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Resveratrol protects rabbit articular chondrocyte against sodium nitroprusside-induced apoptosis via scavenging ROS
Abstract
This study aims to investigate the mechanism by which resveratrol (RV) prevents sodium nitroprusside (SNP)-induced chondrocyte apoptosis, which is a characteristic feature of osteoarthritis (OA). Rabbit articular chondrocytes were pre-incubated with 100 μM RV for 18 h before 1.5 mM SNP co-treatment for 6 h. Cell viability was evaluated by CCK-8. Annexin V/PI double staining and Hoechst 33258 staining were used to determine the fashion of SNP-induced chondrocytes death. Mitochondrial membrane potential (ΔΨm) was measured by using flow cytometry (FCM) with TMRM and Rhodamine 123 staining. Intracellular reactive oxygen species (ROS) and nitric oxide (NO) levels were confirmed by FCM analysis with DCFH-DA and DAF-FM DA staining. Cytoskeleton proteins of chondrocytes co-stained with Actin-Trakcer Green and Tubulin-Trakcer Red were validated by confocal microscopy. SNP induced time- and dose-dependent chondrocytes apoptosis with decline of ΔΨm, activation of caspases as well as cytoskeletal remodeling. SNP induced a significant induction of both ROS and NO. RV remarkably prevented SNP-induced ROS production and apoptosis as well as cytoskeletal remodeling, but did not prevent SNP-induced NO production. Pretreatment with NO scavengers did not significantly prevent SNP-induced apoptosis and cytoskeletal remodeling. SNP induces NO-independent ROS production which dominates rabbit articular chondrocyte apoptosis, and RV protects chondrocytes against SNP-induced apoptosis via scavenging ROS instead of NO.
... Resveratrol (RV; 3,5,4'-trihydroxy-trans-stilbene), a natural polyphenol compound which could be extracted from grapes, has previously been proved to possess anti-inflammatory, antioxidant, and anticancer bioactivities in different kinds of cells and tissues (25)(26)(27)(28)(29). RV can protect IVD from degeneration by reducing levels of proinflammatory cytokines and activating silent mating type information regulator 2 homolog 1 (SIRT1) (30)(31)(32)(33)(34). ...
... Imaging of cytoskeletal and morphological structure. Imaging of cytoskeletal structure was carried out as previously described (29). After staining with Actin-Tracker Green and Tubulin-Trakcer Red, cytoskeletons (x400) were imaged using a fluorescent microscope (LEICA DM4000B; Leica Microsystems GmbH, Wetzlar, Germany). ...
... However, no study focused on effects of SNP on NP cells. Moreover, a recent study show that ROS rather than NO plays the key role in SNP induced in rabbit articular chondrocytes apoptosis, suggesting a new mechanism of SNP induced apoptosis (29). In our study, both intracellular NO and ROS level was raised by SNP treatment. ...
Oxidative stress induced disc cell apoptosis plays an important role in intervertebral disc (IVD) degeneration. The present study aims to investigate effects of resveratrol (RV), a natural polyphenol compound, on sodium nitroprusside (SNP) induced nucleus pulposus (NP) cell apoptosis and related mechanism. Rat NP cells were pretreated with RV, N-acetyl cysteine (NAC) and carboxy-PTIO (PTIO) before SNP treatment. Cell Counting Kit-8 assay was carried out for cell viability evaluation. Annexin V/propidium iodide (PI), Hoechst 33258 and Actin‑Tracker Green and Tubulin-Tracker Red staining were conducted to detect NP cell apoptosis and apoptotic structural changes. Mitochondrial membrane potential (ΔΨm) was analyzed with tetramethylrhodamine methyl ester staining. DCFH-DA and DAF-FM DA staining was used to determine intracellular reactive oxygen species (ROS) and nitric oxide (NO) levels. An ex vivo experiment was also carried out followed by TUNEL assay of sections of discs. SNP induced NP cell apoptosis, excessive production of intracellular ROS and NO, reduction of ΔΨm as well as disruption of cytoskeletal and morphological structure. Meanwhile, organ culture results showed that SNP induced NP cell apoptosis ex vivo. RV and NAC siginificantly inhibited SNP induced NP cell apoptosis, production of intracellular ROS, deline of ΔΨm as well as disruption of cytoskeletal and morphological structure, while RV did not suppress NO production. RV and NAC could also suppress SNP induced NP cell apoptosis ex vivo. However, PTIO did not prevent SNP induced NP cell apoptosis, though it scavenged NO significantly. In conclusion, RV protects against SNP induced NP cell apoptosis by scavenging ROS but not NO, suggesting a promising prospect of RV in IVD degeneration retardation.
... This notion was widely adopted to study the molecular mechanism of SNP-induced apoptosis in various cell lines [20][21][22]. However, we and other research groups found that reactive oxygen species (ROS), the by-product of SNP independent of NO, mediated SNP-induced cytotoxicity in various kinds of cells [23][24][25][26][27][28][29]. Our recent studies firmly demonstrated that hydroxyl radicals ( @BULLET OH) from the Fenton reaction between iron ions and hydrogen peroxide (H 2 O 2 ) dominated the SNP-induced NOindependent chondrocytes apoptosis though SNP induced NO production [23]. ...
... Cell viability was detected by Cell Counting Kit-8 (CCK-8) assay (Dojindo, Kumamoto, Japan) as described previously [29] . HepG2 cells cultured in 96- well plates (1 × 10 4 per well) for 24 h were treated with different stimuli, viable cells were assessed by absorbance measurements at 450 nm using an auto microplate reader (infinite M200, Tecan, Austria). ...
... Hep3B cells cultured in 96-well plates (1×10 4 per well) for 24 h were treated with different stimuli, viable cells were assessed by absorbance measurements at 450 nm using the microplate reader. Cell apoptosis was quantified by flow cytometry (FCM) (FACSCCanto II, BD Biosciences) analysis with Annexin V-FITC/PI apoptosis detection kit (Bestbio, Shanghai, China) as described previously [29]. HepG2 cells cultured in 6-well (5×10 5 per well) plates for 24 h were treated with different stimuli, then the cells were collected and stained with 5 ml of AnnexinV-FITC and 10 ml PI for 15 min at 4°C before FCM analysis. ...
This study aims to explore which radicals dominate sodium nitroprusside (SNP)-induced cytotoxicity in human hepatocellular carcinoma (HCC) cells (HepG2 and Hep3B). Exposure of SNP to cell medium produced abundant nitric oxide (NO), superoxide anion (O2•-), hydrogen peroxide (H2O2) and iron ions. SNP potently induced caspases activation, mitochondrial membrane permeabilization and apoptosis in HCC cells. In Hep3B cells, pretreatment with NO scavenger (PTIO) did not prevent SNP-induced cytotoxicity. However, in HepG2 cells, SNP-induced cytotoxicity was prevented significantly by pretreatment with PTIO and O2•- scavenger, and especially was almost completely blocked by pretreatment with FeTPPS (peroxynitrite scavenger). In contrast, although H2O2 scavenger potently scavenged SNP-induced H2O2 production, it did not prevent SNP-induced cytotoxicity in HepG2 cells. In addition, pretreatment with DFO (iron ions chelator) and iron-saturated DFO respectively completely prevented SNP-induced cytotoxicity in HepG2 cells. Collectively, peroxynitrite from the reaction between NO and O2•- elicited from SNP dominates the SNP-induced apoptosis of HepG2 cells, in which both iron ions and H2O2 are not involved.
... Casp-3 is a biomarker that reflects cellular apoptosis. Numerous studies have demonstrated the protective effects of R in different models of joint pathology, including experimental osteoarthritis and sodium nitroprusside-induced chondrocyte apoptosis, mediated through ROS and NO generation, followed by the activation of caspases and cytoskeletal remodeling [33][34][35]. These studies have shown that R has a protective anti-apoptotic effect on chondrocytes [34]. ...
... Numerous studies have demonstrated the protective effects of R in different models of joint pathology, including experimental osteoarthritis and sodium nitroprusside-induced chondrocyte apoptosis, mediated through ROS and NO generation, followed by the activation of caspases and cytoskeletal remodeling [33][34][35]. These studies have shown that R has a protective anti-apoptotic effect on chondrocytes [34]. ...
Introduction:
Estrogens play a considerable role in maintaining bone and articular cartilage homeostasis. Menopause provokes joint disorders due to metabolic syndrome and altered signaling pathways. Phytoestrogen resveratrol was demonstrated to provide chondroprotective and osteoprotective effects. However, the mechanisms of such action of Resveratrol are still being explored.
Aim:
The study aims to determine the effect of Resveratrol on the joints and its therapeutic mechanism in ovariectomized rats.
Material and methods:
The study was carried out on Wistar female rats that were divided into three groups, including control animals; ovariectomized rats (OVX); and the OVX group treated with an intravaginal gel containing Resveratrol (0.5 % 0.1 mL, daily 28 days). Knee joint tissues (articular cartilage, subchondral plate, subchondral bone) were assessed by histomorphometry. The expression of mTOR, PTEN, Caspase 3 and BCL-2 in articular cartilage and subchondral bone were evaluated immunohistochemically.
Results:
Resveratrol treatment of OVX rats prevented weight gain by 17 % (P < 0.001), demonstrating the systemic effect on metabolic pathways. Although there were no statistically significant differences in the thickness of articular cartilage between groups, OVX rats possessed degenerative changes in chondrocytes, associated with the enhanced expression of mTOR (P < 0.001) and Casp-3 (P = 0.005). Resveratrol decreased mTOR (P = 0.007) and Casp-3 (P = 0.011) expression in chondrocytes, reducing degenerative changes. At the same time, resveratrol attenuated the deterioration of trabecular bone in OVX rats (P = 0.002). This effect was through the up-regulation of BCL-2 (P = 0.018) and down-regulation of Casp-3 expression (P < 0.001).
Conclusions:
Intravaginal administration of resveratrol provided systemic effects and ameliorated joint tissue structure and signaling in OVX rats through stimulation of BCL-2 and reduced Casp-3 expression.
... Some studies indicated that the productions of nitric oxide (NO) and hydrogen peroxide (H2O2) could explain the cytotoxicity of SNP, [20,30] and enhancement of inducible nitric oxide synthase (iNOS) related to NO production [31]. To determine whether NO, H2O2, and iNOS were involved in the effects of SNP on primary chondrocytes, cells were treated with 0.8 mM SNP and/or 30 μM S-Equol for 24 h, and then intracellular NO generation, mitochondrial H2O2 production and iNOS expression were measured. ...
... Some studies indicated that the productions of nitric oxide (NO) and hydrogen peroxide (H 2 O 2 ) could explain the cytotoxicity of SNP [20,30] and enhancement of inducible nitric oxide synthase (iNOS) related to NO production [31]. To determine whether NO, H 2 O 2 , and iNOS were involved in the effects of SNP on primary chondrocytes, cells were treated with 0.8 mM SNP and/or 30 µM S-Equol for 24 h, and then intracellular NO generation, mitochondrial H 2 O 2 production and iNOS expression were measured. ...
Osteoarthritis (OA) is a common chronic disease with increasing prevalence in societies with more aging populations, therefore, it is causing more concern. S-Equol, a kind of isoflavones, was reported to be bioavailable and beneficial to humans in many aspects, such as improving menopausal symptoms, osteoporosis and prevention of cardiovascular disease. This study investigated the effects of S-Equol on OA progress in which rat primary chondrocytes were treated with sodium nitroprusside (SNP) to mimic OA progress with or without the co-addition of S-Equol for the evaluation of S-Equol’s efficacy on OA. Results showed treatment of 0.8 mM SNP caused cell death, and increased oxidative stress (NO and H2O2), apoptosis, and proteoglycan loss. Furthermore, the expressions of MMPs of MMP-2, MMP-3, MMP-9, and MMP-13 and p53 were increased. The addition of 30 μM S-Equol could lessen those caused by SNP. Moreover, S-Equol activates the PI3K/Akt pathway, which is an upstream regulation of p53 and NO production and is associated with apoptosis and matrix degradation. As a pretreatment of phosphoinositide 3-kinases (PI3K) inhibitor, all S-Equol protective functions against SNP decrease or disappear. In conclusion, through PI3K/Akt activation, S-Equol can protect chondrocytes against SNP-induced matrix degradation and apoptosis, which are commonly found in OA, suggesting S-Equol is a potential for OA prevention.
... In this line, Del Carlo et al. concluded that NO-induced cell death required ROS generation to occur in human cultured chondrocytes [214]. In rabbit studies, Liang et al. also proposed that the co-production of ROS with NO could explain the cytotoxicity of SNP, since ROS scavenger resveratrol avoided SNP-induced cell death [226]. Feeding this hypothesis, NOC12, a more potent producer of NO than SNP, was less able to induce cell death compared to SNP [227]. ...
... The apoptosis signaling events leading to cell death after SNP treatment in chondrocytes usually involves mitochondrial perturbation, such as reduction of mδΨ and Complex IV activity [72], release of cyt c and AIF, cleaved Bid mitochondrial translocation [231], Bax and Bak activation [226], as well as blockage of respiration and ATP generation in human articular chondrocytes and TC28 cells [232] and rabbit chondrocytes [233]. The latter studies suggested that mitochondrial perturbation could contribute to matrix loss and cartilage mineralization, or to the inhibition of cartilage matrix synthesis [232,233]. ...
Osteoarthritis (OA) is a joint pathology characterized by progressive cartilage degradation. Medical care is mainly based on alleviating pain symptoms. Compelling studies report the presence of empty lacunae and hypocellularity in cartilage with aging and OA progression, suggesting that chondrocyte cell death occurs and participates to OA development. However, the relative contribution of apoptosis per se in OA pathogenesis appears complex to evaluate. Indeed, depending on technical approaches, OA stages, cartilage layers, animal models, as well as in vivo or in vitro experiments, the percentage of apoptosis and cell death types can vary. Apoptosis, chondroptosis, necrosis, and autophagic cell death are described in this review. The question of cell death causality in OA progression is also addressed, as well as the molecular pathways leading to cell death in response to the following inducers: Fas, Interleukin-1β (IL-1β), Tumor Necrosis factor-α (TNF-α), leptin, nitric oxide (NO) donors, and mechanical stresses. Furthermore, the protective role of autophagy in chondrocytes is highlighted, as well as its decline during OA progression, enhancing chondrocyte cell death; the transition being mainly controlled by HIF-1α/HIF-2α imbalance. Finally, we have considered whether interfering in chondrocyte apoptosis or promoting autophagy could constitute therapeutic strategies to impede OA progression.
... Moreover, an increase in chondrocyte apoptosis has been anatomically linked to proteoglycan depletion [52]. Previous studies have suggested that RVS protects chondrocytes against apoptosis via different mechanisms, such as scavenging ROS [53], inhibiting mitochondrial membrane depolarization and ATP depletion [54], regulating the MALAT1/miR-9/NF-κB signaling pathway [55]. In the current study, tunel staining revealed that a massive amount of apoptotic chondrocytes was found upon DMM intervention compared to sham group, while the number of apoptotic chondrocytes was reduced in resveratrol-treated rats. ...
Our previous studies have confirmed that resveratrol (RSV) can prevent the development of osteoarthritis through a variety of mechanisms, such as apoptosis inhibition, autophagy induction and SIRT 1 activation. However, the pharmaceutical application of RSV is mainly limited by its low bioavailability. Here, we designed and synthesized RSV-loaded poly (D, l-lactide-coglycolide acid) (PLGA)-nanoparticles (NPs). The average particle size, polydispersity index and positive charge of RSV-loaded PLGA NPs were 50.40 nm, 0.217 and 12.57 mV, respectively. These nanoparticles had marked encapsulation efficiency (92.35 %) and drug loading (15.1 %) for RSV. It was found that RSV-loaded PLGA NPs not only inhibited the apoptosis of chondrocytes induced by IL-1, but also rescued GAG loss in vitro. Pharmacokinetic data showed that RSV-loaded PLGA NPs demonstrated a significantly profound and prolonged concentration profile in joint tissues, with quantifiable RSV concentrations over 35 days. The therapeutic effects of RSV-loaded PLGA NPs were then examined in rat osteoarthritis models. In vitro magnetic resonance imaging results showed that RSV-loaded PLGA NPs treatment dramatically reduced both T1ρ and T2 relaxation times at 4, 8, 12 weeks during administration, implying that cartilage destruction was alleviated. Histological assessments showed that RSV-loaded PLGA NPs significantly improved osteoarthritis symptoms. Gene expression analysis revealed that osteoarthritis mediator genes were downregulated in rats treated with RSV-PLGA NPs. Mechanistic studies indicated that RSV-loaded PLGA NPs inhibit apoptosis and promote autophagy. Collectively, this study demonstrates that intra-articular delivery of RSV via PLGA NPs might be an effective therapeutic approach for osteoarthritis.
... We have also investigated the intracellular ROS that in uences the beginning stages of apoptotic signaling [47,60]. Figure 5(b) reveals that the levels of intracellular ROS in MCF-7 breast cancer cells after treatment with Silibinin and LES nanoparticles dramatically increase in a time-dependent manner. ...
It has long been known that Silibinin, a naturally derived herbal phytochemical, is an effective drug for treating toxic thyroid damage; however, its role in cancer treatment is still not approved and is under investigation. In this work, we propose a protocol to prepare liposome-encapsulated Silibinin (LES) with the potential to produce reactive oxygen species (ROS) to treat MCF-7 breast cancer cells. Spherical-shaped LES nanoparticles with an average size of 60 nm and narrow particle size distribution (PDI=0.11) were prepared through the hydration of thin films. Studies of the pharmacokinetics showed that a burst release occurred during the first 12 h, followed by a sustained release over the next 12 days. MTT assays and the analysis of the drug effect determined that LES nanoparticles displayed a significant cytotoxic effect in killing breast cancer cells. IC50 values for LES nanoparticles were experimentally determined to be 20 μМ which was significantly lower than that of the pristine drug (38 μМ). It was also found that LES could remarkably change the expression levels of Caspase-3, Caspase-9, Bax, Bcl-2, and hTERT genes compared to the pristine drug. Therefore, encapsulating Silibilin into liposomes is an effective strategy to enhance breast cancer treatment effectiveness dramatically.
... Dave et al. demonstrated that RES inhibits prostaglandin E2 (PGE 2 )-mediated mitochondrial depolarization and adenosine triphosphate (ATP) production to chondrocyte apoptosis (Dave et al., 2008). Interestingly, in sodium nitroprusside (SNP)-induced chondrocyte apoptosis model, the researchers observed that RES protected chondrocytes from apoptosis by scavenging ROS and preventing abnormal cytoskeletal remodeling, suggesting that RES has the potential to prevent changes in chondrocyte structure (Jin et al., 2014;Liang et al., 2014). Furthermore, in animal models of KOA, RES has also been shown to prevent KOA by inhibiting NO production and reducing the number of TUNEL-positive cells, thereby preventing cartilage destruction Gu et al., 2016). ...
Osteoarthritis (OA) is one of the progressing chronic joint associated with by many complex factors such as age, obesity, and trauma. Knee osteoarthritis (KOA) is the most common type of OA. KOA is characterized by articular cartilage destruction and degeneration, synovial inflammation, and abnormal subchondral bone changes. To date, no practical clinical approach has been able to modify the pathological progression of KOA. Drug therapy is limited to pain control and may lead to serious side effects when taken for a long time. Therefore, searching for safer and more reliable treatments has become necessary. Interestingly, more and more research has focused on natural products, and monomeric compounds derived from natural products have received much attention as drug candidates for KOA treatment. Resveratrol (RES), a natural phenolic compound, has various pharmacological and biological activities, including anti-cancer, anti-apoptotic, and anti-decay. Recently, studies on the effects of RES on maintaining the normal homeostasis of chondrocytes in KOA have received increasing attention, which seems to be attributed to the multi-targeted effects of RES on chondrocyte function. This review summarizes preclinical trials, clinical trials, and emerging tissue engineering studies of RES for KOA and discusses the specific mechanisms by which RES alleviates KOA. A better understanding of the pharmacological role of RES in KOA could provide clinical implications for intervention in the development of KOA.
... Resveratrol (3,5,4′-trihydroxy-trans-stilbene, Rev) is a natural polyphenol, which widely exists in various plants, such as Polygonum cuspidatum, in fruits, including grapes and berries, in peanuts, and in red wine [9]. Resveratrol plays a regulatory role through a series of mechanisms, including scavenging ROS [10], antioxidative [11], and anti-inflammatory activities [12]. Recent studies have established that resveratrol owns the potential in the prevention or treatment of chronic inflammation-related disorders such as cardiovascular diseases, diabetes, obesity, and cancer [13][14][15][16]. ...
Background
hyperlipidemia acute pancreatitis (HTG-AP) is a major hidden danger affecting human health, however, whether there is a protective effect of resveratrol on HTG-AP is unclear. Therefore our study was aimed to investigate the preventive effect and the underlying mechanism of resveratrol in the HTG-AP mice model.
Methods
This research was divided into two parts. In the first part, mice were adaptively fed with normal chow or HFD for 6 weeks. From the second week, resveratrol-treated mice were in intragastric administration with resveratrol (45 mg/kg/d) for 4 weeks. In the second part, the procedures were the same as the first part. After the last intragastric administration with resveratrol, all mice were intraperitoneal injections of cerulean.
Results
We found resveratrol effectively inhibited pancreatic pathological injury in the HFD, AP, and HTG-AP mice. Resveratrol reduced the LPS, IL-6, TNF-α, and MCP-1 expressions in the HFD mice. Resveratrol also reduced TNF-α, MDA, and MCP-1 expressions and increased SOD and T-AOC expressions in the AP and HTG-AP mice. Furthermore, resveratrol suppressed the NF-κB pro-inflammatory signaling pathway in pancreatic tissues in the AP and HTG-AP mice. Moreover, resveratrol improved the gut microbiota in the HFD mice.
Conclusion
The resveratrol pre-treatment could attenuate pancreas injury, inflammation, and oxidative stress in the HTG-AP mice, via restraining the NF-κB signaling pathway and regulating gut microbiota. Therefore, Our study proved that the resveratrol pre-treatment had a preventive effect on HTG-AP.
... The authors indicated that pretreatment with resveratrol (100 µM) significantly lowered the sodium nitroprusside-induced ROS. The study also revealed that resveratrol can contribute to a reduction in apoptosis due to its ROS scavenging effects [57]. ...
Inflammaging, the steady development of the inflammatory state over age is an attributable characteristic of aging that potentiates the initiation of pathogenesis in many age-related disorders (ARDs) including neurodegenerative diseases, arthritis, cancer, atherosclerosis, type 2 diabetes, and osteoporosis. Inflammaging is characterized by subclinical chronic, low grade, steady inflammatory states and is considered a crucial underlying cause behind the high mortality and morbidity rate associated with ARDs. Although a coherent set of studies detailed the underlying pathomechanisms of inflammaging, the potential benefits from non-toxic nutrients from natural and synthetic sources in modulating or delaying inflammaging processes was not discussed. In this review, the available literature and recent updates of natural and synthetic nutrients that help in controlling inflammaging process was explored. Also, we discussed the clinical trial reports and patent claims on potential nutrients demonstrating therapeutic benefits in controlling inflammaging and inflammation-associated ARDs.
... NO produced by SNP increased apoptosis of the rabbit articular chondrocytes. It was consistent with the previous reports that NO initiated chondrocyte apoptosis process [27] similar to that occurred in OA patients. ...
Background
Nitric oxide (NO) and reactive oxygen species (ROS) play an important role in the pathology of human osteoarthritis (OA). Ankylosing spondylitis (AS) and atypical OA have similar clinical manifestations and often require differential diagnosis. The mechanism is however not totally clear yet. This study aims to investigate the effects of excessive NO-ROS in OA patients and the effects of extracellular signal-regulated kinases (ERK) pathway in NO-induced apoptosis of chondrocytes during OA progress.
Methods and results
Serum samples from OA or AS as pathological control patients and healthy controls were collected for NO and related chemical measurements. The rabbit articular chondrocytes were cultured in vitro, and NO was applied by Sodium Nitroprusside (SNP) in culture medium to mimic OA condition in patients. The level of SNP-evoked chondrocyte apoptosis with or without PD98059 (ERK-specific inhibitor) was evaluated by TUNEL assay, Annexin V flow cytometry and Western blotting. The activity and mRNA expression of caspase-3 in chondrocytes were measured by assay kits and RT-PCR. The levels of NO and malondialdehyde (MDA) in serum were significantly higher in OA patients, while only MDA was significantly higher in AS patients. However, the level of superoxide dismutase (SOD) was lower in both OA and AS patients. SNP induced chondrocyte apoptosis was enhanced by PD98059 with increased protein expression and functional activity of caspase-3.
Conclusions
The increase in nitric oxide occurs specifically in OA patients. ERK pathway may play a protective role on the NO-induced chondrocyte apoptosis, and inhibition of ERK pathway enhances the NO-induced apoptosis.
... In recent years, a variety of plant extracts, such as curcumin [4], L-theanine [5], and resveratrol [6], have been found to exert therapeutic or preventive effects on OA progression. Baicalein, a major flavonoid constituent in the plants of genus Scutellaria (Lamiaceae) [7], is found in abundant quantity in the root of S. baicalensis. ...
Baicalein has been shown to have chondroprotective potential in vitro. However, its effect on disease modification in osteoarthritis (OA) is largely unknown. The present study is aimed at determining whether baicalein could slow the progression of OA and inhibit OA-related inflammation in a rat model of destabilization of the medial meniscus (DMM) and the underlying mechanisms. The rats subjected to DMM surgery were treated with baicalein (0.8, 1.6, and 3.2 μg/L, 50 μL, once a week) by intra-articular injection for 6 weeks. Dexamethasone (0.4 mg/mL, 50 μL, once a week) was used as a positive control. Histologic grading of cartilage degeneration was performed using the Osteoarthritis Research Society International (OARSI) recommended grading system (on a scale of 0-6). The expression levels of molecules associated with cartilage homeostasis and inflammatory cytokines were analyzed; moreover, the NLRP3 inflammasome activation and cartilage oxidative stress-associated molecules were determined. Baicalein treatment reduced the OARSI score and slowed OA disease progression in a dose-dependent manner within a certain range. Compared with DMM rats, intra-articular injection of baicalein led to (1) reduced levels of inflammatory mediates such as IL-1β and TNF-α, (2) reduced immunochemical staining of MMP-13 and ADAMTS-5, (3) suppressed immunochemical staining loss of type II collagen, (4) reduced expression of cartilage degradation markers including CTX-II and COMP in urine, and (5) inhibited NLRP3 inflammasome activation rather than regulated expression of SOD, GSH, and MDA. In contrast to the administration of baicalein, dexamethasone injection showed similar effects to slow OA progression, while dexamethasone inhibited NLRP3 inflammasome partly through decreasing levels of SOD, GSH, and MDA. This study indicated that baicalein may have the potential for OA prevention and exerts anti-inflammatory effects partly via suppressing NLRP3 inflammasome activation without affecting oxidative stress-associated molecules, and inhibition of cartilage catabolism enzymes in an OA rat model.
1. Introduction
Osteoarthritis (OA) is the most common arthritis and degenerative disease of the articular joints involving the articular cartilage, subchondral bone, and synovium and characterized by joint dysfunction, chronic pain, and disability [1]. The only current therapeutic option for OA is pain-relief drugs, such as paracetamol and nonsteroidal anti-inflammatory drugs (NSAIDs) [2]. However, safety is focused due to gastrointestinal injury and cardiovascular events for long-term administration of NSAIDs [3].
In recent years, a variety of plant extracts, such as curcumin [4], L-theanine [5], and resveratrol [6], have been found to exert therapeutic or preventive effects on OA progression. Baicalein, a major flavonoid constituent in the plants of genus Scutellaria (Lamiaceae) [7], is found in abundant quantity in the root of S. baicalensis. Baicalein have been found to exhibit several pharmacological activities, such as antioxidant [8], anticancer [9], anticardiovascular [10], antidiabetic [11], and anti-inflammatory [12] activities. Evidence from several studies suggested that baicalein is effective in mitigating OA chondrocyte apoptosis and inflammation in vitro. For example, baicalein has been shown to alleviate the inflammatory process by reducing the expression of inflammatory cytokines such as IL-6, IL-8, and iNOS, in contrast increasing IL-10 production [13] and inhibiting degrading enzymes of the extracellular matrix such as matrix metalloproteinase-3 (MMP-3) and MMP-13 in human chondrocytes and in IL-1β- and TNF-α-treated mouse articular cartilage explants [14]. Moreover, our recent in vitro study determined the effects of baicalein on IL-1β-induced primary chondrocytes of rats. The results showed that baicalein downregulated the elevated caspase-3 levels but promoted the expression of Bcl-2. Baicalein treatment significantly inhibited NF-κB signaling pathway activation and suppressed apoptosis and matrix degradation, indicating that baicalein might exert OA protective effect [15].
As is known, low-grade inflammation is believed to be involved in the development and progression of OA [16]; moreover, inflammatory cytokines such as IL-1β, IL-18, and TNF-α have demonstrated overexpression in posttraumatic osteoarthritis (PTOA) patients [17] and animal models [1]. A number of studies demonstrated that elevated levels of IL-1β and TNF-α suppress the synthesis of type II collagen and aggrecan, furthermore stimulating chondrocytes to release MMP-1, MMP-3, MMP-13, and disintegrin metalloproteinase with thrombospondin motifs (ADAMTS), which are major chondrocyte catabolic components to degrading the cartilage extracellular matrix [18–21]. Interestingly, it was determined that activation of inflammasome plays a key role in proinflammatory cytokine production, especially IL-1β. As a most studied inflammasome, NLRP3 inflammasome (NLR family, pyrin domain containing 3; NLR refers to “nucleotide-binding domain, leucine-rich repeat”) is responsible for the activation of caspase-1 and caspase-5, which leads to the secretion of IL-1β and IL-18, in turn causing pyroptosis [22]. Much higher levels of NLRP3 are detected in the OA synovium than in normal, subsequent activated caspase-1, and the release of IL-1β and IL-18 by pyroptotic chondrocytes triggers the inflammatory cascade, leading to cartilage degradation and chondrocyte death [23, 24]. There are various NLRP3 activators, such as mitochondrial damage, lysosomal damage, cytosolic K+ efflux, and reactive oxygen species- (ROS-) induced cell oxidative stress [25]. Several lines of evidences suggest that oxidative stress, which contributes to the imbalance of the expression of antioxidant enzymes and ROS scavenging systems, causes abnormalities in cartilage and bone metabolism, aggravating cartilage degradation [26]. Whether baicalein exerts its anti-inflammatory effects through NLRP3 inflammasome and the role of oxidative stress in the activation of NLRP3 during baicalein treatment are still unknown.
In a word, the chondrocyte protective effects and the underlying mechanism of baicalein for OA have not been well investigated. In our study, dexamethasone was used as a positive control due to its exact effect on OA disease modification through intra-articular injection [27], and the anti-inflammatory properties of dexamethasone have been considered [28]. Based on the above properties of baicalein, we aim to study the efficacy of baicalein compared to dexamethasone and the possible mechanism using a DMM-induced rat OA model in vivo.
2. Materials and Methods
2.1. Experimental Design
Rats were housed on a standardized pelleted diet and supplied with tap water. All animal experiments were carried out in accordance with the guidelines of the China Ethical Committee for Animal Experiments. OA was induced in 8-week-old male SD rats (bought from the laboratory animal center of Liaoning Changsheng Biotechnology Co., Ltd., China) by DMM surgery of the right knees as described [29]. Sham surgery was performed on the right knee joint using a separated group of rats as the control. Rats were randomly divided into 6 groups (/group): (1) DMM group, (2) sham group (vehicle control), (3) baicalein (0.8 μg/L, 50 μL), (4) baicalein (1.6 μg/L, 50 μL), (5) baicalein (3.2 μg/L, 50 μL), and (6) dexamethasone (DXM, 0.4 mg/mL, 50 μL). After 1 week, rats in the treatment group received baicalein or dexamethasone (once a week) postoperatively via articular cavity injection for 6 weeks. Baicalein (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in DMSO, and further dilutions were prepared in saline. In the control group, rats were injected with 2% DMSO (in saline) only. The dose of baicalein is determined based on our in vitro experiments and preliminary experiments [15]. Three different doses are set to avoid a single dose that is not effective, so as to choose a dose that can work.
After the last treatment, euthanasia was performed on the rats, and serum and urine samples were collected for the ELISA assay. Three out of 8 rats were chosen randomly of each group to observe the gross morphology, and subsequently, the articular cartilage was collected to detect the levels of MMP-13 and ADAMTS-5, and the experiment was repeated three times. Another 5 rats were used for pathological analysis, and the complete knee joints were immobilized in 4% paraformaldehyde.
2.2. Histological Examination
The knee joint in each group (/group) was placed in a bucket which was filled up with decalcification solution (Servicebio Biotechnology Co., Ltd, Wuhan, China, G1105) and then put in a constant temperature shaker. The replacement period of the decalcification solution is 2-3 d. The degree of decalcification is observed every two days. If the needle can be moved, the tissue is cut with a Lycra blade according to the material requirements, which can accelerate the softening speed. The dehydrating process was performed in gradient alcohol, and the intact flexed joint was embedded in paraffin. Then, serial sagittal sections were obtained across the entire knee joint. Slides were stained for sulfated glycosaminoglycans (GAGs) with Safranin O/Fast Green. Histologic grading of cartilage degeneration was performed using the Osteoarthritis Research Society International (OARSI) recommended grading system (on a scale of 0-6) [30], and histological analyses were performed by two experienced observers and double-blinded.
2.3. Immunohistochemical Assessment
After 6-week intra-articular injection, the complete right knee of every group (/group) was collected for the histologic section, and the procedures were in accordance with the histological examination. After deparaffinization, the sections were rinsed with 0.3% H2O2 in 60% methanol for 30 min and permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 20 min. The section then was incubated in goat serum in PBS for nonspecific adsorption. For immunohistochemistry, sections were incubated with (1) anti-rabbit polyclonal antibody directed at collagen II (1 : 400; Abcam, ab34712), (2) anti-MMP-13 mouse monoclonal antibody (1 : 150; Novus, OTI2D8), (3) anti-ADAMTS-5 rabbit polyclonal antibody (1 : 100; Novus, NBP2-15286), (4) anti-NLRP3 rabbit polyclonal antibody (1 : 200; ABclonal, A12694), or anti-caspase-1 rabbit polyclonal antibody (1 : 150; ABclonal, A0964). Horseradish peroxidase- (HRP-) conjugated secondary antibody was applied and stained with a diaminobenzidine (DAB) kit. The positive stained chondrocytes in three central regions of articular cartilage were counted using Image-Pro Plus version 6.0 software.
2.4. Measurements of Inflammatory Cytokines
Serum samples from eight rats were collected, and concentrations of IL-1β and TNF-α were measured using specific rat enzyme-linked immunosorbent assay (ELISA) kits (Huijia Biotechnology Co., Ltd., Nanjing, China) according to the manufacturer’s instructions.
2.5. Measurements of Cartilage Catabolism Protein
The cartilage of knee joints (/group) was collected, and the cartilage was ground with PMSF : RIPI (1 : 99) (Beyotime, China) under low temperature for 30 min and then centrifuged at 12000 rpm. 200 μL of lysis buffer was added per 100 mg cartilage sample. The supernatant was collected for measuring the levels of MMP-13 and ADAMTS-5 via the ELISA kit (Huijia Biotechnology Co., Ltd (China). Rat urine samples (/group) were collected and centrifuged at for 15 min within 1 h. The supernatant was stored at −80°C until future analysis. The concentrations of COMP and CTX-II (Jingmei Co., Ltd., Jiangsu, China) were measured in urine according to the manufacturer’s instructions.
2.6. Measurements of SOD, GSH, and MDA Levels in Serum
Serum levels of superoxide dismutase (SOD), glutathione peroxidase (GSH), and malonaldehyde (MDA) were examined using detection kits (Beyotime Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer’s instructions.
2.7. Statistical Analysis
All statistical analyses were performed using SPSS 22.0 software, and the results are expressed as the . One-way analysis of variance (ANOVA) was used for comparisons between groups, and was considered statistically significant. Multiple comparisons between groups were performed using a post hoc Tukey test. Histological analyses were performed by two experienced investigators who were double-blinded.
3. Results
3.1. Changes in Gross Morphology of the Joints in the DMM-Induced OA Model
A healthy articular surface was observed in both femur and tibia in sham cartilage (Figure 1). There were obvious macroscopic changes in the appearance of the right joints in the OA model at 7 weeks postsurgery. DMM cartilage was abraded and contained fibrotic tissue, and the cartilage showed significant osteophyte formation at the medial tibia plateau (MTP) and the intercondylar space of the femur (Figure 1). Moreover, the cartilage of baicalein-treated rats with 0.8 and 1.6 μg/L exhibited no detectable macroscopic difference compared to DMM cartilage. In contrast, the joints administrated with 3.2 μg/L baicalein or dexamethasone showed significantly milder injuries than DMM joints, including cartilage opacity and roughness (Figure 1).
... Resveratrol activated SIRT1 and suppressed IL-1β induced expression of HIF-2α in human chondrocytes and intraarticular injection of resveratrol slowed the progression of experimental OA in a mouse model of OA [117]. Resveratrol also protected rabbit chondrocytes against sodium nitroprusside induced apoptosis by scavenging the SNP induced ROS and NO [118]. Resveratrol induced the expression of HO-1 via the activation of Nrf2 and suppressed oxidative stress in rat with OA [114]. ...
Osteoarthritis (OA) is the most prevalent joint degenerative disease leading to irreversible structural and functional changes in the joint and is a major cause of disability and reduced life expectancy in ageing population. Despite the high prevalence of OA, there is no disease modifying drug available for the management of OA. Oxidative stress, a result of an imbalance between the production of reactive oxygen species (ROS) and their clearance by antioxidant defense system, is high in OA cartilage and is a major cause of chronic inflammation. Inflammatory mediators, such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) are highly upregulated in OA joints and induce ROS production and expression of matrix degrading proteases leading to cartilage extracellular matrix degradation and joint dysfunction. ROS and inflammation are interdependent, each being the target of other and represent ideal target/s for the treatment of OA. Plant polyphenols possess potent antioxidant and anti-inflammatory properties and can inhibit ROS production and inflammation in chondrocytes, cartilage explants and in animal models of OA. The aim of this review is to discuss the chondroprotective effects of polyphenols and modulation of different molecular pathways associated with OA pathogenesis and limitations and future prospects of polyphenols in OA treatment.
... Autophagy has been demonstrated to interact with the intracellular ROS [21][22][23] that has crucial effects on the destiny of tumor cells. 24 Accumulating evidence indicates that tumor cells usually have abnormal high level of ROS, which benefits their survival and resistance to drugs, 25 and particularly in CML cells, the constitutively activated BCR-ABL ...
Background:
Chronic myeloid leukemia (CML) is a myeloproliferative disorder due to the existence of BCR-ABL fusion protein that allows the cells to keep proliferating uncontrollably. Although tyrosine kinase inhibitors can inhibit the activity of BCR-ABL fusion protein to trigger the cells apoptosis, drug resistance or intolerance exists in part of CML patients. Arsenic sulfide in its raw form (r-As4S4) can be orally administrated and certain therapeutic effects have been found out in the treatment of hematologic malignancies through inducing cell apoptosis.
Methods:
In this work, a water-dissolvable arsenic sulfide nanoformualtion (ee-As4S4) composed of As4S4 particulates with 470 nm in diameter and encapsulated by a kind of hydrophilic polymer was fabricated and applied to the CML cell line K562, K562/AO2 and primary cells from the bone marrow of CML patients.
Results:
Results showed that instead of inhibiting the activity of BCR-ABL, ee-As4S4 induced direct degradation of BCR-ABL in K562 cells within 6 hr incubation, followed by the occurrence of erythroid differentiation in K562 after 72 hr incubation, evidenced by the significantly upregulated CD235a and benzidine staining, which was not detectable with r-As4S4. The ee-As4S4-induced erythroid differentiation was also observed in K562/AO2 cells and bone marrow mononuclear cells of CML patients. Mechanistic studies indicated that ee-As4S4 induced autophagy by downregulating the level of intracellular ROS and hypoxia-inducible factor-1α significantly, which led to the subsequent degradation of BCR-ABL. When the concentration was increased, ee-As4S4 induced much more significant apoptosis and cell cycle arrest than r-As4S4, and the cytotoxicity of the former was about 178 times of the latter.
Conclusion:
ee-As4S4 was capable of inducing significant erythroid differentiation of CML cells by inducing the direct degradation of BCR-ABL; the new effect could improve hematopoietic function of CML patients as well as inhibit the leukemic cell proliferation.
... RSV, a polyphenolic compound in red wine, was well known for its anti-inflammation and antiapoptotic properties [37,38]. However, its protective properties and underlying mechanisms are largely unknown in the occurrence of sepsis-induced ALI. ...
Sepsis is a major cause of death in intensive care units. The purpose of this study was to investigate the effect of resveratrol (RSV) on sepsis-induced acute lung injury (ALI). The underlying molecular mechanisms were deciphered by both in vitro and in vivo experiments. Polymicrobial sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP). RSV pretreatment significantly attenuated CLP-induced acute lung injury, which was associated with enhanced expression of VEGF-B. The protective properties of RSV were assayed in lipopolysaccharide (LPS)-stimulated MH-S cells. We determine that RSV administration inhibited the increased production of TNF-α, IL-6, and IL-1β in LPS-stimulated MH-S cells, which was associated with inhibition of the nuclear factor-κB, P38, and ERK signaling pathways. We also provide evidence that RSV administration reduced LPS-induced apoptosis of MH-S cells by altering the unbalance of Bax/Bcl-2 and inhibiting LPS-induced autophagy. The inhibitory effects of RSV on cytokine levels and apoptosis of alveolar macrophages were both blocked by VEGF-B siRNA. Furthermore, RSV administration regulated LPS-induced C5aR and C5L2 expression, revealing an additional mechanism underlying RSV's anti-inflammatory and anti-apoptosis effects. Collectively, these results demonstrated that RSV was able to protect against sepsis-induced acute lung injury by activating the VEGF-B signaling pathway.
... Considering that ROS are involved in the induction of both processes (autophagy and apoptosis) [35,36], it was necessary to investigate whether ROS blockade would affect the activation of these cell death processes. ...
Triple negative breast cancer (TNBC) is very aggressive and lacks specific therapeutic targets, having limited treatment options and poor prognosis. [6]-gingerol is the most abundant and studied compound in ginger, presenting diverse biological properties such as antitumor activity against several types of cancer, including breast cancer. In this study, we show that the semi-synthetic analogue SSi6, generated after chemical modification of the [6]-gingerol molecule, using acetone-2,4-dinitrophenylhydrazone (2,4-DNPH) reagent, enhanced selective cytotoxic effects on MDA-MB-231 cells. Remarkably, unlike the original [6]-gingerol molecule, SSi6 enabled autophagy followed by caspase-independent apoptosis in tumor cells. We found a time-dependent association between SSi6-induced oxidative stress, autophagy and apoptosis. Initial SSi6-induced reactive oxygen species (ROS) accumulation (1h) led to autophagy activation (2-6h), which was followed by caspase-independent apoptosis (14h) in TNBC cells. Additionally, our data showed that SSi6 induction of ROS plays a key role in the promotion of autophagy and apoptosis. In order to investigate whether the observed cell death induction was dependent on preceding autophagy in MDA-MB-231 cells, we used siRNA to knock down LC3B prior to SSi6 treatment. Our data show that LC3B downregulation decreased the number of apoptotic cells after treatment with SSi6, indicating that autophagy is a key initial step on SSi6-induced caspase-independent apoptosis. Overall, the results of this study show that structural modifications of natural compounds can be an interesting strategy for developing antitumor drugs, with distinct mechanisms of actions, which could possibly be used against triple negative breast cancer cells that are resistant to canonical apoptosis-inducing drugs.
... Several studies previously reported that intracellular ROS production is the byproduct of normal cellular oxidative processes involved in the initiation of apoptotic signaling [23]. We examined the effect of DPT on ROS production using flow cytometry with DCFH-DA, an indicator for ROS. ...
Deoxypodophyllotoxin (DPT) is a naturally occurring flavolignan isolated from Anthriscus sylvestris. Recently, it has been reported that DPT inhibits tubulin polymerization and induces G2/M cell cycle arrest followed by apoptosis through multiple cellular processes. Despite these findings, details regarding the cellular and molecular mechanisms underlying the DPT-mediated cell death have been poorly understood. To define a mechanism of DPT-mediated cell death response, we examined whether DPT activates signaling pathways for autophagy and apoptosis. We demonstrated that DPT inhibited cell viability and induced apoptosis in prostate cancer cell lines, as evidenced by a mitochondrial membrane potential and expression of apoptosis-related proteins. Reactive oxygen species (ROS), primarily generated from the mitochondria, play an important role in various cellular responses, such as apoptosis and autophagy. DPT significantly triggered mitochondrial ROS, which were detected by MitoSOX, a selective fluorescent dye of mitochondria-derived ROS. Furthermore, DPT induced autophagy through an up-regulation of autophagic biomarkers, including a conversion of microtubule-associated protein 1 light chain 3 - I (LC3-I) into LC3-II and a formation of acidic vesicular organelles. Moreover, mitochondrial ROS promoted AKT-independent autophagy and ERK signaling. The inhibition of autophagy with 3-methyladenine or LC3 knockdown enhanced DPTinduced apoptosis, suggesting that an autophagy plays a protective role in cell survival against apoptotic prostate cancer cells. Additionally, the results from an in vivo xenograft model confirmed that DPT inhibited tumor growth by regulating the apoptosis- and autophagy-related proteins.
... Cd induces ROS generation, leading to neuronal apoptosis via targeting the NOX2-derived, ROS-dependent PP5-JNK signaling pathway [35]. Sodium nitroprusside induces NO-independent ROS production, causing rabbit articular chondrocyte apoptosis [36]. Carnosic acid induces apoptosis by promoting ROS production and activating JNK signaling pathways in human cervical cancer cells [37]. ...
Lead (Pb) pollution has become one of the most serious global ecological problems. In animals, Pb ingestion induces apoptosis in many tissues. However, the mechanisms by which Pb induces apoptosis in chicken splenic lymphocytes in vitro via the PI3K/Akt pathway and the antagonistic effect of selenium (Se) on Pb remain unclear. Therefore, we established the in vitro Se-Pb interaction model in chicken splenic lymphocytes and examined the frequency of apoptotic cells using acridine orange/ethidium bromide (AO/EB) staining and the TdT-mediated dUTP nick end labeling assay and detected the activities of glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT), as well as the levels of malondialdehyde (MDA) and reactive oxygen species (ROS). The expression of PI3K/Akt pathway-related genes was also examined by qRT-PCR and western blotting. MDA and ROS levels were markedly increased, whereas the activities of GPx, SOD, and CAT were significantly decreased; the levels of the PI3K, Akt, and Bcl-2 messenger RNAs (mRNAs) and proteins were decreased; and the levels of the p53, Bax, cytochrome c (Cyt-c), caspase 3, and caspase 9 mRNAs and proteins were increased in the Pb group. In addition, the frequency of apoptotic cells was also significantly increased by the Pb treatment. However, Se supplementation during Pb exposure observably attenuated Pb-induced apoptosis; increased the levels of the PI3K, Akt, and Bcl-2 mRNAs and proteins; and decrease the levels of the p53, Bax, Cyt-c, caspase 3, and caspase 9 mRNAs and proteins in the chicken spleen. In conclusion, Pb exposure causes oxidative stress, inhibits the PI3K/Akt pathway, and subsequently induces apoptosis in chicken splenic lymphocytes in vitro, and these effects are partially attenuated by Se supplementation. To the best of our knowledge, this study is the first to reveal the antagonistic effect of Se on Pb-induced apoptosis of chicken splenic lymphocytes in vitro via the activation of the PI3K/Akt pathway.
... The level of intracellular ROS in chondrocytes was detected using a chemical fluorescence method (2,7-dichlorofuorescin diacetate, DCFH-DA) as previously described (Liang, Wang & Chen, 2014). Briefly, the cell culture medium from the 24-well plates was discarded and washed three times with free DMEM. ...
We have previously reported the application of low molecular weight XG(LM-XG), with molecular weights ranging from 1 × 10⁶ Da to 1.5 × 10⁶ Da for treating osteoarthritis. In this study, we investigated the anti-apoptotic activity of LM-XG under oxidative stress conditions, activated by hydrogen peroxide (H2O2)-treated chondrocytes in vitro. Chondrocytes were pretreated with various doses of LM-XG (0, 10, 100, 500, or 1000 μg/mL) or 1000 μg/mL sodium hyaluronate for 12 h, and then exposed to 0.5 mmol/L H2O2 for another 12 h. After treatment, chondrocyte viability was evaluated using a cell counting kit-8; DNA fragmentation was detected using Hoechst33258 staining; the percentage of DNA fragmentation was evaluated using the diphenylamine DNA assay kit; the apoptosis rate was evaluated using flow cytometry; chondrocyte ultra-microscopic morphology was observed using transmission electron microscopy; intracellular reactive oxygen species levels were observed and quantified using 2,7-dichlorofuorescin diacetate, mitochondrial permeability transition analysis was performed using MitoTracker Red CMXRos and 4′,6-diamidino-2-phenylindole staining; and finally, caspase-3 activity was detected by western blot. The results showed that, compared with H2O2-treated chondrocytes, LM-XG improved cell viability, decreased the percentage of DNA fragmentation, reduced the apoptosis rate, decreased the levels of intracellular reactive oxygen species and mitochondrial permeability transition, reverted the morphological damage, and downregulated cleaved caspase-3 levels. These results demonstrate that LM-XG has anti-apoptotic activity in H2O2-treated chondrocytes.
... NO and ROS are responsible for SNP-induced chondrocytes apoptosis through an intrinsic apoptosis pathway. Resveratrol has been demonstrated to scavenge SNPinduced ROS, instead of NO, to remarkably prevent chondrocytes apoptosis [49]. Using atomic force microscopy (AFM), resveratrol potently prevent SNP-induced chondrocytes changes, which include shrunk, round, lamellipodia contraction, and aggregation of the cytoskeleton, decrease in adherent junctions among cells, and decrease in the expression of cytoskeletal proteins [50]. ...
Osteoarthritis (OA) is a chronic degenerative joint disease characterized by articular cartilage destruction, synovial inflammation, and osteophyte formation. No effective treatments are available. The current pharmacological medications such as nonsteroidal anti-inflammatory drugs (NSAIDs) and analgesics, accompanied by possible adverse effects, might ameliorate OA symptoms. But they do not arrest the progression of OA. Traditional Chinese medicine (TCM) provides medical value by modification of disease and symptoms in OA. Valuable work on exploring TCM merits for OA patients has been investigated using modern technologies, although the complicated interacting network among the numerous components indicates the uncertainty of target specification. This review will provide an overview of the action mechanism of TCM in the last 5 years, discussing the TCM activities of anti-inflammation, antiapoptosis, antioxidation, anticatabolism, and proliferation in OA. TCM is a proposed medical option for OA treatment.
... Chondrocyte apoptosis is related to • NO which is considered as the primary inducer mediated by caspase-3 and tyrosine kinase activation [117]. It is reported that resveratrol prevents sodium nitroprusside-induced chondrocyte apoptosis via scavenging ROS [118]. Lee and Yang demonstrated that polychlorinated biphenyl 126 is an initiator of chondrocyte apoptosis via generating ROS, increasing NO production and NF-kB binding activity in the chondrocytes [119]. ...
Chondrocytes communicate with each other mainly via diffusible signals rather than direct cell-to-cell contact. The chondrogenic differentiation of mesenchymal stem cells (MSCs) is well regulated by the interactions of varieties of growth factors, cytokines, and signaling molecules. A number of critical signaling molecules have been identified to regulate the differentiation of chondrocyte from mesenchymal progenitor cells to their terminal maturation of hypertrophic chondrocytes, including bone morphogenetic proteins (BMPs), SRY-related high-mobility group-box gene 9 (Sox9), parathyroid hormone-related peptide (PTHrP), Indian hedgehog (Ihh), fibroblast growth factor receptor 3 (FGFR3), and β-catenin. Except for these molecules, other factors such as adenosine, O2 tension, and reactive oxygen species (ROS) also have a vital role in cartilage formation and chondrocyte maturation. Here, we outlined the complex transcriptional network and the function of key factors in this network that determine and regulate the genetic program of chondrogenesis and chondrocyte differentiation.
... We examined the intracellular ROS involved in the initiation of apoptotic signaling, which are the byproducts of normal cellular oxidative processes [27]. As a result, intracellular ROS levels were significantly increased in a time-dependent manner in PC-3 cells treated with silibinin (Fig. 1a). ...
Background
Silibinin, a biologically active compound of milk thistle, has chemopreventive effects on cancer cell lines. Recently it was reported that silibinin inhibited tumor growth through activation of the apoptotic signaling pathway. Although various evidences showed multiple signaling pathways of silibinin in apoptosis, there were no reports to address the clear mechanism of ROS-mediated pathway in prostate cancer PC-3 cells. Several studies suggested that reactive oxygen species (ROS) play an important role in various signaling cascades, but the primary source of ROS was currently unclear.
Methods
The effect of silibinin was investigated on cell growth of prostate cell lines by MTT assay. We examined whether silibinin induced apoptosis through production of ROS using flow cytometry. Expression of apoptosis-, endoplasmic reticulum (ER)-related protein and gene were determined by western blotting and RT-PCR, respectively.
Results
Results showed that silibinin triggered mitochondrial ROS production through NOX4 expression and finally led to induce apoptosis. In addition, mitochondrial ROS caused ER stress through disruption of Ca2+ homeostasis. Co-treatment of ROS inhibitor reduced the silibinin-induced apoptosis through the inhibition of NOX4 expression, resulting in reduction of both Ca2+ level and ER stress response.
Conclusions
Taken together, silibinin induced mitochondrial ROS-dependent apoptosis through NOX4, which is associated with disruption of Ca2+ homeostasis and ER stress response. Therefore, the regulation of NOX4, mitochondrial ROS producer, could be a potential target for the treatment of prostate cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-016-2516-6) contains supplementary material, which is available to authorized users.
Articular cartilage injury is one of the most common diseases in orthopedic clinics. Following an articular cartilage injury, an inability to resist vascular invasion can result in cartilage calcification by newly formed blood vessels. This process ultimately leads to the loss of joint function, significantly impacting the patient's quality of life. As a result, developing anti‐angiogenic methods to repair damaged cartilage has become a popular research topic. Despite this, tissue engineering, as an anti‐angiogenic strategy in cartilage injury repair, has not yet been adequately investigated. This exhaustive literature review mainly focused on the process and mechanism of vascular invasion in articular cartilage injury repair and summarized the major regulatory factors and signaling pathways affecting angiogenesis in the process of cartilage injury. We aimed to discuss several potential methods for engineering cartilage repair with anti‐angiogenic strategies. Three anti‐angiogenic tissue engineering methods were identified, including administering angiogenesis inhibitors, applying scaffolds to manage angiogenesis, and utilizing in vitro bioreactors to enhance the therapeutic properties of cultured chondrocytes. The advantages and disadvantages of each strategy were also analyzed. By exploring these anti‐angiogenic tissue engineering methods, we hope to provide guidance for researchers in related fields for future research and development in cartilage repair.
Half-sandwich Ru(II) complexes containing nitro-substituted furoylthiourea ligands, bearing the general formula [(η6-p-cymene)RuCl2(L)] (1-6) and [(η6-p-cymene)RuCl(L)(PPh3)]+ (7--12), have been synthesized and characterized. In contrast to the spectroscopic data which revealed monodentate coordination of the ligands to the Ru(II) ion via a "S" atom, single crystal X-ray structures revealed an unusual bidentate N, S coordination with the metal center forming a four-membered ring. Interaction studies by absorption, emission, and viscosity measurements revealed intercalation of the Ru(II) complexes with calf thymus (CT) DNA. The complexes showed good interactions with bovine serum albumin (BSA) as well. Further, their cytotoxicity was explored exclusively against breast cancer cells, namely, MCF-7, T47-D, and MDA-MB-231, wherein all of the complexes were found to display more pronounced activity than their ligand counterparts. Complexes 7-12 bearing triphenylphosphine displayed significant cytotoxicity, among which complex 12 showed IC50 values of 0.6 ± 0.9, 0.1 ± 0.8, and 0.1 ± 0.2 μM against MCF-7, T47-D, and MDA-MB-231 cell lines, respectively. The most active complexes were tested for their mode of cell death through staining assays, which confirmed apoptosis. The upregulation of apoptotic inducing and downregulation of apoptotic suppressing proteins as inferred from the western blot analysis also corroborated the apoptotic mode of cell death. The active complexes effectively generated reactive oxygen species (ROS) in MDA-MB-231 cells as analyzed from the 2',7'-dichlorofluorescein diacetate (DCFH-DA) staining. Finally, in vivo studies of the highly active complexes (6 and 12) were performed on the mice model. Histological analyses revealed that treatment with these complexes at high doses of up to 8 mg/kg did not induce any visible damage to the tested organs.
During osteoarthritis, the oxidation-reduction balance will be broken, resulting in the excessive formation of reactive oxygen species. These reactive oxygen species have been shown to disrupt proteins, lipids, and deoxyribonucleic acid. They induce oxidative strain, which can induce the development of osteoarthritis and lead to worse consequences. To solve this problem, antioxidant molecules have attracted wide attention for their ability to scavenge free radicals and reactive oxygen species. Nevertheless, most antioxidants encounter obstacles that affect their bioavailability, including a narrow absorption capacity, difficulty in penetrating cell membranes, and disintegration during delivery. Therefore, to solve these problems, the excellently targeted delivery, biocompatibility, deformability, and other characteristics of nanoparticles and hydrogels have been considered, and nanoparticles and hydrogels are at present being widely developed as antioxidant carriers. Nanoparticles and hydrogels that target the oxidative stress pathway can be mixed with antioxidants to increase their bioavailability, adhesion, and other related characteristics, which leads to better treatment of osteoarthritis. This research study aims to examine the biological scaffolds of osteoarthritis, which contain antioxidant nanoparticles and antioxidant hydrogels, as well as process controlling reactive oxygen compounds throughout the entire knee joint cavity. In addition, current challenges and potential solutions that can use antioxidant nanoparticles and antioxidant hydrogel biological scaffolds have been proposed to provide a theoretical basis for their future clinical applications.
Articular cartilage injury repair remains a challenge for clinicians and researchers. Mesenchymal stem cells (MSCs) have multiple differentiation potentials and can be induced to differentiate into the chondrogenic lineage for cartilage defect repair; however, the insufficient capacity of chondrogenic differentiation and excess reactive oxygen species (ROS)-mediated oxidative stress, which always lead to differentiation into hypertrophic chondrocytes, still need to be resolved. Accordingly, kartogenin (KGN), which can promote chondrogenic differentiation of MSCs, has shown promise in promoting infected cartilage repair. However, realizing controllable release to prolong its action time and avoid hypertrophic differentiation is critical. We herein developed a mesoporous Prussian blue nanoparticle (mPB)-based near-infrared (NIR) light-responsive controlled nanosystem. KGN was encapsulated in temperature-stimulated responsive phase change materials (PCMs), which were used as excellent gating materials (KGN-PCM@mPBs). In addition, the mPBs could efficiently scavenge ROS by their enzyme-like antioxidative activities. Our study demonstrates that the nanocomposites could efficiently promote chondrogenic differentiation and successfully inhibit the hypertrophic differentiation of MSCs. By intra-articular injection of KGN-PCM@mPBs and NIR-triggered precisely controlled release, satisfactory cartilage repair effects can be achieved in a rat chondral defect model. Thus, this constructed NIR-mediated KGN-PCM@mPB nanoplatform may represent an effective cartilage repair strategy with satisfactory biosafety in clinical applications.
Reactive oxygen species (ROS) play distinct but important roles in physiological and pathophysiological processes. Recent studies on osteoarthritis (OA) have suggested that ROS plays a crucial role in its development and progression, serving as key mediators in the degradation of the extracellular matrix, mitochondrial dysfunction, chondrocyte apoptosis, and OA progression. With the continuous development of nanomaterial technology, the ROS-scavenging ability and antioxidant effects of nanomaterials are being explored, with promising results already achieved in OA treatment. However, current research on nanomaterials as ROS scavengers for OA is relatively non-uniform and includes both inorganic and functionalized organic nanomaterials. Although the therapeutic efficacy of nanomaterials has been reported to be conclusive, there is still no uniformity in the timing and potential of their use in clinical practice. This paper reviews the nanomaterials currently used as ROS scavengers for OA treatment, along with their mechanisms of action, with the aim of providing a reference and direction for similar studies, and ultimately promoting the early clinical use of nanomaterials for OA treatment. STATEMENT OF SIGNIFICANCE: Reactive oxygen species (ROS) play an important role in the pathogenesis of osteoarthritis (OA). Nanomaterials serving as promising ROS scavengers have gained increasing attention in recent years. This review provides a comprehensive overview of ROS production and regulation, as well as their role in OA pathogenesis. Furthermore, this review highlights the applications of various types of nanomaterials as ROS scavengers in OA treatment and their mechanisms of action. Finally, the challenges and future prospects of nanomaterial-based ROS scavengers in OA therapy are discussed.
Aging is a collection of changes that contribute to decline in maximum function and ultimately death of an organism. This process is controlled and initiated by several mechanisms including telomere shortening, oxidative stress, AMP-activated protein kinase and sirt-1. Several therapies have been reported to relieve the process of aging. Among these, diet therapy seems to be the most appropriate approach. Fruits are an important part of regular diet. They contain several compounds which have potential to handle the problem of aging and its related disorders. The present paper provides a comprehensive review on different factors present in various fruits related to the process of aging together with their antiaging mechanisms.
The intergeneric conjugative transfer of antibiotic resistance genes (ARGs) is recognized as an important way to the dissemination of antibiotic resistance. However, it is unknown whether the extensive use of chloroxylenol (para-chloro-meta-xylenol, PCMX) in many pharmaceutical personal care products will lead to the spread of ARGs. In this study, the ability and mechanisms of PCMX to accelerate the intergeneric conjugative transfer were investigated. Results showed that exposure of bacteria to environmental concentrations of PCMX (0.20–1.00 mg/L) can significantly stimulate the increase of conjugative transfer by 8.45–9.51 fold. The phenotypic experiments and genome-wide RNA sequencing revealed that 0.02–5.00 mg/L PCMX exposure could increase the content of alkaline phosphatase and malondialdehyde, which are characteristic products of cell wall and membrane damage. In addition, PCMX could lead to excessive production of reactive oxygen species (ROS) by 1.26–2.00 times, the superoxide dismutase and catalase produced by bacteria in response to oxidative stress were not enough to neutralize the damage of ROS, thus promoting the conjugative transfer. Gene Ontology enrichment analyses indicated that cell membrane permeability, pili, some chemical compounds transport and energy metabolism affected conjugative transfer. This study deepened the understanding of PCMX in promoting propagation of ARGs, and provided a new perspective for use and treatment of personal care products.
Nitrate has been widely used in sewer systems for sulfide control. However, significant chemical consumption and the loss of carbon source were observed in previous studies. To find a feasible and cost-effective control strategy of the sulfide control, the effect of nitrate combined with sodium nitroprusside (SNP) dosage strategy was tested in lab-scale sewer biofilm reactors. Results showed that nitrate and SNP were strongly synergistic, with 30 mg N/L nitrate and 20 mg/L SNP being sufficient for sulfide control in this study. While large amount of nitrate alone (100 mg N/L) is required to achieve the same sulfide control effectiveness. Meanwhile, the nitrate combined with SNP could reduce the organic carbon source loss by 80%. Additionally, the high-throughput sequencing results showed that the relative abundance of autotrophic, nitrate reducing-sulfide oxidizing bacteria genera (a-NR-SOB) such as Arcobacter and Sulfurimonas was increased by around 18%, while the heterotrophic, nitrate-reducing bacteria (hNRB) such as Thauera was substantially reduced. It demonstrated that the sulfide control was mainly due to the a-NR-SOB activity under the nitrate and SNP dosing strategy. The microbial functional prediction further revealed that nitrate and SNP promoted the dissimilatory nitrate reduction process which utilizes sulfide as an effective electron donor. Moreover, economic assessment indicated that using the combination of nitrate and SNP for sulfide control in sewers would lower the chemical costs by approximately 35% compared with only nitrate addition.
Background: The aim of this study was to investigate the therapeutic effect and the underlying mechanism of resveratrol in high fat diet (HFD) and hyperlipidemia AP (HTG-AP) mice model.
Methods: Following successful establishment of the HFD and HTG-AP mice model, resveratrol was administrated. 16sRNA sequencing of gut microbiota in colonic fecal, the LPS, MCP-1, TNF-α, and IL-6 expressions in serum, and MCP-1 expression of the pancreatic tissues were measured in HFD model. The MDA, SOD, T-AOC, TNF-α, and MCP-1 expressions; the NF‑κB proinflammatory signaling pathway‑related proteins in pancreatic tissues were determined. Histopathological examination was evaluated in both models.
Results: Resveratrol effectively inhibited pancreatic pathological injury in both models. It reduced the MDA, SOD, T-AOC, TNF-α, and MCP-1 expressions and changed composition of gut microbiota in feces compared with the HFD model. Resveratrol also reduced oxidative stress by decreasing the level of MDA and increasing the levels of SOD and T-AOC. TNF-α and MCP-1 were decreased following the administration of resveratrol. Furthermore, resveratrol suppressed the NF‑κB proinflammatory signaling pathway in pancreatic tissues.
Conclusions: The study suggested that resveratrol had therapeutic effect on HFD and HTG-AP mice model by regulating the gut microbiota, promoting antioxidant capacity and inhibiting proinflammatory cytokines via the NF‑κB inflammatory pathway. The results can provide evidence that resveratrol might be regarded as a promising therapeutic agent for HTG-AP.
Chronic myeloid leukemia (CML) is one kind of hematological malignancy featured with retarded differentiation that is highly linked to the level of intracellular reactive oxygen species (ROS). In this work, ultra-small platinum nanoparticles deposited on gold nanorods (Au@Pt) were synthesized and applied on the CML cells. It was shown that Au@Pt had multienzyme-like activities that induced a fluctuation of the intracellular ROS level over the incubation time, depending on their temporal locations in the cells. The ROS fluctuation triggered the cellular autophagy and enhanced the level of autophagic protein Beclin-1, which caused the degradation of fusion protein BCR-ABL that is the key factor of the retarded differentiation and led to the downregulation of the phosphorylation of PI3K and AKT. These interactions together broke the retared differentaition and drove the CML cells to differentiate towards megakaryocytes, which is of great significance of enhancing the leukemic cells apoptosis. Therefore, Au@Pt exhibited a novel function and promising therapeutic potential for the CML treatment.
Realgar is a kind of mineral drug and has a long history of medical use in Chinese medicine. The active component of realgar is As4S4 and can be orally administrated. In past decades, several traditional Chinese prescriptions containing realgar has showed certain clinical effects in the treatment of some hematologic malignancies. However, realgar is poorly soluble in neutral or acidic aqueous solution due to its crystal structures, which not only leads to the problem of extremely low bioavailability in clinical practices but also sets up obstacles to biomedical studies to understand molecular mechanisms. Hence, there have been continuous efforts made to seek effective approaches for developing suitable processing techniques of realgar, aiming to increase the bioavailability and efficacy, and meanwhile, also to get insights into pharmaceutical mechanisms at molecular levels. This chapter briefly introduces the history of medical use of realgar in Chinese medicine prescriptions, followed by a comprehensively review of the efforts for developing realgar nanotherapeutics by using various technologies, especially a novel one-step preparation approach to fabricate nanoformulation was addressed. In the last part, therapeutic effects of realgar nanoformulations in cells and animals of acute and chronic myeloid leukemia and solid tumors are demonstrated, and molecular mechanisms are portrayed as well.
Even through many physiological pathways of resveratrol have been established since its association with the “French paradox” in 1992, its exact pathological roles are still ambiguous and disputable. To understand how resveratrol exerts its effects would not only enlighten the pathogenesis study of related diseases, but also promote the development of more potent molecules. Fo-cusing on the reported cellular directly and indirectly ROS scavenging processes of resveratrol, we evaluated the activation effect of the inherent antioxidation thiols system in sub-cellular level by two cysteine (Cys) specific fluorescent probes mitochondria targetable Mito-1 and lysosomes targetable Ly-1. We found that 50 μM resveratrol treatment could induce distinct Cys up-regulation in both mitochondria and lysosomes which might be a general biological phenomenon in various cell lines for the first time.
Osteoarthritis (OA) is a whole joint disease driven by abnormal biomechanics and attendant cell-derived and tissue-derived factors. The disease is multifactorial and polygenic, and its progression is significantly related to oxidative stress and reactive oxygen species (ROS). Augmented ROS generation can cause the damage of structural biomolecules of the joint and, by acting as intracellular signaling component, ROS are associated with various inflammatory responses. By activating several signaling pathways, ROS have a vital importance in the patho-physiology of OA. This review is focused on the mechanism of ROS which regulate intracellular signaling processes, chondrocyte senescence and apoptosis, extracellular matrix synthesis and degradation, along with synovial inflammation and dysfunction of the subcondral bone, targeting the complex oxidative stress signaling
Aims:
Oxidative stress is an important risk factor in development and progression of type 2 diabetes. Resveratrol (RSV), as a natural antioxidant, reduces intracellular reactive oxygen species (ROS) and oxidative stress.
Materials and methods:
The study investigated the effects of RSV treatment on high-fat diet (HFD)-fed mice and muscle, adipose, and hepatic cells of insulin resistance. HFD-fed mice were treated with RSV for 10 weeks. Blood glucose, plasma triglyceride (TG), body weight and glucose-lipid metabolism of skeletal muscle, fat and liver were examined. We further assessed the metabolic regulation of RSV in C2C12 myotubes, 3T3-L1 adipocytes and HepG2 cells of insulin resistance.
Key findings:
We found that RSV reduced blood glucose, plasma TG and body weight, ameliorated insulin resistance in HFD-fed mice. RSV reduced lipid accumulation and increased glycogen storage in muscle and hepatic cells, promoted lipolysis in adipocytes. We further found RSV reduced ROS levels in muscle, adipose, and hepatic cells of insulin resistance, contributing to improvement of metabolic abnormalities in HFD-fed mice.
Significance:
The study reveals that RSV ameliorates metabolic disorders and insulin resistance in HFD-fed mice, which provides further demonstration in RSV-treated type 2 diabetes.
Aims:
Reactive oxygen species (ROS) bidirectionally regulate insulin sensitivity in skeletal muscle. Insulin-induced ROS generation elevates insulin-regulated metabolic effects; however, chronic oxidative stress causes severe insulin resistance in skeletal muscle. Resveratrol (RV), as a natural antioxidant, eliminates intracellular ROS. It's unclear that whether it has different roles in insulin signaling pathway in skeletal muscle.
Main methods:
C57BL/6J mice and C2C12 myotubes were used to assess metabolic regulation effects of RV. Protein activation was detected using Immunofluorescence and Western Blot analysis. ROS were analyzed using confocal microscope and flow cytometry sorting (FACS). Intracellular reducing molecules were detected using an enzymatic method. Glucose uptake was measured using a fluorescent deoxyglucose analog (2-NBDG).
Key findings:
We found that RV attenuated insulin-stimulated AKT phosphorylation via elimination of insulin-induced ROS generation in skeletal muscle, suggesting that RV decreased activation of the insulin-induced AKT signaling. In skeletal muscle of insulin resistance, RV reduced oxidative stress, restored intracellular glutathione (GSH) level, and enhanced insulin-induced AKT activation and glucose absorption. These results suggested that RV ameliorated insulin resistance by change of redox levels in skeletal muscle.
Significance:
This study revealed bidirectional regulation effects of RV on insulin-stimulated metabolism in skeletal muscle through alternation of intracellular redox homeostasis, which might provide a guidance role for treatment of metabolic diseases.
Ischemic preconditioning and pharmacological preconditioning are common strategies to prevent lethal myocardial injury, especially nutritional preconditioning (NPC). In this study, we investigated the effects of astragaloside IV (Ast), as an NPC agent, on myocardium suffered anoxia/reoxygenation (A/R) injury. Rats received 5 mg/kg Ast daily for 3 weeks by intragastric administration. Then, hearts were harvested and underwent A/R treatment using a Langendorff apparatus. Ast- pretreatment significantly promoted functional recovery of the myocardium, reduced infarct size, and oxidative stress, and decreased the apoptotic index. Similar findings were demonstrated in H9c2 cardiomyocytes that were pretreated with Ast for 24 h. Moreover, Ast-pretreatment significantly upregulated Bcl-2 expression, especially in mitochondria. The effects of Ast treatment against A/R injury were also reflected by increased antioxidant potential, inhibited reactive oxygen species (ROS) burst, increased oxygen consumption rate, maintained mitochondrial membrane potential (MMP), inhibited mitochondrial permeability transition pore (mPTP) opening, and prevented apoptosis. Selective inhibition of Bcl-2 by ABT-737 decreased myocardial injury protection of Ast. Ast-pretreatment resulted in NPC- related effects against A/R, and mitochondria may be the target of a cascade of events elicited by upregulating Bcl-2 expression, promoting translocation of Bcl-2 into mitochondria, maintaining MMP, inhibiting ROS bursts, thereby leading to recovery of mitochondrial respiration, preventing mPTP opening, decreasing cytochrome C release, preventing apoptosis, and ultimately alleviating myocardial injury.
Rheumatoid arthritis (RA) is a systemic autoimmune disease primarily affecting joints and is featured by chronic synovial inflammation and angiogenesis. We employed a bovine type-II collagen (BIIC)-induced Sprague-Dawley rat arthritis model and an in vitro RA model based on interleukin (IL)-1β-stimulated rat synovial cells (RSC-364) to explore the preventive effect of resveratrol on RA and the underlying mechanisms. We found that resveratrol ameliorated BIIC-elicited synovitis and RA-related pathological hallmarks such as inflammatory cell infiltration and angiogenesis in the synovial tissue. Also, BIIC-stimulated rats displayed increased serum levels of proinflammatory cytokines and reactive oxygen species (ROS), as manifested by elevated serum malonaldehyde contents combined with reduced superoxide dismutase activity. It is noteworthy that resveratrol abolished BIIC-induced ROS and inflammation, confirming the antioxidative and anti-inflammatory actions of resveratrol in the context of RA. Furthermore, immunoblotting indicated that resveratrol downregulated the increase in the levels of hypoxia-inducible factor-1α (HIF-1α) and that of the activated phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase in IL-1β-stimulated RSC-364 cells. Moreover, we observed that resveratrol-treated RSC-364 cells displayed both G0/G1 cell-cycle arrest and enhanced levels of apoptosis. Altogether, the present evidence established the preventive role of resveratrol in RA progression. Mechanistically, resveratrol inhibits MAPK signaling pathways, likely by reducing ROS accumulation, to suppress the inflammatory response and cell proliferation and to provoke cell apoptosis in the synovial tissue, along with mitigation of HIF-1α-mediated angiogenesis. Thus resveratrol appears to hold great potential for clinical translation as a novel RA therapeutic.
Nitric oxide (NO) is an important mediator in physiologic and pathophysiologic pathways in the body. NO is believed to be mainly a catabolic player in joint physiology. In pathological situations such as osteoarthritis (OA), additional reactive molecules, so-called reactive oxygen species (ROS), are abnormally produced and contribute directly or indirectly to cartilage degradation, matrix metalloproteinase (MMP) activation and cell death induction. MMPs play a key role in the progression of various joint diseases and influence joint inflammation and matrix degradation. Most actions of MMPs in joints are catabolic in nature. Various studies have detected MMPs in cranial cruciate ligament (CrCL) tissue during aging, degeneration, or in torn CrCLs. CrCL has been shown to produce more NO compared to other ligaments. In vitro experiments have demonstrated that different NO donors induce apoptosis in CrCL cells.
The preventive and therapeutic effects of curcumin on degeneration of articular (joint) cartilage diseases have rarely been investigated. In the present study, the protective effects of curcumin against sodium nitroprusside (SNP)-induced chondrocyte apoptosis were evaluated and the underlying molecular mechanisms were elucidated. Curcumin was used to as a co-treatment with SNP in chondrocytes, and changes occurring in the cells were observed and evaluated. It was shown using a cell counting kit-8 (CCK-8) assay that curcumin protected the viability of chondrocytes against SNP damage. NO (nitric oxide) from SNP could be scavenged by curcumin. Flow cytometry and Hoechst 33342 staining showed that curcumin not only inhibited the cell apoptosis in a concentration-dependent pattern but also ameliorated the SNP-induced nuclear chromatin damage and reduction of the mitochondrial membrane potential in chondrocytes. In SNP-treated chondrocytes, curcumin downregulated the expression of Bax and cleaved caspase-3 but upregulated the expression of Bcl-2, as shown by western blot. Meanwhile, curcumin administration also protected extracellular matrix (ECM) synthesis and prevented its degradation. Taken together, these results support the hypothesis that curcumin exerts its protective effect on chondrocytes against SNP-induced apoptosis, at least partly, via blocking the mitochondrial-dependent apoptotic pathway and maintaining the metabolic balance of ECM. Thus, curcumin may be a potential candidate to be used as a unique biological agent for the prevent and treatment of osteoarthritis (OA).
The quinolone compounds have been reported for many biological properties, especially as potent antioxidants. This study investigated the antioxidant effect of 7-chloro-4-phenylselenyl-quinoline (PSQ), a quinolone derivative with organoselenium group, against oxidative stress induced by sodium nitroprusside (SNP) in brains of mice. A second objective was to verify the importance of phenylselenyl group presents at position 4 of the quinoline structure to antioxidant effect of compound. So, it was compared the antioxidant effect of PSQ with a quinoline without organoseleniun group (7-chloroquinoline [QN]). Swiss mice were used and received SNP (0.335 μmol/site, intracerebroventricular) 30 min after treatment with PSQ or QN, at the doses of 50 mg/kg (intragastrically). After 1 h, animals were sacrificed and the brains were removed to biochemistry analysis. Thiobarbituric acid reactive species (TBARS), protein carbonyl (PC) and non-protein thiol (NPSH) levels, as well as catalase (CAT), glutathione S transferase (GST) and δ -aminolevulinic acid (δ-ALA-D) activities were determined. SNP increased TBARS and PC levels, and reduced the enzymatic (CAT and GST activity) and non-enzymatic (NPSH levels) antioxidant defenses and inhibited the δ-ALA-D activity. PSQ avoided the increase in the lipid peroxidation and PC levels, as well as the decrease in the NPSH levels, CAT, GST and δ-ALA-D activities QN partially avoided the increase in lipid peroxidation, but it not protected against alterations induced by SNP. In conclusion, phenylselenyl group present in quinoline structure is critical for antioxidant activity of PSQ.
Osteoarthritis is characterized by a chronic, progressive and irreversible degradation of the articular cartilage associated with joint inflammation and a reparative bone response. More than 100 million people are affected by this condition worldwide with significant health and welfare costs. Our available treatment options in osteoarthritis are extremely limited. Chondral or osteochondral grafts have shown some promising results but joint replacement surgery is by far the most common therapeutic approach. The difficulty lies on the limited regeneration capacity of the articular cartilage, poor blood supply and the paucity of resident progenitor stem cells. In addition, our poor understanding of the molecular signalling pathways involved in the senescence and apoptosis of chondrocytes is a major factor restricting further progress in the area. This review focuses on molecules and approaches that can be implemented to delay or even rescue chondrocyte apoptosis. Ways of modulating the physiologic response to trauma preventing chondrocyte death are proposed. The use of several cytokines, growth factors and advances made in altering several of the degenerative genetic pathways involved in chondrocyte apoptosis and degradation are also presented. The suggested approaches can help clinicians to improve cartilage tissue regeneration.
Over the past years, several studies have found that foods rich in polyphenols protect against age-related disease, such as atherosclerosis, cardiovascular disease, cancer, arthritis, cataracts, osteoporosis, type 2 diabetes (T2D), hypertension and Alzheimer's disease. Resveratrol and pterostilbene, the polyphenol found in grape and blueberries, have beneficial effects as anti-aging compounds through modulating the hallmarks of aging, including oxidative damage, inflammation, telomere attrition and cell senescence. In this review, we discuss the relationship between resveratrol and pterostilbene and possible aging biomarker, including oxidative stress, inflammation, and high-calorie diets. Moreover, we also discuss the positive effect of resveratrol and pterostilbene on lifespan, aged-related disease, and health maintenance. Furthermore, we summarize a variety of important mechanisms modulated by resveratrol and pterostilbene possibly involved in attenuating age-associated disorders. Overall, we describe resveratrol and pterostilbene potential for prevention or treatment of several age-related diseases by modulating age-related mechanisms. © 2017 BioFactors, 2017.
The low bioavailability and short biological half-life of berberine chloride (BBR) negatively affect the protective role of this compound against osteoarthritis (OA). The present study was performed to evaluate the effectiveness of sustained BBR release system. Novel BBR-loaded chitosan microspheres (BBR-loaded CMs) were successfully synthesized using an ionic cross-linking method for sustained release. The basic characteristics of the prepared microspheres were subsequently evaluated by scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) techniques, encapsulation efficiency (EE), and in vitro release experiments. BBR-loaded CMs displayed spherical forms to encapsulate a considerable quantity of BBR (100.8 ± 2.7 mg/g); these microspheres also exhibited an ideal releasing profile. The FT-IR spectra and XRD results revealed that BBR-loaded CMs were successfully synthesized via electrostatic interaction. In vitro experiments further showed that BBR-loaded CMs significantly inhibited sodium nitroprusside (SNP)-stimulated chondrocyte apoptosis as well as cytoskeletal remodeling, and led to increasing mitochondrial membrane potential and maintaining the nuclear morphology. BBR-loaded CMs exerted markedly higher anti-apoptotic activity in the treatment of OA, and markedly inhibited the protein expression levels of caspase-3, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 and matrix metalloproteinase (MMP)-13 induced by SNP in rat articular chondrocytes, compared with free BBR at equivalent concentration. Therefore, novel BBR-loaded CMs may offer potential for application in the treatment of OA.
Berberine chloride (BBR) is an isoquinoline derivative alkaloid isolated from medicinal herbs, including Coptis chinensis and Berberis aristate. This compound plays significant roles in the treatment of osteoarthritis (OA). The purpose of this study was to investigate the effects of BBR on the proliferation of sodium nitroprusside (SNP)-stimulated chondrocytes in vitro, the articular cartilage in a rat OA model, as well as to discuss the molecular mechanisms underlying these effects. In vitro, we demonstrated that BBR led to cell proliferation, increased the cell population in S-phase and decreased that in G0/G1-phase; moreover, the F-actin remodeling in SNP-stimulated chondrocytes were prevented. In addition, BBR markedly up-regulated P-catenin, c-Myc, and cyclin Dl expression of genes and proteins, and down regulated glycogen synthase kinase-3 beta (GSK-3 beta) and matrix metalloproteinase-7 (MMP-7) expression. Notably, inhibition of the Wnt/beta-catenin pathway by XAV939 partially blocked these effects. The in vivo results suggested that BBR promoted P-catenin protein level and enhanced proliferating cell nuclear antigen (PCNA) expression in osteoarthritic rat cartilage. In conclusion, these findings indicate that BBR promotes SNP-stimulated chondrocyte proliferation by promoting G1/S phase transition and synthesis of PCNA in cartilage through activation of Wnt/beta-catenin signaling pathway.
OBJECTIVE To determine the effect of resveratrol administration in performance horses with lameness localized to the distal tarsal joints.
DESIGN Randomized, blinded, placebo-controlled clinical trial.
ANIMALS 45 client-owned horses with lameness localized to the distal tarsal joints.
PROCEDURES All horses received injections of triamcinolone acetonide in the centrodistal and tarsometatarsal joints of both hind limbs. A placebo or a supplement containing resveratrol was fed twice daily by owners for 4 months. Primary outcomes were horse performance as determined by rider opinion (better, worse, or the same) and change in lameness severity from the enrollment examination.
RESULTS Complete data were obtained for 21 horses that received resveratrol and 20 that received the placebo. Percentage of riders who reported that the horse's performance was better, compared with worse or the same, was significantly higher for the resveratrol group than for the placebo group after 2 (20/21 [95%] vs 14/20 [70%]) and 4 (18/21 [86%] vs 10/20 [50%]) months. The change in A1:A2 ratio between the enrollment and 4-month recheck examinations was significantly better for horses in the resveratrol versus placebo group. However, subjective lameness scores and degree of asymmetry of pelvis movement did not differ between groups.
CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that in performance horses with lameness localized to the distal tarsal joints, injection of triamcinolone in the centrodistal and tarsometatarsal joints of both hind limbs followed by oral supplementation with resveratrol for 4 months resulted in reduced lameness, compared with triamcinolone injection and supplementation with a placebo.
Berberine chloride (BBR) is an isoquinoline derivative alkaloid isolated from medicinal herbs, including Coptis chinensis and Berberis aristate. This compound plays significant roles in the treatment of osteoarthritis (OA). The purpose of this study was to investigate the effects of BBR on the proliferation of sodium nitroprusside (SNP)-stimulated chondrocytes in vitro, the articular cartilage in a rat OA model, as well as to discuss the molecular mechanisms underlying these effects. In vitro, we demonstrated that BBR led to cell proliferation, increased the cell population in S-phase and decreased that in G0/G1-phase; moreover, the F-actin remodeling in SNP-stimulated chondrocytes were prevented. In addition, BBR markedly up-regulated β-catenin, c-Myc, and cyclin D1 expression of genes and proteins, and down-regulated glycogen synthase kinase-3β (GSK-3β) and matrix metalloproteinase-7 (MMP-7) expression. Notably, inhibition of the Wnt/β-catenin pathway by XAV939 partially blocked these effects. The in vivo results suggested that BBR promoted β-catenin protein level and enhanced proliferating cell nuclear antigen (PCNA) expression in osteoarthritic rat cartilage. In conclusion, these findings indicate that BBR promotes SNP-stimulated chondrocyte proliferation by promoting G1/S phase transition and synthesis of PCNA in cartilage through activation of Wnt/β-catenin signaling pathway.
Resveratrol (trans-3,4'-trihydroxystillbene), a naturally occurring polyphenolic antioxidant found in grapes and red wine, elicits diverse biochemical responses and demonstrates anti-aging, anti-inflammatory, and anti-proliferative effects in several cell types. Previously, resveratrol was shown to regulate differentiation and inflammation in rabbit articular chondrocytes, while the direct production of nitric oxide (NO) in these cells by treatment with the NO donor sodium nitroprusside (SNP) led to apoptosis. In this study, the effect of resveratrol on NO-induced apoptosis in rabbit articular chondrocytes was investigated. Resveratrol dramatically reduced NO-induced apoptosis in chondrocytes, as determined by phase-contrast microscopy, the MTT assay, FACS analysis, and DAPI staining. Treatment with resveratrol inhibited the SNP-induced expression of p53 and p21 and reduced the expression of procaspase-3 in chondrocytes, as detected by western blot analysis. SNP-induced degradation of I-kappa B alpha (IκB-α) was rescued by resveratrol treatment, and the SN50 peptide-mediated inhibition of NF-kappa B (NF-κB) activity potently blocked SNP-induced caspase-3 activation and apoptosis. Our results suggest that resveratrol inhibits NO-induced apoptosis through the NF-κB pathway in articular chondrocytes.
Background
Activated microglial cells are an important pathological component in brains of patients with neurodegenerative diseases. The purpose of this study was to investigate the effect of He-Ne (632.8 nm, 64.6 mW/cm2) low-level laser therapy (LLLT), a non-damaging physical therapy, on activated microglia, and the subsequent signaling events of LLLT-induced neuroprotective effects and phagocytic responses.
Methods
To model microglial activation, we treated the microglial BV2 cells with lipopolysaccharide (LPS). For the LLLT-induced neuroprotective study, neuronal cells with activated microglial cells in a Transwell™ cell-culture system were used. For the phagocytosis study, fluorescence-labeled microspheres were added into the treated microglial cells to confirm the role of LLLT.
Results
Our results showed that LLLT (20 J/cm2) could attenuate toll-like receptor (TLR)-mediated proinflammatory responses in microglia, characterized by down-regulation of proinflammatory cytokine expression and nitric oxide (NO) production. LLLT-triggered TLR signaling inhibition was achieved by activating tyrosine kinases Src and Syk, which led to MyD88 tyrosine phosphorylation, thus impairing MyD88-dependent proinflammatory signaling cascade. In addition, we found that Src activation could enhance Rac1 activity and F-actin accumulation that typify microglial phagocytic activity. We also found that Src/PI3K/Akt inhibitors prevented LLLT-stimulated Akt (Ser473 and Thr308) phosphorylation and blocked Rac1 activity and actin-based microglial phagocytosis, indicating the activation of Src/PI3K/Akt/Rac1 signaling pathway.
Conclusions
The present study underlines the importance of Src in suppressing inflammation and enhancing microglial phagocytic function in activated microglia during LLLT stimulation. We have identified a new and important neuroprotective signaling pathway that consists of regulation of microglial phagocytosis and inflammation under LLLT treatment. Our research may provide a feasible therapeutic approach to control the progression of neurodegenerative diseases.
Molecular oxygen is required for the production of nitric oxide (NO), a pro-inflammatory mediator that is associated with osteoarthritis and rheumatoid arthritis. To date there has been little consideration of the role of oxygen tension in the regulation of nitric oxide production associated with arthritis. Oxygen tension may be particularly relevant to articular cartilage since it is avascular and therefore exists at a reduced oxygen tension. The superficial zone exists at approximately 6% O2, while the deep zone exists at less than 1% O2. Furthermore, oxygen tension can alter matrix synthesis, and the material properties of articular cartilage in vitro. The increase in nitric oxide associated with arthritis can be caused by pro-inflammatory cytokines and mechanical stress. Oxygen tension significantly alters endogenous NO production in articular cartilage, as well as the stimulation of NO in response to both mechanical loading and pro-inflammatory cytokines. Mechanical loading and pro-inflammatory cytokines also increase the production of prostaglandin E2 (PGE2). There is a complex interaction between NO and PGE2, and oxygen tension can alter this interaction. These findings suggest that the relatively low levels of oxygen within the joint may have significant influences on the metabolic activity, and inflammatory response of cartilage as compared to ambient levels. A better understanding of the role of oxygen in the production of inflammatory mediators in response to mechanical loading, or pro-inflammatory cytokines, may aid in the development of strategies for therapeutic intervention in arthritis.
Accumulation of advanced glycation end products (AGEs) in joints contributes to the pathogenesis of cartilage damage in osteoarthritis (OA). We aim to explore the potential chondroprotective effects of resveratrol on AGEs-stimulated porcine chondrocytes and cartilage explants.
Chondrocytes were isolated from pig joints. Activation of the IκB kinase (IKK)-IκBα-nuclear factor-kappaB (NF-κB) and c-Jun N-terminal kinase (JNK)/extracellular signal-regulated kinase (ERK)-activator protein-1 (AP-1) pathways was assessed by electrophoretic mobility shift assay (EMSA), Western blot and transfection assay. The levels of inducible nitric oxide synthase (iNOS)-NO and cyclooxygenase-2 (COX-2)-prostaglandin E2 (PGE2) were measured by Western blot, Griess reaction or ELISA. The expression and enzyme activity of matrix metalloproteinase-13 (MMP-13) were determined by real time RT/PCR and gelatin zymography, respectively.
We show that AGEs-induced expression of iNOS and COX-2 and production of NO and PGE2 were suppressed by resveratrol. Such effects of resveratrol were likely mediated through inhibiting IKK-IκBα-NF-κB and JNK/ERK-AP-1 signaling pathways induced by AGEs. By targeting these critical signaling pathways, resveratrol decreased AGEs-stimulated expression and activity of MMP-13 and prevented AGEs-mediated destruction of collagen II. Histochemistry analysis further confirms that resveratrol could prevent AGEs-induced degradation of proteoglycan and aggrecan in cartilage explants.
The present study reveals not only the effects and mechanisms regarding how resveratrol may protect cartilage from AGEs-mediated damage but also the potential therapeutic benefit of resveratrol in the treatment of OA.
Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, isolated from the traditional Chinese herb Artemisia annua, is recommended as the first-line anti-malarial drug with low toxicity. DHA has been shown to possess promising anticancer activities and induce cancer cell death through apoptotic pathways, although the molecular mechanisms are not well understood.
In this study, cell counting kit (CCK-8) assay was employed to evaluate the survival of DHA-treated ASTC-a-1 cells. The induction of apoptosis was detected by Hoechst 33258 and PI staining as well as flow cytometry analysis. Collapse of mitochondrial transmembrane potential (DeltaPsim) was measured by dynamic detection under a laser scanning confocal microscope and flow cytometry analysis using Rhodamine123. Caspase-3 activities measured with or without Z-VAD-fmk (a broad spectrum caspase inhibitor) pretreatment by FRET techniques, caspase-3 activity measurement, and western blotting analysis.
Our results indicated that DHA induced apoptotic cell death in a dose- and time-dependent manner, which was accompanied by mitochondrial morphology changes, the loss of DeltaPsim and the activation of caspase-3.
These results show for the first time that DHA can inhibit proliferation and induce apoptosis via caspase-3-dependent mitochondrial death pathway in ASTC-a-1 cells. Our work may provide evidence for further studies of DHA as a possible anticancer drug in the clinical treatment of lung adenocarcinoma.
Five hundred subjects with symptomatic limb joint osteoarthritis, who had been referred to a rheumatologist, were enrolled into a continuing study. They comprised 342 women (mean age 65.3) and 158 men (mean age 59.7), with a mean symptom duration of 15.4 years at entry. Only 31 patients (6%) had symptomatic osteoarthritis of one joint alone; however, in a further 205 (41%) the disease was limited to one site. One hundred and eighty two (36.4%) had two sites affected and 82 (16.4%) three or more sites of symptomatic osteoarthritis. Of 847 affected joints the most commonly involved were 349 (41.2%) knees, 254 (30%) hands, and 161 (19%) hips. Hip disease stood out as a separate entity, often occurring alone, and having a stronger male preponderance and different associations than osteoarthritis at other joint sites. Knee and hand disease were significantly associated in women. Obesity, hypertension, and Heberden's nodes were common. The number of sites affected, as well as the distribution, was strongly related to age as well as sex, suggesting that polyarticular osteoarthritis arises from slow acquisition of new joint sites in a non-random distribution. 'Generalised' osteoarthritis did not emerge as a distinct entity.
Chondrocytes stimulated with IL-1 produce high levels of nitric oxide (NO), which inhibits proliferation induced by transforming growth factor-beta or serum. This study analyzes the role of NO and IL-1 in the induction of chondrocyte cell death. NO generated from sodium nitroprusside induced apoptosis in cultured chondrocytes as demonstrated by electron microscopy, 4',6-dianidino-2-phenylindole dihydrochloride staining, FACS analysis, and histochemical detection of DNA fragmentation. Similar results were obtained with two other NO donors, 3-morpholinosynonimide-hydrochloride and s-nitroso-N-acetyl-D-L-penicillamine. In contrast, oxygen radicals generated by hypoxanthine/xanthine oxidase caused necrosis but did not induce chondrocyte apoptosis. To analyze whether endogenously generated NO induces apoptosis, chondrocytes were stimulated with IL-1, but there was no evidence for apoptotic changes. Combinations of NO inducers such as IL-1, lipopolysaccharide, tumor necrosis factor, and interferon-gamma also failed to trigger apoptosis. IL-1-stimulated chondrocytes are known to produce oxygen radicals that react with NO to form products that can induce cell death in other systems. We thus tested IL-1 in combination with the oxygen radical scavengers N-acetyl cysteine, dimethyl sulfoxide, or 5,5'-dimetylpyrroline 1-oxide. Under these conditions IL-1 was able to induce apoptosis, which was inhibited in a dose-dependent manner by the NO synthase inhibitor N-monomethyl L-arginine. Conversely, endogenous oxygen radicals induced by inflammatory mediators caused necrosis under conditions in which the simultaneous production of NO was reduced. These results suggest that NO, but not oxygen radicals, is the primary inducer of apoptosis in human articular chondrocytes.
Tyrosine nitration is a widely used marker of peroxynitrite (ONOO−) produced from the reaction of nitric oxide with superoxide. Pfeiffer and Mayer (Pfeiffer, S., and Mayer, B. (1998) J. Biol. Chem. 273, 27280–27285) reported that superoxide produced from hypoxanthine plus xanthine oxidase in combination with nitric oxide
produced from spermine NONOate did not nitrate tyrosine at neutral pH. They suggested that nitric oxide and superoxide at
neutral pH form a less reactive intermediate distinct from preformed alkaline peroxynitrite that does not nitrate tyrosine.
Using a stopped-flow spectrophotometer to rapidly mix potassium superoxide with nitric oxide at pH 7.4, we report that an
intermediate spectrally and kinetically identical to preformed alkalinecis-peroxynitrite was formed in 100% yield. Furthermore, this intermediate nitrated tyrosine in the same yield and at the same
rate as preformed peroxynitrite. Equivalent concentrations of nitric oxide under aerobic conditions in the absence of superoxide
did not produce detectable concentrations of nitrotyrosine. Carbon dioxide increased the efficiency of nitration by nitric
oxide plus superoxide to the same extent as peroxynitrite. In experiments using xanthine oxidase as a source of superoxide,
tyrosine nitration was substantially inhibited by urate formed from hypoxanthine oxidation, which was sufficient to account
for the lack of tyrosine nitration previously reported. We conclude that peroxynitrite formed from the reaction of nitric
oxide with superoxide at physiological pH remains an important species responsible for tyrosine nitration in vivo.
Nitric oxide (NO) causes apoptosis and dedifferentiation of articular chondrocytes by the modulation of extracellular signal-regulated kinase (ERK), p38 kinase, and protein kinase C (PKC) alpha and -zeta. In this study, we investigated the effects and mechanisms of non-steroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, ketoprofen, ibuprofen, sulindac sulfide, and flurbiprofen, in NO-induced apoptosis and dedifferentiation of articular chondrocytes. We found that all of the examined NSAIDs inhibited apoptosis and dedifferentiation. NO production in chondrocytes caused activation of ERK-1/2 and p38 kinase, which oppositely regulate apoptosis and dedifferentiation. NO production also caused inhibition of PKCalpha and -zeta independent of and dependent on, respectively, p38 kinase, which is required for apoptosis and dedifferentiation. Among the signaling molecules modulated by NO, NSAIDs blocked NO-induced activation of p38 kinase, potentiated ERK activation, and blocked inhibition of PKCalpha and -zeta. NSAIDs also inhibited some of the apoptotic signaling that is downstream of p38 kinase and PKC, such as NFkappaB activation, p53 accumulation, and caspase-3 activation. The inhibitory effects of NSAIDs on apoptosis and dedifferentiation were independent of the inhibition of cyclooxygenase (COX)-2 and prostaglandin E(2) (PGE(2)) production, as evidenced by the observation that specific inhibition of COX-2 activity and PGE(2) production or exogenous PGE(2) did not affect NO-induced apoptosis and dedifferentiation. Taken together, our results indicate that NSAIDs block NO-induced apoptosis and dedifferentiation of articular chondrocytes by the modulation of ERK, p38 kinase, and PKCalpha and -zeta in a manner independent of their ability to inhibit COX-2 and PGE(2) production.
This study investigated the molecular mechanisms underlying inhibition of protein kinase C (PKC) zeta by p38 kinase during nitric oxide (NO)-induced apoptosis of chondrocytes. Coimmunoprecipitation experiments showed that activation of p38 kinase following addition of an NO donor resulted in a physical association between PKCzeta and p38 kinase. Direct interaction of p38 kinase with PKCzeta was confirmed in vitro using p38 kinase and PKCzeta recombinant proteins. p38 kinase interacts with the regulatory domain of PKCzeta and its association blocked PKCzeta autophosphorylation. Micro LC-MS/MS analysis using recombinant proteins indicated that the interaction of p38 kinase with PKCzeta blocked autophosphorylation of PKCzeta on Thr-560, which is required for PKCzeta activation. Collectively, our results demonstrate a novel mechanism of PKCzeta regulation: following activation by the production of NO, p38 kinase binds directly to the PKCzeta regulatory domain, preventing PKCzeta autophosphorylation on Thr-560, thereby inhibiting PKCzeta activation.
To investigate the effect of nitric oxide (NO) on mitochondrial activity and its relation with the apoptosis of human articular chondrocytes.
Mitochondrial function was evaluated by analysing respiratory chain enzyme complexes, citrate synthase (CS) activities, and mitochondrial membrane potential (Delta psi m). The activities of the mitochondrial respiratory chain (MRC) complexes (complex I: NADH CoQ(1) reductase, complex II: succinate dehydrogenase, complex III: ubiquinol cytochrome c reductase, complex IV: cytochrome c oxidase) and CS were measured in human articular chondrocytes isolated from normal cartilage. The Delta psi m was measured by 5,5',6,6'-tetracholoro-1,1',3,3'-tetraethylbenzimidazole carbocyanide iodide (JC-1) using flow cytometry. Apoptosis was analysed by flow cytometry. The mRNA expression of caspases was analysed by ribonuclease protection analysis and the detection of protein synthesis by western blotting. Sodium nitroprusside (SNP) was used as an NO compound donor.
SNP at concentrations higher than 0.5 mmol/l for 24 hours induced cellular changes characteristic of apoptosis. SNP elicited mRNA expression of caspase-3 and caspase-7 and down regulated bcl-2 synthesis in a dose and time dependent manner. Furthermore, 0.5 mM SNP induced depolarisation of the mitochondrial membrane at 5, 12, and 24 hours. Analysis of the MRC showed that at 5 hours, 0.5 mM SNP reduced the activity of complex IV by 33%. The individual inhibition of mitochondrial complex IV with azide modified the Delta psi m and induced apoptosis.
This study suggests that the effect of NO on chondrocyte survival is mediated by its effect on complex IV of the MRC.
Growth factors are important in the development, maintenance and repair of cartilage. The principal aim of this study was to test the capacity of three growth factors with established roles in cartilage, namely insulin-like growth factor (IGF)-1, fibroblast growth factor (FGF) and transforming growth factor (TGF)-beta 1, to alter intracellular reactive oxygen species (ROS) levels. Explants of articular cartilage from young, mature, and aged rats were pretreated with IGF-1, FGF, or TGF-beta 1 and intracellular ROS levels were quantified using the free radical sensing probe dihydrorhodamine 123 (DHR 123), confocal microscopy, and densitometric image analysis. Viability of chondrocytes following ROS stress and growth factor treatment was assessed using the live/dead cytotoxicity assay, and the activities of the antioxidant enzymes--catalase (CAT), total superoxide dismutase (SOD), and glutathione peroxidase (GPX)--were measured spectrophotometrically by decay of the substrate from the reaction mixture. The effect of IGF-1 on ROS levels in cultured human chondrocytes also was examined. In rat cartilage, FGF did not significantly affect ROS levels or antioxidant enzyme activity in any age group. TGF-beta1 significantly increased cellular ROS levels in mature and old cartilage whereas in marked contrast, IGF-1 significantly and age-dependently reduced ROS levels. IGF-1 also had a potent antioxidant effect on cultured human chondrocytes. Pretreatment of rat cartilage with IGF-1 significantly enhanced the activity of GPX, without altering the activity of SOD or CAT, and protected chondrocytes against ROS-induced cell death. TGF-beta 1 had no significant effect on the activity of the antioxidant enzymes. Despite promoting ROS production, TGF-beta 1 was not cytotoxic. We concluded that TGF-beta 1 exhibits an acute pro-oxidant effect in cartilage that is not cytotoxic, suggesting a role in physiological cell signalling. In marked contrast, IGF-1 is a potent antioxidant in mature and aged rat and human chondrocytes, protecting cells against ROS-induced cell death probably through the enhancement of the activity of the antioxidant enzyme GPX.
Chondrogenesis and endochondral ossification are the cartilage differentiation processes that lead to skeletal formation and growth in the developing vertebrate as well as skeletal repair in the adult. The exquisite regulation of these processes, both in normal development and in pathologic situations, is impacted by a number of different types of stress. These include normal stressors such as mechanical loading and hypoxia as well pathologic stressors such as injury and/or inflammation and environmental toxins. This article provides an overview of the processes of chondrogenesis and endochondral ossification and their control at the molecular level. A summary of the influence of the most well-understood normal and pathologic stressors on the differentiation program is also presented.
Objective
To explore the signaling pathways by which the proinflammatory cytokine interleukin‐17 (IL‐17) may contribute to cartilage catabolism in osteoarthritis (OA) by inducing inducible nitric oxide synthase (iNOS) expression in chondrocytes.
Methods
We examined the IL‐17–induced NO production in human OA chondrocytes, in combination with the proinflammatory cytokines IL‐1β, tumor necrosis factor α (TNFα), and leukemia inhibitory factor (LIF); the antiinflammatory cytokines IL‐4, IL‐10, and IL‐13; and IL‐1 receptor antagonist (IL‐1Ra). Further, we explored the major intracellular signaling pathways through which IL‐17 induced iNOS expression and NO production.
Results
Treatment with IL‐17 induced a dose‐dependent increase in the level of NO. When IL‐17 was combined with the above factors, it resulted in a synergistic effect with TNFα, an additive effect with LIF, and no further effect than when used alone with IL‐1β. IL‐4, IL‐10, IL‐13, and IL‐1Ra had no true effect on IL‐17–induced NO production. The cAMP mimetics, 3‐isobutyl‐1‐methyl xanthine plus forskolin, completely blocked IL‐17–induced NO production. KT‐5720, genistein, and Calphostin C, inhibitors of protein kinase A (PKA), tyrosine kinase, and protein kinase C, respectively, reduced the IL‐17–induced NO production by 72%, 56%, and 42%, respectively. Within minutes, IL‐17 induced the phosphorylation of mitogen‐activated protein kinase kinase‐1/2 (MEK‐1/2), ‐3/6 (MKK‐3/6), p44/42, p38, and inhibitor of nuclear factor κB (IκB)‐α, as well as the activation of mitogen‐activated protein kinase–activated protein kinase‐1 and ‐2 (MAPKAPK‐1 and ‐2). Interestingly, IL‐17 induced phosphorylation of the stress‐activated protein kinase/Jun N‐terminal kinase (SAPK/JNK) (p54/46) only when PKA was inhibited. Specific protein kinase inhibitors for MEK‐1/2 (PD98059), p38 (SB202190), and nuclear factor κB (NF‐κB) (pyrrolidine dithiocarbamate) each markedly decreased the IL‐17–increased iNOS level and NO production. Inhibiting MAPK, including MEK‐1/2 and p38, had no effect on the IL‐17–induced activation of IκB‐α, but reversed the IL‐17 activation of MAPKAPK‐1 and ‐2, respectively.
Conclusion
These findings show that the stimulation of NO production by IL‐17 is mediated mainly by a complex activation of kinases, especially PKA, NF‐κB, and MAPK. NF‐κB appears to require MAPK activation, with downstream activation of MAPKAPK probably acting as a transactivating factor, to induce iNOS expression.
Objective
To explore the signaling pathways by which the proinflammatory cytokine interleukin-17 (IL-17) may contribute to cartilage catabolism in osteoarthritis (OA) by inducing inducible nitric oxide synthase (iNOS) expression in chondrocytes.Methods
We examined the IL-17–induced NO production in human OA chondrocytes, in combination with the proinflammatory cytokines IL-1β, tumor necrosis factor α (TNFα), and leukemia inhibitory factor (LIF); the antiinflammatory cytokines IL-4, IL-10, and IL-13; and IL-1 receptor antagonist (IL-1Ra). Further, we explored the major intracellular signaling pathways through which IL-17 induced iNOS expression and NO production.ResultsTreatment with IL-17 induced a dose-dependent increase in the level of NO. When IL-17 was combined with the above factors, it resulted in a synergistic effect with TNFα, an additive effect with LIF, and no further effect than when used alone with IL-1β. IL-4, IL-10, IL-13, and IL-1Ra had no true effect on IL-17–induced NO production. The cAMP mimetics, 3-isobutyl-1-methyl xanthine plus forskolin, completely blocked IL-17–induced NO production. KT-5720, genistein, and Calphostin C, inhibitors of protein kinase A (PKA), tyrosine kinase, and protein kinase C, respectively, reduced the IL-17–induced NO production by 72%, 56%, and 42%, respectively. Within minutes, IL-17 induced the phosphorylation of mitogen-activated protein kinase kinase-1/2 (MEK-1/2), -3/6 (MKK-3/6), p44/42, p38, and inhibitor of nuclear factor κB (IκB)-α, as well as the activation of mitogen-activated protein kinase–activated protein kinase-1 and -2 (MAPKAPK-1 and -2). Interestingly, IL-17 induced phosphorylation of the stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) (p54/46) only when PKA was inhibited. Specific protein kinase inhibitors for MEK-1/2 (PD98059), p38 (SB202190), and nuclear factor κB (NF-κB) (pyrrolidine dithiocarbamate) each markedly decreased the IL-17–increased iNOS level and NO production. Inhibiting MAPK, including MEK-1/2 and p38, had no effect on the IL-17–induced activation of IκB-α, but reversed the IL-17 activation of MAPKAPK-1 and -2, respectively.Conclusion
These findings show that the stimulation of NO production by IL-17 is mediated mainly by a complex activation of kinases, especially PKA, NF-κB, and MAPK. NF-κB appears to require MAPK activation, with downstream activation of MAPKAPK probably acting as a transactivating factor, to induce iNOS expression.
Objective
To determine which kind of cell death occurs in cartilage from patients with osteoarthritis (OA).Methods
Seven normal and 16 OA cartilage samples were collected at autopsy or during joint replacement surgery, respectively. A piece of cartilage was cryopreserved until histologic studies were done. The rest of the cartilage was used to isolate chondrocytes. Apoptotic chondrocytes were analyzed by light and fluorescence microscopy using nuclear 4′,6-diamidino-2-phenylindole dihydrochloride stain. Apoptotic chondrocytes were quantified by fluorescence-activated cell sorter (FACS) analysis. The TUNEL technique was used to study histologic apoptosis in situ. Superficial cartilage was processed for ultrastructural study by electron microscopy.ResultsOA chondrocytes displayed nuclear and cytoplasmic changes consistent with apoptotic cell death. FACS analysis showed that the OA cartilage had a higher proportion of apoptotic chondrocytes than did normal tissue (51% versus 11%; P < 0.01). In situ study of DNA fragmentation in the cartilage showed that apoptotic cells were located in the superficial and middle zones. Ultrastructural analysis of the superficial OA cartilage revealed some empty lacunae, lysosomal-like structures, matrix vesicle-like structures, fragmented chondrocytes, and nuclear condensation.Conclusion
Chondrocytes in OA cartilage demonstrated morphologic changes that are characteristic features of apoptosis. This mechanism of cell death plays an important role in the pathogenesis of OA and could be targeted for new treatment strategies.
Although caspases have been demonstrated to be involved in artemisinin (ARTE)-induced apoptosis, their exact functions are not well understood. The aim of this report is to explore the roles of caspase-8, -9 and -3 during ARTE-induced apoptosis in human lung adenocarcinoma (ASTC-a-1) cells. ARTE treatment induces a rapid generation of reactive oxygen species (ROS), and ROS-dependent apoptosis as well as the activation of caspase-8, -9 and -3 via time- and dose-dependent fashion. Of upmost importance, inhibition of caspase-8 or -9, but not caspase-3, almost completely blocks the ARTE-induced not only activation of the caspase-8, -9 and -3 but also apoptosis. In addition, the apoptotic process triggered by ARTE does not involve the Bid cleavage, tBid translocation, significant loss of mitochondrial membrane potential and cytochrome c release from mitochondria. Moreover, silencing Bax/Bak does not prevent the ATRE-induced cell death as well as the activation of caspase-8, -9 and -3. Collectively, our data firstly demonstrate that ARTE triggers a ROS-mediated positive feedback amplification activation loop between caspase-8 and -9 independent of mitochondria, which dominantly mediated the ARTE-induced apoptosis via a caspase-3-independent apoptotic pathway in ASTC-a-1 cells. Our findings imply a potential to develop new derivatives from artemisinin to effectively initiate the amplification activation loop of caspases.
The natural phytoestrogen resveratrol (RSV) may have therapeutic potential for arthritic conditions. RSV is chondroprotective for articular cartilage in rabbit models for arthritis, but its biological effects on human articular cartilage and chondrosarcoma cells are unknown. Effects of RSV on human articular cartilage homeostasis were studied by assessing production of matrix-degrading enzymes (MMP-13, ADAMTS4, and ADAMTS5), as well as proteoglycan production and synthesis. The counteractions of RSV against catabolic factors (e.g., FGF-2 or IL-1β) were examined by in vitro and ex vivo using monolayer, three-dimensional alginate beads and cartilage explants cultures, respectively. RSV improves cell viability of articular chondrocytes and effectively antagonizes cartilage-degrading protease production that was initiated by catabolic and/or anti-anabolic cytokines in human articular chondrocytes. RSV significantly also enhances BMP7-promoted proteoglycan synthesis as assessed by (35) S-sulfate incorporation. Protein-DNA interaction arrays suggest that RSV inhibits the activation of transcription factors involved in inflammation and cartilage catabolic signaling pathways, including direct downstream regulators of MAPK (e.g., AP-1, PEA3) and NFκB. RSV selectively compromises survival of human chondrosarcoma cells, but not primary articular chondrocytes, revealing cell-specific activity of RSV on non-tumorigenic versus tumor-derived cells. We propose that RSV exerts its chondroprotective functions, in part, by deactivating p53-induced apoptosis in human primary chondrocytes, but not human chondrosarcoma. Our findings suggest that RSV has potential as a unique biologic treatment for both prevention and treatment of cartilage degenerative diseases.
In chondrocytes, resveratrol, a natural SIRT1 activator, exerts an anti-inflammatory response via inhibition of nuclear factor kappaB (NF-κB). Given that SIRT1 inhibits the transactivation potential of NF-κB by deacetylating acetylated lysines in p65, the NF-κB subunit, we investigated the effects of resveratrol-activated SIRT1 on articular chondrocytes. We found that when chondrocytes were stimulated with interleukin 1β (IL-1β), the time- and dose-dependent expression of inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production was suppressed by resveratrol. Resveratrol-activated SIRT1 mediated this suppression. SIRT1 suppressed not only the nuclear translocation of NF-κB but also the acetylation of p65. Furthermore, acetylated Lys310 in p65, which must be present for transactivation activity, was the immediate downstream target of SIRT1. Therefore, SIRT1 protects against the inflammatory response induced by IL-1β in articular chondrocytes. Resveratrol, as an activator of SIRT1, merits consideration as a therapeutic agent in the treatment and prevention of osteoarthritis.
Interleukin-1 beta (IL-1) is implicated in cartilage destruction in arthritis through promotion of matrix metalloproteinase production. Upregulation of collagenase gene expression by IL-1 is known to require the transactivators Fos and Jun. Recently, reactive oxygen species (ROS) have been suggested to act as intracellular signaling molecules mediating the biological effects of cytokines. Here, we demonstrated ROS production by IL-1-stimulated bovine chondrocytes and that neutralizing ROS activity by the potent antioxidant, N-acetylcysteine, or inhibiting endogenous ROS production by diphenyleneiodonium (DPI), significantly attenuated IL-1-induced c-fos and collagenase gene expression. The inhibitory effect of DPI implicates enzymes such as NADPH oxidase in the endogenous production of ROS. Chondrocytes were also found to produce nitric oxide (NO) upon IL-1 stimulation. That NO may mediate part of the inducing effects of IL-1 was supported by the observation that L-NG-monomethylarginine, a NO synthase inhibitor, partially inhibited IL-1-regulated collagenase expression. Moreover, treatment of chondrocytes with the NO-producing agent, S-nitroso-N-acetylpenicillamine, was sufficient to induce collagenase mRNA levels. In summary, our results suggest that ROS released in response to IL-1 may function as second messengers transducing extracellular stimuli to their targets in the nucleus, leading to augmentation of gene expression.
We investigated whether N-acetylcysteine (NAC), a precursor of glutathione, could protect rabbit articular chondrocytes against nitric oxide (NO)-induced apoptosis and could prevent cartilage destruction in an experimental model of osteoarthritis (OA) in rats. Isolated chondrocytes were treated with various concentrations of NAC (0-2 mM). Apoptosis was induced by 0.75 mM sodium nitroprusside (SNP) dehydrate, which produces NO. Cell viability was assessed by MTT assay, while apoptosis was evaluated by Hoechst 33342 and TUNEL staining. Intracellular reactive oxygen species (ROS) and glutathione levels were measured, and expression of p53 and caspase-3 were determined by Western blotting. To determine whether intraarticular injection of NAC prevents cartilage destruction in vivo, cartilage samples of an OA model were subjected to H&E, Safranin O, and TUNEL staining. NAC prevented NO-induced apoptosis, ROS overproduction, p53 up-regulation, and caspase-3 activation. The protective effects of NAC were significantly blocked by buthionine sulfoximine, a glutathione synthetase inhibitor, indicating that the apoptosis-preventing activity of NAC was mediated by glutathione. Using a rat model of experimentally induced OA, we found that NAC also significantly prevented cartilage destruction and chondrocyte apoptosis in vivo. These results indicate that NAC inhibits NO-induced apoptosis of chondrocytes through glutathione in vitro, and inhibits chondrocyte apoptosis and articular cartilage degeneration in vivo.
SIRT1 is known to inhibit apoptosis and to promote survival of various types of cells. However, the roles of SIRT1 in apoptosis of human chondrocytes have never been reported. We undertook this study to investigate the relationship of SIRT1 to apoptosis of human chondrocytes, which is a characteristic feature of osteoarthritis (OA).
The expression of SIRT1 in human chondrocytes was examined by reverse transcription-polymerase chain reaction, immunoblotting, and immunohistology of human cartilage samples. The expression of SIRT1 under catabolic, mechanical, and nutritional stresses was investigated by immunoblotting. To examine the effect of SIRT1 on apoptosis, SIRT1 was inhibited by small interfering RNA (siRNA) and activated by resveratrol during nitric oxide (NO)-induced apoptosis. TUNEL staining and immunoblotting of cleaved poly(ADP-ribose) polymerase (PARP) were performed to detect apoptosis. To examine the mechanisms of apoptosis, we used immunoblotting to determine the levels of cleaved caspases and mitochondria-related apoptotic signaling proteins, Bax and Bcl-2, in the mitochondrial fraction.
SIRT1 expression was confirmed in human chondrocytes and human cartilage samples. All catabolic, mechanical, and nutritional stresses inhibited SIRT1 expression. SIRT1 inhibition by siRNA for SIRT1 increased the percentage of TUNEL-positive cells and increased the amounts of cleaved PARP and cleaved caspases 3 and 9 induced by NO. In contrast, treatment with resveratrol decreased the percentage of TUNEL-positive cells and decreased the amounts of cleaved PARP and cleaved caspases 3 and 9 induced by NO. Furthermore, in the mitochondrial fraction, SIRT1 inhibition by siRNA for SIRT1 increased the amount of Bax but reduced the amount of Bcl-2, while resveratrol reduced the amount of Bax but increased the amount of Bcl-2.
These results indicate that SIRT1 regulates apoptosis in human chondrocytes through the modulation of mitochondria-related apoptotic signals. Further research on SIRT1 might contribute to resolving the pathogenesis of OA.
Osteoarthritis is often a progressive and disabling disease, which occurs in the setting of a variety of risk factors--such as advancing age, obesity, and trauma--that conspire to incite a cascade of pathophysiologic events within joint tissues. An important emerging theme in osteoarthritis is a broadening of focus from a disease of cartilage to one of the 'whole joint'. The synovium, bone, and cartilage are each involved in pathologic processes that lead to progressive joint degeneration. Additional themes that have emerged over the past decade are novel mechanisms of cartilage degradation and repair, the relationship between biomechanics and biochemical pathways, the importance of inflammation, and the role played by genetics. In this review we summarize current scientific understanding of osteoarthritis and examine the pathobiologic mechanisms that contribute to progressive disease.
To determine the effects of the antioxidant resveratrol on the functions of human chondrocytes in osteoarthritis (OA).
Chondrocytes and cartilage explants were isolated from OA patients undergoing knee replacement surgery. Effects of resveratrol in the presence or absence of interleukin-1beta (IL-1beta) stimulation were assessed by measurement of prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4)) synthesis, cyclooxygenase (COX) activity, matrix metalloproteinase (MMP) expression, and proteoglycan production. To explore the mechanisms of action of resveratrol, its effects on mitochondrial function and apoptosis were examined by assessing mitochondrial membrane potential, ATP levels, cytochrome c release, and annexin V staining.
Resveratrol inhibited both spontaneous and IL-1beta-induced PGE(2) production by >20% (P < 0.05) and by 80% (P < 0.001), respectively; similarly, LTB(4) production was reduced by >50% (P < 0.05). The production of PGE(2) was inhibited via a 70-90% suppression of COX-2 expression and enzyme activity (P < 0.05). Resveratrol also promoted anabolic effects in OA explant cultures, by elevating proteoglycan synthesis and decreasing production of MMPs 1, 3, and 13. Pretreatment of OA chondrocytes with resveratrol blocked mitochondrial membrane depolarization, loss of mitochondrial biomass, and IL-1beta-induced ATP depletion. Similarly, IL-1beta-mediated induction of the apoptotic markers cytochrome c and annexin V was also inhibited by resveratrol. Exogenous addition of PGE(2) abolished the protective effects of resveratrol on mitochondrial membrane integrity, ATP levels, expression of apoptotic markers, and DNA fragmentation.
Resveratrol protects against IL-1beta-induced catabolic effects and prevents chondrocyte apoptosis via its inhibition of mitochondrial membrane depolarization and ATP depletion. These beneficial effects of resveratrol are due, in part, to its capacity to inhibit COX-2-derived PGE(2) synthesis. Resveratrol may therefore protect against oxidant injury and apoptosis, which are main features of progressive OA.
IL-1 inhibits chondrocyte proliferation induced by TGF beta or serum. This study analyzed the role of nitric oxide (NO), which is induced at high levels by IL-1 in chondrocytes. NO, when administered through sodium nitroprusside (SNP), inhibited TGF beta or serum-induced chondrocyte proliferation. To determine whether IL-1-induced NO is responsible for growth inhibition by IL-1, chondrocytes were cultured in the presence of the nitric oxide synthase inhibitor N-monomethyl-L-arginine (NMA), which dose-dependently reduced the antiproliferative effects of IL-1. Analysis of interactions between PGE2, a known chondrocyte growth inhibitor, and NO showed that PGE2 does not induce NO and is not required for NO induction by IL-1. However, SNP induced high levels of PGE2, and NMA reduced IL-1-induced PGE2. This raised the possibility that PGE2 is a downstream mediator of the antiproliferative effects of NO. This was confirmed in experiments where the growth inhibitory effects of SNP were reduced by indomethacin. These results suggest that the chondrocyte growth inhibition by IL-1 in chondrocytes is due to the induction of NO, which stimulates the production of PGE2 as a mediator of its antiproliferative effects.
Up to 2% of the oxygen consumed by the mitochondrial respiratory chain undergoes one electron reduction, typically by the semiquinone form of coenzyme Q, to generate the superoxide radical, and subsequently other reactive oxygen species such as hydrogen peroxide and the hydroxyl radical. Under conditions in which mitochondrial generation of reactive oxygen species is increased (such as in the presence of Ca2+ ions or when the mitochondrial antioxidant defense mechanisms are compromised), these reactive oxygen species may lead to irreversible damage of mitochondrial DNA, membrane lipids and proteins, resulting in mitochondrial dysfunction and ultimately cell death. The nature of this damage and the cellular conditions in which it occurs are discussed in this review article.
Activated articular chondrocytes produce large amounts of nitric oxide (NO), and there is increasing evidence that this is involved in the etiopathogenesis of osteoarthritis (OA). Because of its short half-life, the biological effects of endogenously produced NO are likely to occur locally within the cartilage. We have observed that inhibitors of NO synthases relieve the inhibition of matrix synthesis that otherwise occurs in response to IL-1. To avoid the use of inhibitors, we have recently transduced chondrocytes with the iNOS (NOS-2) gene and confirmed the ability of the endogenously produced NO to inhibit matrix synthesis. Despite the high levels of NO made by these cells, there was no evidence of apoptosis or other forms of cell death. NO was also shown to inhibit the production of TGF-beta(1)by cells treated with IL-1, as well as to decrease matrix production in response to IGF-1. The hypothesis that NO inhibits matrix production by interfering with important autocrine and paracrine factors should be entertained.
Chondrocyte cell death, possibly related to increased production of endogenous nitric oxide (NO), has been observed during the pathogenesis of osteoarthritis and rheumatoid arthritis. The purpose of this study was to investigate the potential role of NO in causing chondrocyte cell death and to determine the contribution of other reactive oxygen species (ROS).
Cell death and cytotoxicity were evaluated in human articular chondrocytes in response to various NO donor compounds with and without agents that stimulate or inhibit the production of additional ROS using both the alginate bead and the monolayer culture systems. Cell death was quantified by a total cell count with fluorescent labels, and cytotoxicity was measured as a function of cellular NADH- and NADPH-dependent dehydrogenase activity. To determine if the redox status of the chondrocyte could influence the observed effect of NO, cells were preincubated for 24 hours in L-cystine- and glutathione (GSH)-depleted media to reduce intracellular GSH levels, a major defense mechanism against oxidative stress. Apoptosis was analyzed with the quantification of histone-associated DNA fragments.
Treatment of chondrocytes with peroxynitrite (ONOO-), 3-morpholinosydnonimine (SIN-1), and sodium nitroprusside (SNP) resulted in apoptotic cell death at concentrations of 0.5 mM, 1.0 mM, and 0.5 mM, respectively. In contrast, treatment of chondrocytes with diazeniumdiolates (or the "NOC" compounds, NOC-5 and NOC-12) at concentrations as high as 2.0 mM did not cause cell death. Furthermore, NOC-5 and NOC-12, at all concentrations tested (0.125-2.0 mM), could prevent cell death caused by oxidative stress. Selective ROS scavengers protected against cell death caused by either SIN-1 or ONOO-; however, no protection could be afforded against the cytotoxicity of SNP with any of the ROS scavengers tested.
These results show that NO by itself is not cytotoxic to cultured chondrocytes and can even be protective under certain conditions of oxidative stress. Chondrocyte cell death from NO occurs under conditions where other ROS are also generated.
During apoptosis, mitochondrial membrane permeability (MMP) increases and the release into the cytosol of pro-apoptotic factors (procaspases, caspase activators and caspase-independent factors such as apoptosis-inducing factor (AIF)) leads to the apoptotic phenotype. Apart from this pivotal role of mitochondria during the execution phase of apoptosis (documented in other reviews of this issue), it appears that reactive oxygen species (ROS) produced by the mitochondria can be involved in cell death. These toxic compounds are normally detoxified by the cells, failing which oxidative stress occurs. However, ROS are not only dangerous molecules for the cell, but they also display a physiological role, as mediators in signal transduction pathways. ROS participate in early and late steps of the regulation of apoptosis, according to different possible molecular mechanisms. In agreement with this role of ROS in apoptosis signaling, inhibition of apoptosis by anti-apoptotic Bcl-2 and Bcl-x(L) is associated with a protection against ROS and/or a shift of the cellular redox potential to a more reduced state. Furthermore, the fact that active forms of cell death in yeast and plants also involve ROS suggests the existence of an ancestral redox-sensitive death signaling pathway that has been independent of caspases and Bcl-2.
To determine whether oxidative damage to cartilage proteins can be detected in aging and osteoarthritic (OA) cartilage, and to correlate the results with the local production of interleukin-1beta (IL-1beta) and the responsiveness of isolated chondrocytes to stimulation with insulin-like growth factor 1 (IGF-1).
The presence of nitrotyrosine was used as a measure of oxidative damage. Histologic sections of knee articular cartilage, obtained from young adult and old adult cynomolgus monkeys, which develop age-related, naturally occurring OA, were evaluated. Each cartilage section was graded histologically on a scale of 0-7 for the presence of OA-like changes, and serial sections were immunostained using antibodies to nitrotyrosine and IL-1beta. Chondrocytes isolated and cultured from cartilage adjacent to the sections used for immunostaining were tested for their response to IGF-1 stimulation by measuring sulfate incorporation in alginate cultures. For comparison with the monkey tissues, cartilage sections from human tissue donors and from tissue removed at the time of OA-related joint replacement surgery were also immunostained for nitrotyrosine and IL-1beta.
The presence of nitrotyrosine was associated with aging and with the development of OA in cartilage samples from both monkeys and humans. All sections that were highly positive for IL-1beta also showed staining for nitrotyrosine. However, in a few sections from older adult monkeys and humans, nitrotyrosine was present but IL-1beta was absent, suggesting that some age-related oxidative damage is independent of IL-1beta. In chondrocytes that were isolated from monkey cartilage positive for nitrotyrosine or IL-1beta, the response to stimulation with IGF-1 was significantly reduced. In some samples from older adult monkeys, IGF-1 resistance was seen in cells isolated from tissue that did not stain for nitrotyrosine or IL-1beta.
Oxidative damage due to the concomitant overproduction of nitric oxide and other reactive oxygen species is present in both aging and OA cartilage. This damage can contribute to the resistance of chondrocytes to IGF-1 stimulation, but it is unlikely to be the sole cause of IGF-1 resistance in these chondrocytes.
Our previous studies showed that reactive oxygen species (ROS) are required for the pro-inflammatory cytokine interleukin-1 beta (IL-1) to induce the activity of the Nuclear transcription Factor-kappa B (NF-kappa B) and the expression of the inducible isoform of the nitric oxide synthase (iNOS) in bovine articular chondrocytes. This study aimed at elucidating the role of hydrogen peroxide (H(2)O(2)) and the superoxide radical, two major ROS, in mediating those IL-1-induced responses. The results obtained show that chondrocytes produce both H(2)O(2) and superoxide radical in response to IL-1. Treatment of the chondrocyte cultures with H(2)O(2) alone did not induce NF-kappa B activation or iNOS expression. Addition of H(2)O(2) simultaneously with IL-1 did neither enhance nor inhibit NF-kappa B activation and iNOS expression, relatively to treatment with IL-1 alone. Accordingly, treatment with catalase did not inhibit those IL-1-induced responses. Treatment with superoxide dismutase, however, effectively prevented IL-1-induced I kappa B-alpha degradation and iNOS expression. Taken together, the results obtained indicate that superoxide mediates IL-1-induced I kappa B-alpha degradation and the consequent NF-kappa B activation and iNOS expression in chondrocytes, whereas H(2)O(2) does not seem to participate in those IL-1-induced responses. In conclusion, the present study identifies the superoxide radical as the ROS involved in mediating the IL-1-induced signaling pathway that leads to NF-kappa B activation and to the expression of NF-kappa B-dependent genes in bovine articular chondrocytes.
The metabolism of cells in articular joint tissues in normal and pathological conditions is subject to a complex environmental control. In addition to soluble mediators such as cytokines and growth factors, as well as mechanical stimuli, reactive oxygen species (ROS) emerge as major factors in this regulation. ROS production has been found to increase in joint diseases, such as osteoarthritis and rheumatoid arthritis, but their role in joint diseases initiation and progression remains questionable.
This review is focused on the role of ROS, mainly nitric oxide, peroxynitrite and superoxide anion radicals, in the signaling mechanisms implied in the main cellular functions, including synthesis and degradation of matrix components. The direct effects of ROS on cartilage matrix components as well as their inflammatory and immunomodulatory effects are also considered.
Some intracellular signaling pathways are redox sensitive and ROS are involved in the regulation of the production of some biochemical factors involved in cartilage degradation and joint inflammation. Further, ROS may cause damage to all matrix components, either by a direct attack or indirectly by reducing matrix components synthesis, by inducing apoptosis or by activating latent metalloproteinases. Finally, we have highlighted the uncoupling effect of ROS on tissue remodeling and synovium inflammation, suggesting that antioxidant therapy could be helpful to treat structural changes but not to relieve symptoms.
This review of the literature supports the concept that ROS are not only deleterious agents involved in cartilage degradation, but that they also act as integral factors of intracellular signaling mechanisms. Further investigation is required to support the concept of antioxidant therapy in the management of joint diseases.
To examine the role of oxidative stress in mediating cell death in chondrocytes isolated from the articular cartilage of young and old adult human tissue donors.
Cell death induced by the oxidant SIN-1 was evaluated in the alginate bead culture system using fluorescent probes to assess membrane integrity. Generation of peroxynitrite by the decomposition of SIN-1 was confirmed by positive immunostaining of treated cells for 3-nitrotyrosine. Determinations of oxidized glutathione (GSSG) and reduced glutathione (GSH) were performed in monolayer cultures using an enzyme- recycling assay. Cells were depleted of intracellular glutathione either by the addition of DL-buthionine-(S,R)-sulfoximine or by removal of L-cystine from the culture media. The activity of cellular antioxidant enzymes was determined spectrophotometrically by the decay of substrate from the reaction mixture.
More chondrocytes (>2-fold) from old donors (>/=50 years) died after exposure to 1 mM SIN-1 relative to those derived from young donors (18-49 years). Although autocrine production of insulin-like growth factor 1 (IGF-1) promotes chondrocyte survival, pretreatment with IGF-1 could not prevent the cell death induced by SIN-1 exposure. Cells isolated from old donors had a higher ratio of GSSG to GSH. Glutathione reductase is the principal enzyme involved in the regeneration of GSH from GSSG. Treatment of chondrocytes with SIN-1 to induce oxidative stress in vitro resulted in the decreased activity of glutathione reductase and thioredoxin reductase, but not catalase. Cells depleted of intracellular glutathione were more susceptible to cell death induced by SIN-1.
These results provide evidence that increased oxidative stress with aging makes chondrocytes more susceptible to oxidant-mediated cell death through the dysregulation of the glutathione antioxidant system. This may represent an important contributing factor to the development of osteoarthritis in older adults.
Reactive oxygen species (ROS) are potent inducers of oxidative damage and have been implicated in the regulation of specific cellular functions, including apoptosis. Mitochondrial ROS increase markedly after proapoptotic signals, though the biological significance and the underlying molecular mechanisms remain undetermined. P66Shc is a genetic determinant of life span in mammals, which regulates ROS metabolism and apoptosis. We report here that p66Shc is a redox enzyme that generates mitochondrial ROS (hydrogen peroxide) as signaling molecules for apoptosis. For this function, p66Shc utilizes reducing equivalents of the mitochondrial electron transfer chain through the oxidation of cytochrome c. Redox-defective mutants of p66Shc are unable to induce mitochondrial ROS generation and swelling in vitro or to mediate mitochondrial apoptosis in vivo. These data demonstrate the existence of alternative redox reactions of the mitochondrial electron transfer chain, which evolved to generate proapoptotic ROS in response to specific stress signals.
Aged mammalian tissues show a decreased capacity to produce ATP by oxidative phosphorylation due to dysfunctional mitochondria. The mitochondrial content of rat brain and liver is not reduced in aging and the impairment of mitochondrial function is due to decreased rates of electron transfer by the selectively diminished activities of complexes I and IV. Inner membrane H(+) impermeability and F(1)-ATP synthase activity are only slightly affected by aging. Dysfunctional mitochondria in aged rodents are characterized, besides decreased electron transfer and O(2) uptake, by an increased content of oxidation products of phospholipids, proteins and DNA, a decreased membrane potential, and increased size and fragility. Free radical-mediated oxidations are determining factors of mitochondrial dysfunction and turnover, cell apoptosis, tissue function, and lifespan. Inner membrane enzyme activities, such as those of complexes I and IV and mitochondrial nitric oxide synthase, decrease upon aging and afford aging markers. The activities of these three enzymes in mice brain are linearly correlated with neurological performance, as determined by the tightrope and the T-maze tests. The same enzymatic activities correlated positively with mice survival and negatively with the mitochondrial content of lipid and protein oxidation products. Conditions that increase survival, as vitamin E dietary supplementation, caloric restriction, high spontaneous neurological activity, and moderate physical exercise, ameliorate mitochondrial dysfunction in aged brain and liver. The pleiotropic signaling of mitochondrial H(2)O(2) and nitric oxide diffusion to the cytosol seems modified in aged animals and to contribute to the decreased mitochondrial biogenesis in old animals.
During inflammation, overproduction of nitric oxide (NO) can damage chondrocytes. In this study, we separately evaluated the toxic effects of exogenous and endogenous NO on human chondrocytes and their possible mechanisms. Human chondrocytes were exposed to sodium nitroprusside (SNP), an NO donor, or a combination of lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) as the exogenous and endogenous sources of NO, respectively. Administration of SNP or a combination of LPS and IFN-gamma in human chondrocytes increased cellular NO levels but decreased cell viability. Exposure to exogenous or endogenous NO significantly induced apoptosis of human chondrocytes. When treated with exogenous or endogenous NO, the mitochondrial membrane potential time-dependently decreased. Exposure to exogenous or endogenous NO significantly enhanced cellular reactive oxygen species (ROS) and cytochrome c (Cyt c) levels. Administration of exogenous or endogenous NO increased caspase-3 activity and consequently induced DNA fragmentation. Suppression of caspase-3 activation by Z-DEVD-FMK decreased NO-induced DNA fragmentation and cell apoptosis. Similar to SNP, exposure of human chondrocytes to S-nitrosoglutathione (GSNO), another NO donor, caused significant increases in Cyt c levels, caspase-3 activity, and DNA fragmentation, and induced cell apoptosis. Pretreatment with N-monomethyl arginine (NMMA), an inhibitor of NO synthase, significantly decreased cellular NO levels, and lowered endogenous NO-induced alterations in cellular Cyt c amounts, caspase-3 activity, DNA fragmentation, and cell apoptosis. Results of this study show that NO from exogenous and endogenous sources can induce apoptotic insults to human chondrocytes via a mitochondria-dependent mechanism.
Nitric oxide (NO) can regulate chondrocyte activities. This study was aimed to evaluate the molecular mechanisms of NO donor sodium nitroprusside (SNP)-induced insults to human chondrocytes. Exposure of human chondrocytes to SNP increased cellular NO levels but decreased cell viability in concentration- and time-dependent manners. SNP time dependently induced DNA fragmentation and cell apoptosis. Treatment with 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide, an NO scavenger, significantly lowered SNP-induced cell injuries. Administration of SNP interrupted F-actin and microtubule cytoskeletons and stimulated phosphorylation of mitogen-activated protein kinase kinase kinase-1 (MEKK1) and c-Jun N-terminal kinase (JNK). Similar to SNP, cytochalasin D, an inhibitor of F-actin formation, disturbed F-actin polymerization and increased MEKK1 and JNK activations. Overexpression of a dominant negative mutant of MEKK1 (dnMEK1) in human chondrocytes significantly ameliorated SNP-induced cell apoptosis. Exposure to SNP promoted Bax translocation from the cytoplasm to mitochondria, but application of dnMEKK1 lowered the translocation. SNP time dependently decreased the mitochondrial membrane potential, complex I NADH dehydrogenase activity, and cellular ATP levels, but increased the release of cytochrome c from mitochondria to the cytoplasm. Activities of caspase-9, -3, and -6 were sequentially increased by SNP administration. This study shows that SNP can induce apoptosis of human chondrocytes through sequential events, including cytoskeletal remodeling, activation of MEKK1/JNK, Bax translocation, mitochondrial dysfunction, cytochrome c release, caspase activation, and DNA fragmentation.
To examine whether cilostazol, a selective phosphodiesterase type III inhibitor, protects rat articular chondrocytes against nitric oxide (NO)-induced apoptosis and prevents cartilage destruction in mono-iodoacetate-induced osteoarthritis (OA) in a rat model in which inducible nitric oxide synthase (iNOS) is expressed.
The NO donor sodium nitroprusside was administered to rat articular chondrocytes that had been pretreated with cilostazol. Induction of apoptosis was evaluated by DNA electrophoresis and pulsed-field gel electrophoresis. The expression level and the subcellular location of apoptosis-associated factors were examined by Western blot analysis and confocal microscopy, respectively. Protein kinase CK2 (PKCK2) activity was also assayed. To examine whether orally administered cilostazol prevents cartilage destruction in vivo, cartilage samples obtained from rats with experimentally induced OA were subjected to hematoxylin and eosin, Safranin O, and TUNEL staining and immunohistochemical analysis of iNOS expression.
Cilostazol prevented NO-induced reduction in viability, in a dose-dependent manner. It also prevented the up-regulation of phosphorylated p53 and p38, the down-regulation of heme oxygenase 1, the subcellular translocation of apoptosis-inducing factor and cytochrome c, and the activation of caspases 3, 7, and 8 induced by NO treatment, indicating that cilostazol prevented NO-induced cell death by blocking apoptosis. In addition, cilostazol prevented NO-induced translocation of cleaved Bid onto mitochondria, and caused phosphorylated Bid to accumulate in the nucleus and cytosol. Cilostazol prevented the down-regulation of PKCK2 and the reduction in PKCK2 activity induced by NO, indicating that its apoptosis-preventing activity was mediated via PKCK2. It also prevented chondrocyte apoptosis and cartilage destruction in a rat model of experimentally induced OA.
Our findings indicate that cilostazol prevents NO-induced apoptosis of chondrocytes via PKCK2 in vitro and prevents cartilage destruction in a rat model of OA.
Osteoarthritis is an inflammatory disease of load-bearing synovial joints that is currently treated with drugs that exhibit numerous side effects and are only temporarily effective on pain, the main symptom of the disease. Consequently, there is an acute need for novel, safe and more effective chemotherapeutic agents for the treatment of osteoarthritis and related arthritic diseases. Resveratrol is a phytoalexin stilbene produced naturally by plants including red grapes, peanuts and various berries. Recent research in various cell models has demonstrated that resveratrol is safe and has potent anti-inflammatory properties. However, its potential for treating arthritic conditions has not been explored. In this study we provide experimental evidence that resveratrol inhibits the expression of VEGF, MMP-3, MMP-9 and COX-2 in human articular chondrocytes stimulated with the pro-inflammatory cytokine IL-1beta. Since these gene products are regulated by the transcription factor NF-kappaB, we investigated the effects of resveratrol on IL-1beta-induced NF-kappaB signaling pathway. Resveratrol, like N-Ac-Leu-Leu-norleucinal (ALLN) suppressed IL-1beta-induced proteasome function and the degradation of IkappaBalpha (an inhibitor of NF-kappaB) without affecting IkappaBalpha kinase activation, IkappaBalpha-phosphorylation or IkappaBalpha-ubiquitination which suppressed nuclear translocation of the p65 subunit of NF-kappaB and its phosphorylation. Furthermore, we observed that resveratrol as well as ALLN inhibited IL-1beta-induced apoptosis, caspase-3 activation and PARP cleavage in human articular chondrocytes. In summary, our results suggest that resveratrol suppresses apoptosis and inflammatory signaling through its actions on the NF-kappaB pathway in human chondrocytes. We propose that resveratrol should be explored further for the prophylactic treatment of osteoarthritis in humans and companion animals.