG to explant media. Proteoglycan synthesis was assessed by quantification of 35S-sulfate incorporation into proteoglycan. Explant media PGE2 concentrations were evaluated using a PGE2-specific enzyme-linked immunoassay. Data were collected at 48-hour intervals and normalized by DNA content.
Proteoglycan release was induced by rEqIL-1alpha and rEqIL-1beta at concentrations > or =0.1 ng/ml, with 38 to 76% and 88 to 98% of total GAG released by 4 and 6 days, respectively. Inhibition of proteoglycan synthesis (42 to 64%) was observed at IL-1 concentrations > or = 0.1 ng/ml at 2 and 4 days. Increased PGE2 concentrations were observed at IL-1 concentrations > or = 0.1 ng/ml at 2 and 4 days.
The rEqIL-1 induced potent concentration-dependent derangement of equine chondrocyte metabolism in vitro. These findings suggest this model may be suitable for the in vitro study of the pathogenesis and treatment of joint disease in horses.
Interleukin-1 (IL-1), the main cytokine instigator of cartilage degeneration in arthritis, induces matrix metalloproteinase-3 (MMP-3) and MMP-13 RNA and protein in chondrocytes. The molecular mechanisms of this induction were investigated with specific inhibitors of mitogen-activated protein kinase (MAPK) signaling pathways and activating protein (AP-1) and nuclear factor kappa B (NF-kappa B) transcription factors. IL-1 rapidly induced the activation of extracellular-signal regulated kinase (ERK), protein 38 (p38) and c-Jun N-terminal kinase (JNK) MAPKs in the first-passage human femoral head OA chondrocytes. The ERK-MAPK pathway inhibitor, PD98059, attained 46-53% (MMP-3) and 59-66% (MMP-13) inhibition of RNA induction in human OA and 47-52% (MMP-3) and 69-73% (MMP-13) inhibition in bovine chondrocytes. U0126 conferred 37-77% (MMP-3) and 43-73% (MMP-13) suppression in human and 77-100% (MMP-3) and 96-100% (MMP-13) in bovine chondrocytes. P38 and JNK inhibitor, SB203580 caused 35-37% reduction of MMP-3 and MMP-13 RNA in human and 36-46% (MMP-3) and 60-88% (MMP-13) in bovine chondrocytes. Inhibitor of JNK, AP-1 and NF-kappa B, curcumin, achieved 48-99% suppression of MMP-3 and 45-97% of MMP-13 in human and 8-100% (MMP-3) and 32-100% (MMP-13) in bovine chondrocytes. NF-kappaB inhibitor, pyrrolidine dithiocarbamate yielded 83-84% reduction of MMP-3 and 38-55% for MMP-13 in human chondrocytes. In bovine chondrocytes, the induction decreased by 54-64% for MMP-3 and 74-93% for MMP-13 RNA. These results suggest the involvement of MAPKs, AP-1 and NF-kappa B transcription factors in the IL-1 induction of MMPs in chondrocytes. Inhibition of IL-1 signal transduction by these agents could be useful for reducing cartilage resorption by MMPs in arthritis.
Tumor necrosis factor alpha (TNF-alpha), a major proinflammatory cytokine, induces arthritic joint inflammation and resorption of cartilage by matrix metalloproteinase-13 (MMP-13). RNA for MMP-13 is increased in human arthritic femoral cartilage. Mechanisms of this induction were investigated by pretreating primary human osteoarthritic (OA) femoral head chondrocytes or chondrosarcoma cells with the potential inhibitors of TNF-alpha signal transduction and downstream target transcription factors followed by stimulation with TNF-alpha and analysis of MMP-13 RNA/protein. TNF-alpha rapidly activated phosphorylation of extracellular signal-regulated kinases (ERKs), p38, and c-jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinases in human chondrocytes. Inhibitors of ERK (U0126, PD98059, and ERK1/2 antisense phosphorothioate oligonucleotide), JNK (SB203580, SP600125, and curcumin), and p38 (SB203580 and SB202190) pathways down-regulated the TNF-stimulated expression of MMP-13. Inhibitors of the transcription factors AP-1 (nordihydroguaiaretic acid, NDGA) and NF-kappaB (curcumin, proteasome inhibitors, and Bay-11-7085) suppressed TNF-alpha-induced MMP-13 expression in primary chondrocytes and SW1353 cells. These results suggest that induction of the MMP-13 gene by TNF-alpha is mediated by ERK, p38, and JNK MAP kinases as well as AP-1 and NF-kappaB transcription factors. Blockade of TNF-alpha signaling and its target transcription factors by the approaches tested here may be beneficial for reducing cartilage breakdown by MMP-13 in arthritis.
Interleukin (IL)-17 promotes cartilage breakdown by inducing matrix metalloproteinases (MMPs) and aggrecanases (a disintegrin and metalloproteinase with thrombospondin motif, ADAMTS) in arthritic joints. We investigated IL-17 signaling pathways inducing MMP-3, MMP-13 and ADAM-TS4 genes in bovine articular chondrocytes. IL-17 stimulated phosphorylation of extracellular signal-regulated kinase (ERK), protein 38 (p38) and c-Jun N-terminal kinase (JNK). ERK pathway inhibitors, PD98059 and U0126, down-regulated IL-17-induced MMP and ADAM-TS4 gene expression. Protein 38 and JNK pathway inhibitors, SB203580 and SP600125, also reduced induction of these genes. Antioxidants and activating protein-1 transcription factor inhibitors, nordihydroguaiaretic acid and N-acetyl-L-cysteine (NAC) suppressed MMP and ADAM-TS4 genes. Similarly, nuclear factor kappa B (NF-kappaB) pathways inhibitors curcumin and Bay-11-7085 also blocked their induction. Thus MMP-3, MMP-13 and ADAM-TS4 genes are coordinately up-regulated by IL-17 via MAP kinases, activating protein-1 (AP-1) and NF-kappaB mediators, which could be targeted for reducing IL-17-triggered cartilage damage.
To determine whether interleukin-1 (IL-1) or tumor necrosis factor alpha (TNFalpha), or both, plays a role in the excessive degradation that is observed in cultured osteoarthritic (OA) articular cartilage.
Antagonists of IL-1 and TNFalpha, namely, IL-1 receptor antagonist and the PEGylated soluble TNFalpha receptor I, respectively, were added at different concentrations to explant cultures of nonarthritic (5 obtained at autopsy) and OA (15 obtained at arthroplasty) articular cartilage. The cleavage of type II collagen (CII) by collagenase was measured by an immunoassay in cartilage and culture media. Proteoglycan (mainly aggrecan) content and degradation were measured by a colorimetric assay for glycosaminoglycan (GAG) content in cartilage and culture media. Reverse transcriptase-polymerase chain reaction was used to analyze gene expression of matrix metalloproteases (MMPs) 1, 3, and 13, CII, aggrecan, IL-1, and TNFalpha.
Antagonists of IL-1 and TNFalpha inhibited the increase in CII cleavage by collagenase as well as the increase in GAG release observed in OA cartilage compared with normal cartilage. Inhibition was significant in tissue from some patients but not from others, although significant inhibition was observed when all the results were analyzed together. An increase in the GAG content in cartilage was seen in 4 of 15 cases. However, this increase was not significant when all the data were combined. Preliminary results indicated no effect of these antagonists on nonarthritic cartilage from 3 different donors. Independent analyses of gene expression in cultured cartilage from 9 other OA patients revealed that IL-1 or TNFalpha blockade, either alone and/or in combination, frequently down-regulated MMP-1, MMP-3, and MMP-13 expression. Expression of IL-1 and TNFalpha was inhibited by either antagonist or by the combination in essentially half the cases. The combined blockade up-regulated aggrecan and CII gene expression in approximately half the cases.
These results suggest that the autocrine/paracrine activities of TNFalpha and IL-1 in articular cartilage may play important roles in cartilage matrix degradation in OA patients but not in all patients. Inhibition of either or both of these cytokines may offer a useful therapeutic approach to the management of OA by reducing gene expression of MMPs involved in cartilage matrix degradation and favoring its repair.
Curcumin (diferuloylmethane) is a nontoxic dietary pigment in tumeric and curry and a potent inhibitor of the common transcription factor Nuclear Factor kappaB (NF-kappaB) in several cell types. It is well established that some of the catabolic effects of the proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha in osteoarthritis are regulated by the activation of NF-kappaB. Therefore, the aim of this study was to determine whether curcumin modifies the catabolic response of chondrocytes to IL-1beta. Human articular chondrocytes were prestimulated with 10 ng/mL IL-1beta for 0, 4, 8, 12, or 24 h and then cotreated with 50 microM curcumin for 0, 12, 24, 36, or 48 h. Synthesis of the cartilage-specific collagen type II and matrix-degrading enzyme matrix metalloproteinase-3 (MMP-3) was investigated in chondrocytes by Western blot analysis. Activation and nuclear translocation of NF-kappaB were observed by immunofluorescence microscopy. IL-1beta induced a decrease in collagen type II and upregulation of MMP-3 in a time-dependent manner. Upregulation of MMP-3 was inhibited by curcumin in a time-dependent manner. In addition, IL-1beta-induced a decrease in type II collagen, which was relieved by curcumin treatment. In response to IL-1beta, NF-kappaB translocated to the nucleus, but translocation was inhibited by curcumin, as revealed by immunofluorescence microscopy. Taken together, these results confirmed an IL-1beta-mediated upregulation of proinflammatory MMP-3 in chondrocytes via an NF-kappaB activation mechanism. Curcumin protected chondrocytes from the catabolic effects of IL-1beta, such as MMP-3 upregulation, and interestingly also relieved cytokine-induced suppression of matrix protein synthesis. Therefore, curcumin antagonizes crucial catabolic effects of IL-1beta signaling that are known to contribute to the pathogenesis of osteoarthritis.