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Nerolidol Protects Against LPS-induced Acute Kidney Injury via Inhibiting TLR4/NF-κB Signaling: NEROLIDOL PREVENTS LPS-INDUCED AKI
Abstract
Acute kidney injury (AKI) is a critical care syndrome, resulting in acute reduction of renal function and up to 22% mortality of hospitalized patients. Nerolidol is a major component in several essential oils that possesses various pharmacological properties. The present study aimed to investigate the potential effect of nerolidol on lipopolysaccharide (LPS)-induced AKI. Nerolidol dose-dependently reduced the pathological injuries of kidney induced by LPS in rats. Nerolidol significantly decreased the levels of blood urea nitrogen and creatinine in LPS-treated rats in a dose-dependent manner. In addition, nerolidol inhibited LPS-induced decrease of cell viability in NRK-52E rat proximal tubular cells, which effect was concentration dependent. Nerolidol notably inhibited the increase of TNFα and IL-1β in LPS-treated rats and the mRNA expression of TNFα and IL-1β in LPS-treated NRK-52E cells. Nerolidol suppressed the increase of toll-like receptor 4 (TLR4) expression, phosphorylation and nuclear translocation of p65 NF-κB in kidneys of LPS-treated rats and LPS-treated NRK-52E cells. Overexpression of TLR4 and p65 NF-κB significantly suppressed nerolidol-induced inhibition of TNFα and IL-1β expression and increase of cell viability in LPS-treated cells. In summary, we found that nerolidol played a critical anti-inflammatory effects through inhibition of TLR4/NF-κB signaling and protected against LPS-induced AKI. Copyright © 2017 John Wiley & Sons, Ltd.
... Owing to its pharmacological and biological activities, nerolidol has shown promise in treating various conditions affecting the kidney. For instance, nerolidol demonstrated therapeutic potential against lipopolysaccharide-induced acute kidney injury in rats [15]. The administration of nerolidol led to a reduction in kidney tissue injury markers and mitigation of the alterations in the pro-inflammatory cytokines, and ultimately resulted in an improvement in renal functions. ...
... The exact mechanism for this attenuation is difficult to ascertain from the current study, but could possibly be attributed to the inhibition of TLR4 stimulation by nerolidol, which has been previously demonstrated in other organs [29]. The ability of nerolidol to target the TLR4/NF-κB signaling cascade might interfere with the translocation of phosphorylated nuclear factor-κB (p-NF-κB), an important agent which triggers the synthesis of pro-inflammatory cytokines including TNF-α [15,30]. Regardless of the exact mechanism of the effect of nerolidol on TNF-α changes, the findings of this study are consistent with the findings reported in other models of acute kidney injury, such as lipopolysaccharide-and ischemia-reperfusion-induced acute kidney injury [15,17]. ...
... The ability of nerolidol to target the TLR4/NF-κB signaling cascade might interfere with the translocation of phosphorylated nuclear factor-κB (p-NF-κB), an important agent which triggers the synthesis of pro-inflammatory cytokines including TNF-α [15,30]. Regardless of the exact mechanism of the effect of nerolidol on TNF-α changes, the findings of this study are consistent with the findings reported in other models of acute kidney injury, such as lipopolysaccharide-and ischemia-reperfusion-induced acute kidney injury [15,17]. ...
Background/Objectives: Obstructive uropathy is a common cause of renal impairment. Recently, there has been a burgeoning interest in exploring natural products as potential alternative remedies for many conditions due to their low toxicity, affordability and wide availability. Methods: We investigated the effect of nerolidol in a rat model of bilateral ureteral obstruction (BUO) injury. Nerolidol, dissolved in a vehicle, was administered orally as a single daily dose of 200 mg/kg to Wistar rats. Sham group (n = 12) underwent sham surgery, whereas the BUO (n = 12) and BUO/NR groups (n = 12) underwent reversible 24-h BUO and received the vehicle or nerolidol, respectively. The treatment started 9 days prior to the BUO/sham surgery and continued for 3 days after reversal. Renal functions were assessed before starting the treatment, just prior to the intervention and 3 days after BUO reversal. Results: Neither nerolidol nor the vehicle affected the basal renal functions. Nerolidol resulted in a significant attenuation in the BUO-induced alterations in renal functional parameters such as serum creatinine and urea, creatinine clearance and urinary albumin-creatinine ratio. Nerolidol also attenuated the changes in several markers associated with renal injury, inflammation, apoptosis and oxidative stress and mitigated the histological alterations. Conclusions: The findings of this study demonstrated the potent reno-protective effects of nerolidol in mitigating the adverse renal effects of bilateral ureteral obstruction. This is attributed to its anti-inflammatory, anti-fibrotic, anti-apoptotic and anti-oxidant properties. These effects were reflected in the partial recovery of renal functions and histological features. These findings may have potential therapeutic implications.
... 9,10 In addition, inhibition of TLR4 signalling protects against SA-AKI development in mice. 11,12 Thus, agents that are capable of suppressing TLR4 signalling might be effective on treating SA-AKI. Tacrolimus (TAC) is an effective immunosuppressant. ...
... The study was approved by the Committee on Ethical Use of Animals of Xuzhou Medical College. At 12,24 and 48 h after LPS treatment, mice were sacrificed under anaesthesia with 1% pentobarbital. We collected the kidney tissues, which were processed for standard histological examination, including fixation in 10% formalin, dehydration in graded alcohol solution, embedding in paraffin, etc. ...
... The renal cortex tissue was also used for immunoblotting assay after being lysed with RIPA buffer as described below. At 12,24 and 48 h after LPS treatment, blood samples were collected for biochemical analysis. All samples were kept at -80°C, blood urea nitrogen (BUN) and creatinine levels were measured using Nanjing Jiancheng Urea Assay kit and Creatinine (Cr) Assay kit (sarcosine oxidase). ...
Lipopolysaccharide (LPS)‐induced sepsis‐associated acute kidney injury (SA‐AKI) is a model of clinical serious care syndrome, with high morbidity and mortality. Tacrolimus (TAC), a novel immunosuppressant that inhibits inflammatory response, plays a pivotal role in kidney diseases. In this study, LPS treated mice and cultured podocytes were used as the models of SA‐AKI in vivo and in vitro, respectively. Medium‐ and high‐dose TAC administration significantly attenuated renal function and renal pathological manifestations at 12, 24 and 48 h after LPS treatment in mice. Moreover, the Toll‐like receptor 4 (TLR4)/myeloid differential protein‐88 (MyD88)/nuclear factor‐kappa (NF‐κB) signalling pathway was also dramatically inhibited by medium‐ and high‐dose TAC administration at 12, 24 and 48 h of LPS treatment mice. In addition, TAC reversed LPS‐induced podocyte cytoskeletal injury and podocyte migratory capability. Our findings indicate that TAC has protective effects against LPS‐induced AKI by inhibiting TLR4/MyD88/NF‐κB signalling pathway and podocyte dysfunction, providing another potential therapeutic effects for the LPS‐induced SA‐AKI.
... NRD has been shown to possess neuroprotective [23], hepatoprotective [24], cardioprotective [25], genoprotective [26], anti-nociceptive [27] and wound-healing properties [28], which is mainly attributed to its action in reducing inflammation. Studies have shown that NRD ameliorates drug-and toxicant-induced oxidative stress and inflammatory events in body organs such as brain [23], kidney [29], liver [24], heart [25], lungs [30], testes [31], eye [32], stomach [33], and uterus [34]. Despite the potent antioxidant and anti-inflammatory roles in various organs, there are no reports available on its role in colonic tissues to demonstrate its therapeutic potential in colitis. ...
... NRD is a lipophilic and highly bioavailable agent that makes it able to achieve required concentration for its therapeutic effects and restores redox balance by abolishing oxidative stress and subsequent inflammatory responses involving many pathways [29,30]. In a preliminary study, NRD was shown to have a protective effect against ethanol-, indomethacin-, and stress-induced gastric ulcer and to significantly inhibit ulcer formation [33]. ...
... NRD has been shown to have anti-inflammatory effects by downregulating the expression of inflammatory mediators and signaling pathways such as inhibition of LOX-1/ IL-1β [32] and TLR4/NF-κB signaling [29] and AMPK/ Nrf-2/HO-1 pathway [30]. The anti-inflammatory activities observed in the present study are in line with previous reports wherein NRD showed potent anti-inflammatory activity by inhibiting the release and secretion of proinflammatory cytokines in brain [23], kidney [29], liver [24], heart [25], lungs [30], testes [31], uterus [34], stomach [33], and ocular [32] tissues. ...
Targeting oxidative stress and inflammation by novel dietary compounds of natural origin convincingly appears to be one of the most important therapeutic strategies to keep inflammatory bowel diseases (IBD) such as ulcerative colitis disease in remission. It is imperative to investigate naturally occuring plant-derived dietary phytochemicals that are receiving attention for their therapeutic benefits to overcome the debilitating conditions of IBD. In the present study, the effect of nerolidol (NRD), a monocyclic sesquiterpene found in German Chamomile tea, was investigated in acetic acid-induced colitis model in Wistar rats. NRD was orally administered at a dose of 50 mg/kg/day either for 3 days before or 30 min after induction of IBD for 7 days, after intrarectal administration of acetic acid. The body weight, macroscopic, and microscopic analyses of the colon in different experimental groups were observed on days 0, 2, 4, and 7. Acetic acid caused significant reduction in body weight and induced macroscopic and microscopic ulcer along with a significant decline of antioxidants, concomitant to increased malondialdehyde (MDA), a marker of lipid peroxidation, and myeloperoxidase (MPO) activity, a marker of neutrophil activation. Treatment with NRD significantly improved IBD-induced reduction in body weight, improved histology, inhibited MDA formation, and restored antioxidants along with reduced MPO activity. Acetic acid also induced the release of pro-inflammatory cytokines and increased calprotectin, released by neutrophils under inflammatory conditions. NRD treatment significantly reduced calprotectin and pro-inflammatory cytokines. NRD treatment showed potential to improve disease activity and inhibit oxidative stress, lipid peroxidation, and inflammation along with histological preservation of the colon tissues.
... The TLR4/NF-κB signaling pathway has a key role in inflammation (Kim et al. 2017;Liu et al. 2019;Yang et al. 2022). LPS treatment stimulates the TLR4/NF-κB signal, while recruits proinflammatory cytokines and results in cellular injury (Akcay et al. 2009;Zhang et al. 2017). We wished to investigate if J147 therapy had an impact on the TLR4/NF-κB signaling pathway in the hippocampus of LPS-induced mice, so hippocampal expression of TLR4 and NF-κB was measured by western blotting. ...
... TLR4 has been studied in multiple diseases, and its role in the pathogenesis of inflammatory liver disease has been demonstrated (Soares et al. 2010). Expression of TLR4 and NF-κB has been shown to be increased markedly in the kidneys of LPS-treated rats and LPS-treated NRK-52E cells (Zhang et al. 2017). TLR4 regulates neuronal death (Zhong et al. 2020). ...
Neuroinflammation is associated with the pathophysiology of depression. The molecular mechanism of depressive-like behavior caused by sepsis-associated encephalopathy (SAE) is incompletely understood. J147 (an analog of curcumin) has been reported to improve memory and has neuroprotective activity, but its biological function in the depressive-like behavior observed in SAE is not known. We investigated the effects of J147 on lipopolysaccharide (LPS)-induced neuroinflammatory, depressive-like behaviors, and the toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signal pathway in the mouse hippocampus and microglia (BV2 cells). The forced-swimming test (FST) and tail-suspension test (TST) were undertaken for assessment of depressive-like behaviors. Expression of the proinflammatory genes interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α were measured using RT-qPCR and ELISA. Microglia activation was detected using immunofluorescence staining. The TLR4/NF-κB signaling pathway was studied using western blotting and immunofluorescence staining. J147 pretreatment markedly downregulated expression of IL-6, IL-1β, and TNF-α, and the mean fluorescence intensity of ionized calcium-binding adapter protein-1 in microglia. J147 restrained LPS-induced nuclear translocation of nuclear factor-kappa B (NF-κB), inhibitor of nuclear factor kappa B (IκB) degradation, and TLR4 activation in microglia. J147 administration inhibited bodyweight loss, mortality, microglia activation, and depressive-like behaviors in LPS-treated mice. In conclusion, J147 ameliorated the sepsis-induced depressive-like behaviors induced by neuroinflammation through attenuating the TLR4/NF-κB signaling pathway in microglia.
... Moreover, nerolidol exhibits antimicrobial activity against serval microbial agents, such as Staphylococcus Aureus, Candida Albicans and Escherichia Coli [16]. In the kidney, nerolidol was reported to have a renoprotective effect in lipopolysaccharide-induced acute kidney injury in rats, as it attenuated the rise in blood urea nitrogen (BUN) and creatinine and inhibited an increase in TNF-α and IL-1β [17]. In a thioacetamide-induced kidney injury model, nerolidol was also shown to ameliorate oxidative damage in kidney tissues [18]. ...
... PAI-1 is also a profibrotic cytokine, as it is considered one of the major inhibitors of fibrinolysis [34,35]. All these cytokines are upregulated in the majority of renal diseases [23,24,26], and the effect of nerolidol on these cytokines in renal IRI appears to be similar to its effect in other conditions [17]. ...
Efforts to decrease the deleterious effects of renal ischemia–reperfusion injury (IRI) are ongoing. Recently, there has been increasing interest in using natural phytochemical compounds as alternative remedies in several diseases. Nerolidol is a natural product extracted from plants with floral odors and has been proven to be effective for the treatment of some conditions. We investigated the effect of nerolidol in a rat model of renal IRI. Nerolidol was dissolved in a vehicle and administered orally as single daily dose of 200 mg/kg for 5 days prior to IRI and continued for 3 days post IRI. G-Sham (n = 10) underwent sham surgery, whereas G-IRI (n = 10) and G-IRI/NR (n = 10) underwent bilateral warm renal ischemia for 30 min and received the vehicle/nerolidol, respectively. Renal functions and histological changes were assessed before starting the medication, just prior to IRI and 3 days after IRI. Nerolidol significantly attenuated the alterations in serum creatinine and urea, creatinine clearance, urinary albumin and the urinary albumin–creatinine ratio. Nerolidol also significantly attenuated the alterations in markers of kidney injury; proinflammatory, profibrotic and apoptotic cytokines; oxidative stress markers; and histological changes. We conclude that nerolidol has a renoprotective effect on IRI-induced renal dysfunction. These findings might have clinical implications.
... Sepsis is prone to AKI, and the mechanism is related to the activation of toll-like receptor 4 (TLR4)/nuclear factor-kappaB (NF-jB) signaling pathway by bacterial products to produce a large number of inflammatory factors [31][32][33][34]. TLR4 is a pattern recognition receptor that acts as a sensor of LPS induced inflammation, activating inflammatory factors [35][36][37] to further activate the NF-jB pathway, which leads to nucleation of p65 egg white and increase of the pro-inflammatory factor tumor necrosis factor alpha (TNF-a), interleukin 1-beta (IL-1b), and interleukin 6 (IL-6) expression [38][39][40][41][42]. TLR4/ NF-jB signaling pathway is one of the most important mechanisms leading to sepsis associated AKI [43]. ...
... Furthermore, TLR4 works as an LPS sensor, and its activation of inflammatory factors in turn causes renal injury; it also activates the NF-jB pathway associated with the production of pro-inflammatory cytokines TNF-a, IL-6, and IL-1b [32]. In the present study, the levels of inflammatory factors TNF-a, IL-6, and IL-1b were dramatically elevated by LPS induction. ...
Renal injury is a fatal complication in critically ill patients with sepsis. As an ultrashort-acting synthetic opioid derivative, remifentanil has been reported to mitigate renal injury and sepsis. Nevertheless, whether remifentanil also suppresses sepsis-triggered renal injury is uncertain. The aim of this study was to investigate the effect of remifentanil on endoplasmic reticulum stress (ERS) and inflammatory response in an in vitro lipopolysaccharide (LPS)-stimulated renal tubular epithelial cell (HK-2) model and its mechanism. The viability of HK-2 cells with the absence or presence of LPS treatment was surveyed by cell counting kit-8 assay. Under the condition of LPS treatment, apoptosis was appraised by TUNEL assay and western blot. Levels of inflammatory factors were estimated though corresponding kits. Western blot tested the expression of toll-like receptor 4 (TLR4)/nuclear factor-kappaB (NF-κB) signaling-associated proteins. Also, the expression of ERS-related proteins was detected by western blot. Further, ERS inducer tunicamycin (TM) was added and the aforementioned experiments were conducted again. The results underlined the protective effects of remifentanil on LPS-evoked viability injury, inflammation, activation of TLR4/NF-κB signaling and ERS in HK-2 cells. Moreover, the impacts of remifentanil on the biological events of LPS-insulted HK-2 cells were all reversed by TM administration. To conclude, remifentanil might have a remarkable ameliorative effect on sepsis-induced renal injury, which implied the potential of remifentanil-based drug therapy in sepsis-induced renal injury.
... TLR4 is known as one of the most prominent receptors mediating the LPS response. When TLR4 binds to LPS ligands, the signal is transduced to the TIR (Toll/IL-1 receptor) region, which then further activates the NF-κB and MAPK pathways [44,45]. When NF-κB is activated and translocated to the nucleus by binding to the promoters of target genes, it can regulate the expression of cytokines such as IL-6, IL-18, and TNF-α [46]. ...
Pyocyanin (PCN) is an extracellular toxin secreted by Pseudomonas aeruginosa (PA), which has redox capacity and disrupts the redox balance of host cells, affecting cell function and leading to cell death. The aim of this experiment was to compare the degree of apoptosis, inflammation, and oxidative stress of bovine mammary epithelium cells (bMECs) induced by lipopolysaccharide (LPS) and pyocyanin (PCN) and to examine whether PCN can promote the apoptosis, inflammation, and oxidative stress of bMECs induced by LPS. In this study, 1 µg/mL LPS and 1 µg/mL PCN were finally selected for subsequent experiments through dose-dependent experiments. In this study, cells were not given any treatment and were used as the control group (NC). The cells were treated with PCN or LPS individually for 6 h as the PCN group (PCN) or the LPS group (LPS), and the combination of LPS and PCN challenge for 6 h as the LPS + PCN (LPS + PCN) group. Compared with the control and LPS groups, PCN resulted in a significantly upregulated expression of genes related to pro-inflammatory (IL-6, TNF-α, MyD88), apoptotic (Bax, Caspase3, Caspase9), as well as protein expression of components in the TLR4/NF-κB signaling pathway (TLR4, p-p65, p65), and p53 signaling pathway (p-p53, p53, Caspase9) (p < 0.05). Moreover, the expression of genes and proteins was significantly upregulated after PCN treatment combined with LPS compared to either LPS or PCN challenge alone (p < 0.05). The stimulation of PCN combined with LPS significantly increased reactive oxygen species (ROS) and malondialdehyde (MDA) production in bovine mammary epithelial cells (bMECs), as well as decreased glutathione peroxidase (GSH-Px) and total antioxidant capacity (T-AOC). Moreover, cells in the LPS + PCN group aggravated oxidative stress and antioxidant inhibition in cells. In addition, the expression of the corresponding genes and proteins related to the Nrf2 pathway (Nrf2, HO-1) was significantly down-regulated in the PCN group as compared to the control group (p < 0.05). Altogether, PCN stimulation exacerbates inflammatory reactions, apoptosis, and oxidative stress reactions, as well as when combined with LPS challenge in bMECs. Therefore, this study indicates that PCN manifests a role in promoting apoptosis, inflammation, and oxidative stress and interacting with LPS to enhance more serious biological stress responses.
... Toll-like receptor signaling plays an essential role in immune responses to various intracellular pathogens [38]. TLR4 acts as an upstream receptor for NF-κB and specifically recognizes pathogen-associated molecules, such as LPS [39,40]. LPS has been reported to induce inflammatory responses through the TLR4/NF-κB pathway [41]. ...
Dairy farming is the most important economic activity in animal husbandry. Mastitis is the most common disease in dairy cattle and has a significant impact on milk quality and yield. The natural extract allicin, which is the main active ingredient of the sulfur-containing organic compounds in garlic, has anti-inflammatory, anticancer, antioxidant, and antibacterial properties; however, the specific mechanism underlying its effect on mastitis in dairy cows needs to be determined. Therefore, in this study, whether allicin can reduce lipopolysaccharide (LPS)-induced inflammation in the mammary epithelium of dairy cows was investigated. A cellular model of mammary inflammation was established by pretreating bovine mammary epithelial cells (MAC-T) with 10 µg/mL LPS, and the cultures were then treated with varying concentrations of allicin (0, 1, 2.5, 5, and 7.5 µM) added to the culture medium. MAC-T cells were examined using RT–qPCR and Western blotting to determine the effect of allicin. Subsequently, the level of phosphorylated nuclear factor kappa-B (NF-κB) was measured to further explore the mechanism underlying the effect of allicin on bovine mammary epithelial cell inflammation. Treatment with 2.5 µM allicin considerably decreased the LPS-induced increase in the levels of the inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α) and inhibited activation of the NOD-like receptor protein 3 (NLRP3) inflammasome in cow mammary epithelial cells. Further research revealed that allicin also inhibited the phosphorylation of inhibitors of nuclear factor kappa-B-α (IκB-α) and NF-κB p65. In mice, LPS-induced mastitis was also ameliorated by allicin. Therefore, we hypothesize that allicin alleviated LPS-induced inflammation in the mammary epithelial cells of cows probably by affecting the TLR4/NF-κB signaling pathway. Allicin will likely become an alternative to antibiotics for the treatment of mastitis in cows.
... TLR4 is a receptor that recruits inflammatory factors and subsequently causes kidney and liver damage, and by increasing the level of TLR4, it stimulates the expression of proinflammatory cytokines IL-6 and TNF-α, thereby activating the NF-kB pathway. Studies have reported that increased NF-kB activation is tightly involved to organ damage (Inandiklioglu et al., 2021;Zhang et al., 2017). ...
In this study, the protective effect of melatonin was investigated in lipopolysaccharide induced sepsis model. 28 rats were randomly divided; Control, Melatonin, LPS and LPS + Melatonin. After LPS application, surgically remove kidney and liver tissues. The level of malondialdehyde (MDA) an oxidative stress marker and the immunoreactivity of Toll‐like receptor‐4 (TLR4), tumor necrosis factor‐α (TNF‐α), transcription factor NF‐κB were evaluated immunohistochemically. Expression levels for TLR4, TNF‐α, NF‐kB, IL‐1β (interleukin 1 beta) and IL‐6 (interleukin 6) evaluated. Additionally, AgNOR staining was performed in tissues. Vacuolization and inflammation were more intense in the kidney and liver sections in the LPS group compared to the other groups. It was observed that vacuolization and inflammation were decreased in LPS+Melatonin applied groups. It was determined that glomerular damage was increased in the LPS and LPS‐melatonin groups, but the damage rate LPS‐Melatonin group was decrease in the LPS group. It was determined that the MDA level in tissues of the LPS group was importantly increased compared to other groups. Additionally, TAA/NA ratio statistically significant differences were discovered between the groups. This study supports the potential protective effects of 10 mg/kg melatonin by modulating critical markers of local immune reaction in a model of LPS‐induced sepsis. In this study, the protective effect of melatonin was investigated in lipopolysaccharide induced sepsis model. 28 rats were randomly divided; Control, Melatonin, LPS and LPS + Melatonin. oxidative stress marker and the immunoreactivity of Toll‐like receptor‐4 (TLR4), tumor necrosis factor‐α (TNF‐α), transcription factor NF‐κB were evaluated immunohistochemically. Expression levels for TLR4, TNF‐α, NF‐kB, IL‐1β (interleukin 1 beta) and IL‐6 (interleukin 6) evaluated.
... Acute kidney injury (AKI) is recognized as a typical emergency and a high-risk factor endangering the morbidity and mortality of hospitalized patients; strategies on how to impede its progression have caught numerous researchers' attention [1]. Pathogenesis of AKI is yet to be fully understood, and renal ischemic/reperfusion (I/R) injury is broadly known as an independent risk factor for its progression. ...
Toll-like receptor 4 (TLR4) can mediate innate activation and inflammation, and it is typically expressed within the ischemic kidney. Augmenter of liver regeneration (ALR) acts as an immunoregulator with a high expression in the kidney induced by renal ischemia/reperfusion (I/R) injury. Exogenous ALR has indicated a role in protecting the kidney from I/R injury. The protective effect of ALR is due to the immune regulatory function which remains to be elucidated. In this study, rats induced by renal I/R were treated with recombinant human ALR (rhALR) and demonstrated that the animals were protected from kidney I/R injury, implying that the rhALR-treated rats had less tubular damage than those untreated rats. Meanwhile, tubular epithelial cell apoptosis, neutrophil (24 h) and macrophage (72 h) infiltration to tubulointerstitium, and levels of inflammatory cytokines were decreased considerably in the rhALR-treated rats as compared to control. Additionally, rhALR could downregulate mRNA expression of TLR4 endogenous ligands and restrain its activation in renal I/R injury rats. It has also been proved that anti-rhALR antibody blocked the inhibition of rhALR of the immune inflammatory response in hypoxia/reoxygenation (H/R) injury in vitro. In rhALR+anti-rhALR antibody-intervened H/R cells, the expression of inflammatory cytokines was upregulated compared with the rhALR-treated cells. Taken together, rhALR could regulate the TLR4 signaling pathway to relieve inflammatory response, thereby protecting renal I/R injury, indicating that ALR is likely to be introduced to develop novel immune therapies for renal I/R injury.
... The sesquiterpene alcohol nerolidol, also called peruviol, occurs naturally in the essential oils of several plants with floral odors including Baccharis dracunculifolia [1][2][3]. Several reports showed that it has protective effects in different conditions and organs such as the heart and kidney [4][5][6]. For instance, nerolidol has been shown to have antiulcer [1], anti-tumor [7,8], and skin permeation enhancing effects [9,10]. ...
Recently, there has been a growing interest in using natural products as treatment alternatives in several diseases. Nerolidol is a natural product which has been shown to have protective effects in several conditions. The low water solubility of nerolidol and many other natural products limits their delivery to the body. In this research, a drug delivery system composed of alginate and chitosan was fabricated and loaded with nerolidol to enhance its water solubility. The chitosan–alginate nanoparticles were fabricated using a new method including the tween 80 pre-gelation, followed by poly-ionic crosslinking between chitosan negative and alginate positive groups. Several characterization techniques were used to validate the fabricated nanoparticles. The molecular interactions between the chitosan, alginate, and nerolidol molecules were confirmed using the Fourier transform infrared spectroscopy. The ultraviolet spectroscopy showed an absorbance peak of the blank nanoparticles at 200 nm and for the pure nerolidol at 280 nm. Using both scanning and transmission electron microscopy, the nanoparticles were found to be spherical in shape with an average size of 12 nm and 35 nm for the blank chitosan–alginate nanoparticles and the nerolidol-loaded chitosan–alginate nanoparticles, respectively. The nanoparticles were also shown to have a loading capacity of 51.7% and an encapsulation efficiency of 87%. A controlled release profile of the loaded drug for up to 28 h using an in vitro model was also observed, which is more efficient than the free form of nerolidol. In conclusion, chitosan–alginate nanoparticles and nerolidol loaded chitosan–alginate nanoparticles were successfully fabricated and characterized to show potential encapsulation and delivery using an in vitro model.
... TLR4 is considered the canonical LPS sensor (Fu et al., 2016;Wang et al., 2020). Studies have shown that LPS can induce AKI via the TLR4 signaling pathway (Dou et al., 2022;Huynh et al., 2020;Zhang et al., 2017). The increase in TLR2 and TLR4 was observed under LPS stimulation in our experiment. ...
Defensins represent an integral part of the innate immune system to ward off potential pathogens. The study used a rat model to investigate mechanisms by which sodium butyrate (NaB) regulates β‐defensin to inhibit lipopolysaccharide (LPS)‐induced nephrotoxicity. We found that NaB alleviated LPS‐induced renal structural damage, as judged by reduced renal lesions and improved glomerular vascular structure. In addition, elevated levels of indicators of kidney damage creatinine and blood urine nitrogen, inflammatory mediators TNF‐α, and IL‐6 dropped after NaB administration. Rat β‐defensin 2 (rBD2), as estimated by mRNA level, was significantly higher in LPS‐treated kidneys, whereas the changes of rBD2 reduced in NaB‐treated kidneys. In addition, NaB alleviated LPS‐induced increase in TLRs mRNA expression. Mechanistically, the present study indicates that NaB has nephroprotective activity resulting from modulation of TLR2/4 to regulate rBD2 expression hence curbing inflammation.
Practical applications
In practice, adding NaB to diet can improve animal performance. Our results suggest that dietary supplementation of NaB increases animal feed intake and improves the body's defense ability to relieve inflammation caused by bacteria. Especially in the age of resistance prohibition, sodium butyrate can partially replace antibiotics to induce the expression of body defensin. It may become a health care product to enhance the body's immunity.
... Nuclear factor-κB (NF-κB) is a family of transcription factors, and has long been considered the central mediator of the process in inflammation and immunity (Kim et al., 2017;Zhang et al., 2017). TLR4 is one of the most critical upstream signaling axis molecules for NF-κB pathway activation, which in turn manage the secretion of varieties of proinflammatory cytokines, chemokines, and adhesion molecules (Yoshimura, 2006;Gong et al., 2021). ...
Flavonoids isolated from medicinal herbs have been utilized as valuable health-care agents due to their virous biological applications. Alpinetin is a natural flavonoid that emerges in many widely used medicinal plants, and has been frequently applied in Chinese patent drugs. Accumulated evidence has demonstrated that alpinetin possesses a broad range of pharmacological activities such as antitumor, antiinflammation, hepatoprotective, cardiovascular protective, lung protective, antibacterial, antiviral, neuroprotective, and other properties through regulating multiple signaling pathways with low systemic toxicity. However, pharmacokinetic studies have documented that alpinetin may have poor oral bioavailability correlated to its extensive glucuronidation. Currently, the reported pharmacological properties and pharmacokinetics profiles of alpinetin are rare to be scientifically reviewed. In this article, we aimed to highlight the mechanisms of action of alpinetin in various diseases to strongly support its curative potentials for prospective clinical applications. We also summarized the pharmacokinetics properties and proposed some viable strategies to convey an appreciable reference for future advances of alpinetin in drug development.
... Proposed molecular targets and mechanisms Preclinical models and in vivo effects b-Caryophyllene -A putative CB2 receptor full agonist (some authors question this though) -It activates JNK, Erk, and PPARs and inhibits the toll-like receptor CD-14/TLR4/MD2 axis -It reduces the expression and production of proinflammatory cytokines such as IL-1b, IL-6, IL-8, and TNF-a -It may also interact with the TLR4 -Antiallodynic, antinociceptive, anti-inflammatory, and neuroprotective in various pain models -In cerebral ischemia-reperfusion injury model, it rescues neurons, inhibits microglial activation, and decreases the release of proinflammatory cytokines Nerolidol -It decreases TNF-a and IL-1b in LPS-stimulated peritoneal macrophages -Possible involvement of TLR4, Nrf2, and/or NF-jB as signaling mechanisms -Dose-dependent antinociception and anti-inflammatory properties in various animal models (acetic acid writhing, formalin, edema, peritonitis, and hot-plate) without impaired motor function -Opioid-insensitive, GABAergic mechanisms without the involvement of ATP-sensitive (K 1 ) channels Fonseca et al., 2016Iqubal et al., 2019Khodabakhsh et al., 2015Ni et al., 2019Ogunwande et al., 2019Pinheiro et al., 2011Zhang et al., 2017 found that c-terpinene, which is a terpinene isoform, did not show toxicity at an oral dose of 2 g/kg and induced measurable antinociception in formalin, glutamate, and capsaicin-treated animals. To investigate the mechanism of action, animals were also pretreated with the general opioid antagonist naloxone, glibenclamide (ATP-sensitive potassium channel inhibitor), atropine (muscarinic acetylcholine receptor antagonist), or mecamylamine (nonselective, noncompetitive antagonist at the nicotinic acetylcholine receptor). ...
Pain prevalence among adults in the United States has increased 25% over the past two decades, resulting in high health-care costs and impacts to patient quality of life. In the last 30 years, our understanding of pain circuits and (intra)cellular mechanisms has grown exponentially, but this understanding has not yet resulted in improved therapies. Options for pain management are limited. Many analgesics have poor efficacy and are accompanied by severe side effects such as addiction, resulting in a devastating opioid abuse and overdose epidemic. These problems have encouraged scientists to identify novel molecular targets and develop alternative pain therapeutics. Increasing preclinical and clinical evidence suggests that cannabis has several beneficial pharmacological activities, including pain relief. Cannabis sativa contains more than 500 chemical compounds, with two principle phytocannabinoids, Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD). Beyond phytocannabinoids, more than 150 terpenes have been identified in different cannabis chemovars. Although the predominant cannabinoids, Δ9-THC and CBD, are thought to be the primary medicinal compounds, terpenes including the monoterpenes β-myrcene, α-pinene, limonene, and linalool, as well as the sesquiterpenes β-caryophyllene and α-humulene may contribute to many pharmacological properties of cannabis, including anti-inflammatory and antinociceptive effects. The aim of this review is to summarize our current knowledge about terpene compounds in cannabis and to analyze the available scientific evidence for a role of cannabis-derived terpenes in modern pain management. SIGNIFICANCE STATEMENT: Decades of research have improved our knowledge of cannabis polypharmacy and contributing phytochemicals, including terpenes. Reform of the legal status for cannabis possession and increased availability (medicinal and recreational) have resulted in cannabis use to combat the increasing prevalence of pain and may help to address the opioid crisis. Better understanding of the pharmacological effects of cannabis and its active components, including terpenes, may assist in identifying new therapeutic approaches and optimizing the use of cannabis and/or terpenes as analgesic agents.
... Studies showed that M2 macrophage depletion could induce TNF-α secretion during acute kidney damage caused by sepsis, and could also suppress the proliferation of tubular cells at significant levels [44], which suggested that TNF-α was associated with tubular cell necrosis [45]. TLR4 is a receptor acting as an LPS sensor, whose activation collects inflammatory factors, then causing kidney damage [46]. Also, the activation of TLR4 makes the NF-κB pathway, which is associated with the initiation of the expression of proinflammatory cytokines IL-1β, IL-6 and TNF-α become sensitive. ...
Aims
In this study, we aimed to investigate the protective effect of apilarnil on kidney damage in the sepsis model induced by LPS.
Main methods
64 Sprague Dawley adult male rats were randomly divided into eight groups; control group, groups in which 0.2, 0.4 and 0.8 g/kg/bw apilarnil (API) was applied by oral gavage method for 10 days, LPS group in which 30 mg/kg/bw lipopolysaccharide (LPS) administered as intraperitoneally, groups in which LPS + 0.2, LPS+ 0.4 and LPS +0,8 API was applied. Six hour after the last administration the rats were anesthetized for euthanasia and kidney tissues were removed for RT-PCR analysis, immunohistochemical analysis and histopathologic analysis.
Key finding
According to the results of RT-PCR expression levels of IL-6, IL-1β, NF-κB, TNF-α and TLR4 were significantly reduced in the LPS + 0,8 API group. Immunoreactivity of TLR4, pNF-κB and TNF-α levels in the LPS + 0.8 apilarnil group were significantly lower than in the LPS and LPS + 0.2 apilarnil groups. Histologically, compared to the LPS group the glomerular damage score tended to decrease in the LPS + 0,4 API and LPS+ 0,8 API groups, while the tubulointerstitial injury score decreased especially in the LPS + 0,8 API group.
Significance
In the present study, 0,8 g/kg dose of apilarnil promoted potential renoprotective effects which were achieved, at least in part, by the modulation of important markers of the local immune response in the model of LPS-induced sepsis.
... Lipopolysaccharide (LPS) is an endotoxin released by Gram-negative (G-) bacteria. Generally, LPS binds to toll-like receptor expressed in macrophages and induces the secretion of inflammatory cytokines by activating multiple inflammatory signaling pathways (9,10). It has been reported that LPS translocates to the liver, and activated macrophages play a central role in the development of alcoholic hepatitis. ...
Background:
Fisetin, a natural potent flavonoid, has various beneficial, pharmacological activities. In this study, we investigated expression changes of the fisetin regulating genes in lipopolysaccharide (LPS)-treated RAW264.7 cells and explored the role of fisetin in inflammation and autophagy.
Methods and results:
Microarray analysis identified 1,071 genes that were regulated by fisetin in LPS-treated RAW264.7 cells, and these genes were mainly related to the process of immune system response. Quantitative real-time polymerase chain reaction and Bio-Plex analysis indicated that fisetin decreased the expression and secretion of several inflammatory cytokines in cells administered with LPS. Western blot analysis and immunofluorescence assay showed that fisetin decreased microtubule-associated protein 1 light-chain 3B (LC3B) and lysosome-associated membrane protein 1 (LAMP1) expression in LPS-treated cells, while the autophagy inhibitor chloroquine (CQ) could partially reverse this effect. In addition, fisetin reduced the elevated expression of p-PI3K, p-AKT and p-mTOR induced by LPS in a concentration-dependent manner.
Conclusions:
Fisetin diminished the expression and secretion of inflammatory cytokines and facilitated autophagosome-lysosome fusion and degradation in LPS-treated RAW264.7 cells via inhibition of the PI3K/AKT/mTOR signaling pathway. Overall, the results of this study provide new clues for the anti-inflammatory mechanism of fisetin and explain the crosstalk between autophagy and inflammation to some extent.
... Furthermore, immunostaining for VCAM-1, a vascular inflammation marker, showed that LPS-AKI kidneys exhibited higher VCAM-1 expression in the cortex compared to the medulla (Figure 3e). Prussian blue-positive iron also co-localized with dilated proximal tubules as shown on Periodic acid-Schiff (PAS) staining (Figure 3f), with the tubular damage being a hallmark of the LPS-AKI model (Zhang et al., 2017). In contrast, kidneys from control mice injected with either magneto-EVs or SPIO-His particles exhibited fewer hypointense areas, and those hypointense areas on T 2 *w images (Figure 3g) correlated well with the negative Prussian blue staining (Figure 3h). ...
Human stem‐cell‐derived extracellular vesicles (EVs) are currently being investigated for cell‐free therapy in regenerative medicine applications, but the lack of noninvasive imaging methods to track EV homing and uptake in injured tissues has limited the refinement and optimization of the approach. Here, we developed a new labelling strategy to prepare magnetic EVs (magneto‐EVs) allowing sensitive yet specific MRI tracking of systemically injected therapeutic EVs. This new labelling strategy relies on the use of ‘sticky’ magnetic particles, namely superparamagnetic iron oxide (SPIO) nanoparticles coated with polyhistidine tags, to efficiently separate magneto‐EVs from unencapsulated SPIO particles. Using this method, we prepared pluripotent stem cell (iPSC)‐derived magneto‐EVs and subsequently used MRI to track their homing in different animal models of kidney injury and myocardial ischemia. Our results showed that iPSC‐derived EVs preferentially accumulated in the injury sites and conferred substantial protection. Our study paves a new pathway for preparing highly purified magnetic EVs and tracking them using MRI towards optimized, systemically administered EV‐based cell‐free therapies.
... TLR4 plays a pivotal role in the pathogenesis of LPS-induced SA-AKI. 34 Consistent with previous studies, 27,28,34 we found that the expression of TLR4 and the downstream signalling molecules was significantly up-regulated in the SA-AKI model in vivo and in vitro. ...
Sepsis‐associated acute kidney injury (SA‐AKI) is a common clinical critical care syndrome. It has received increasing attention due to its high morbidity and mortality; however, its pathophysiological mechanisms remain elusive. LIGHT, the 14th member of the tumour necrosis factor (TNF) superfamily and a bidirectional immunoregulatory molecule that regulates inflammation, plays a pivotal role in disease pathogenesis. In this study, mice with an intraperitoneal injection of LPS and HK‐2 cells challenged with LPS were employed as a model of SA‐AKI in vivo and in vitro, respectively. LIGHT deficiency notably attenuated kidney injury in pathological damage and renal function and markedly mitigated the inflammatory reaction by decreasing inflammatory mediator production and inflammatory cell infiltration in vivo. The TLR4‐Myd88‐NF‐κB signalling pathway in the kidney of LIGHT knockout mice was dramatically down‐regulated compared to the controls. Recombinant human LIGHT aggravated LPS‐treated HK‐2 cell injury by up‐regulating the expression of the TLR4‐Myd88‐NF‐κB signalling pathway and inflammation levels. TAK 242 (a selective TLR4 inhibitor) reduced this trend to some extent. In addition, blocking LIGHT with soluble receptor fusion proteins HVEM‐Fc or LTβR‐Fc in mice attenuated renal dysfunction and pathological damage in SA‐AKI. Our findings indicate that LIGHT aggravates inflammation and promotes kidney damage in LPS‐induced SA‐AKI via the TLR4‐Myd88‐NF‐κB signalling pathway, which provide potential strategies for the treatment of SA‐AKI.
... This increase in COX-2 expression was significantly reduced by NED treatment. In a previous study, NED was shown to suppress TNF-α and IL-6 [42]; therefore, the observed inhibition of COX-2 activity may be facilitated through the suppression of these cytokines. Inducible nitric oxide synthase (iNOS) is expressed in response to bacterial/proinflammatory stimuli and results in nitric oxide (NO) production, which provides cytoprotection. ...
Nerolidol (NED) is a naturally occurring sesquiterpene alcohol present in various plants with potent anti-inflammatory effects. In the current study, we investigated NED as a putative anti-inflammatory compound in an experimental model of colonic inflammation. C57BL/6J male black mice (C57BL/6J) were administered 3% dextran sodium sulfate (DSS) in drinking water for 7 days to induce colitis. Six groups received either vehicle alone or DSS alone or DSS with oral NED (50, 100, and 150 mg/kg body weight/day by oral gavage) or DSS with sulfasalazine. Disease activity index (DAI), colonic histology, and biochemical parameters were measured. TNF-α-treated HT-29 cells were used as in vitro model of colonic inflammation to study NED (25 µM and 50 µM). NED significantly decreased the DAI and reduced the inflammation-associated changes in colon length as well as macroscopic and microscopic architecture of the colon. Changes in tissue Myeloperoxidase (MPO) concentrations, neutrophil and macrophage mRNA expression (CXCL2 and CCL2), and proinflammatory cytokine content (IL-1β, IL-6, and TNF-α) both at the protein and mRNA level were significantly reduced by NED. The increase in content of the proinflammatory enzymes, COX-2 and iNOS induced by DSS were also significantly inhibited by NED along with tissue nitrate levels. NED promoted Nrf2 nuclear translocation dose dependently. NED significantly increased antioxidant enzymes activity (Superoxide dismutase (SOD) and Catalase (CAT)), Hemeoxygenase-1 (HO-1), and SOD3 mRNA levels. NED treatment in TNF-α-challenged HT-29 cells significantly decreased proinflammatory chemokines (CXCL1, IL-8, CCL2) and COX-2 mRNA levels. NED supplementation attenuates colon inflammation through its potent antioxidant and anti-inflammatory activity both in in vivo and in vitro models of colonic inflammation.
... Vanillin (#7) may improve psoriatic skin inflammation (Cheng et al. 2017), inflammatory bowel disease (Wu et al. 2009) via oral delivery and promises to combat oxidative brain injury (Makni et al. 2012) as well as sickle cell disease (Zhang et al. 2004) and relax coronary and basilar arteries (Raffai et al. 2015) when injected. Nerolidol (#9) has been shown to protect kidneys from injury in mice when administered interperitoneally (Zhang et al. 2017). Utilizing Peru Balsam in these ways is likely unsafe not only due to its complex chemical composition that may cause unpredictable effects, but especially because of the toxicity of some of the constituents. ...
Peru Balsam, a resinous substance derived from Myroxylon balsamum var. pereirae, has historically been used as a topical ointment for various skin conditions such as scabies, poorly healing wounds, eczema, and haemorrhoids. The ingredients responsible for these properties are not fully elucidated. We investigated the chemical composition of two Peru Balsam samples, one historical and one modern, using gas chromatography/mass spectrometry to identify the active ingredients responsible for its pharmaceutical properties. Both Peru Balsam specimens investigated had similar compositions, showing the stability of the substance. Components identified are effective against scabies, exhibit antimicrobial activity and aid skin penetration. These properties are consistent with historical uses of Peru Balsam. Several ingredients are also known allergens. This study, combining chemical information with scientific literature related to pharmaceutical properties of natural substances, represents a breakthrough in the elucidation of active ingredients in Peru Balsam.
... Neutrophil mobilisation is stimulated by MIP-2 (Burdon, Martin, & Rankin, 2005). LPS-induced expression of IL-1β and TNFα was inhibited by nerolidol in the kidneys of rats with acute kidney injury, NRK-52E proximal tubular cells of rats, and peritoneal macrophages (Fonsêca et al., 2016;Zhang et al., 2017). Nerolidol inhibits rotenone, which increases the levels of IL-1β, IL-6, and TNF-α in rat brains (Javed, Azimullah, Abul Khair, Ojha, & Haque, 2016). ...
Acute lung injury (ALI) is a severe syndrome, and there are no effective therapeutic appropriate medicines. Nerolidol, which exists in the essential oils of aromatic plants and flowers, exhibits antioxidative and anti-inflammatory activities. The present study evaluated the potential protective effect of nerolidol in ALI and the related mechanism in lipopolysaccharide (LPS)-treated mice. Here, nerolidol inhibited the LPS-induced neutrophil and other leukocyte infiltration of the alveolar space. LPS increased cytokines, chemokines, and adhesion molecules, and proinflammatory protein production was inhibited by nerolidol. LPS-induced phosphorylation of NF-κB p65, p38 MAPK, JNK, ERK were inhibited by nerolidol. The inhibitory concentration of nerolidol for the phosphorylation of NF-κB p65 and its upstream factors, p38 MAPK and JNK, was similar to the inflammatory responses of ALI. In conclusion, nerolidol is a potential protective agent in ALI via the inhibition of NF-κB activation and its upstream factors phosphorylation of p38 MAPK and JNK.
... A study has shown that nerolidol blocked LPS-induced acute kidney injury by inhibiting the TLR4/NF-κB signaling pathway. Specifically, nerolidol markedly prevented the rise of nitrogen and creatinine levels in LPS-treated rats, and also inhibited the increase of inflammatory mediators, like TNF, IL-1β, and NF-κB in LPS-treated NRK-52E cells [214]. Further, de Souza et al. demonstrated that nerolidol nanoencapsulation improved its anti-inflammatory effect on zymosan-induced arthritis in mice. ...
Medicinal use of Cannabis sativa L. has an extensive history and it was essential in the discovery of phytocannabinoids, including the Cannabis major psychoactive compound—Δ9-tetrahydrocannabinol (Δ9-THC)—as well as the G-protein-coupled cannabinoid receptors (CBR), named cannabinoid receptor type-1 (CB1R) and cannabinoid receptor type-2 (CB2R), both part of the now known endocannabinoid system (ECS). Cannabinoids is a vast term that defines several compounds that have been characterized in three categories: (i) endogenous, (ii) synthetic, and (iii) phytocannabinoids, and are able to modulate the CBR and ECS. Particularly, phytocannabinoids are natural terpenoids or phenolic compounds derived from Cannabis sativa. However, these terpenoids and phenolic compounds can also be derived from other plants (non-cannabinoids) and still induce cannabinoid-like properties. Cannabimimetic ligands, beyond the Cannabis plant, can act as CBR agonists or antagonists, or ECS enzyme inhibitors, besides being able of playing a role in immune-mediated inflammatory and infectious diseases, neuroinflammatory, neurological, and neurodegenerative diseases, as well as in cancer, and autoimmunity by itself. In this review, we summarize and critically highlight past, present, and future progress on the understanding of the role of cannabinoid-like molecules, mainly terpenes, as prospective therapeutics for different pathological conditions.
... TLR4 is a key member of TLRs family. Activated TLR4 can induce inflammatory response by activating NF-rB [26]. TLR4 can also regulate the expression of TGF-b1 and promote the recruitment of fibroblasts in the kidney, leading to the occurrence of renal fibrosis [27]. ...
Objective
Nephrotoxicity is the main side effect of cyclosporine A and finding an effective combating method is urgent. The present study investigates the improving effect of erythropoietin (EPO) on cyclosporine A induce renal injury in rats and further explores its possible mechanism.
Methods
Recombinant adenovirus for expression of EPO was constructed and injected into kidney with multipoint. Levels of blood urea nitrogen (BUN) and serum creatinine (SCr) were detected by kits. HE staining and Masson’s trichrome staining were used to evaluate pathological changes. ELISA was performed to detect the levels of transforming growth factor (TGF)-β1, interleukin (IL)-1β, and IL-6 in serum. Levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in kidney were detected according to manufacturer’s instruction. Western blotting was performed to observe the protein expression levels of peroxisome proliferator-activated receptor γ (PPAR γ), Toll-like receptor (TLR) 4, and TGF-β1.
Results
Results showed that EPO overexpression in rat kidney could significantly improve renal injury and fibrosis, suppress the release of inflammatory factors and reduce oxidative stress induced by cyclosporine A. Western blotting results showed that EPO overexpression could up-regulate the expression of PPARγ and down-regulate the expression of TLR4 and TGF-β1. Interestingly, when PPARγ activity was inhibited by T0070907, an effective and specific PPARγ inhibitor, the therapeutic effect of EPO was significantly attenuated.
Conclusion
Taken together, above results shown the protective effect of EPO on cyclosporine A-induced renal injury and confirmed that EPO’s anti-inflammation and antioxidative stress involving the PPAR γ/TLR4/TGFβ1 axis.
... AKI is a frequent complication of sepsis in critically ill patients, and often requires renal replacement therapy. Nowadays more and more attention has been focused on discovering compounds of natural origin in preventing or treating septic AKI [38][39][40][41]. DHA is one of the bioactive derivatives of a traditional anti-malarial drug artemisinin. ...
Septic acute kidney injury (AKI) is a frequent and serious complication of sepsis in critically ill patients associated with high morbidity and mortality. However, the treatment of septic AKI has still been beyond satisfaction. Dihydroartemisinin (DHA) is a semisynthetic derivative of artemisinin and is proposed as a potential agent for treating cancer and inflammatory diseases. In the present study, we aimed to investigate the effect of DHA on lipopolysaccharide (LPS)-induced AKI and the underlying mechanism. Male C57BL/6 mice were pretreated with or without DHA (20 mg/kg/d) for two days, and then were treated with one dose LPS (10 mg/kg) intraperitoneal injection to induce septic AKI. Twenty-four hours after LPS injection, blood samples and kidneys were collected for evaluation. The results indicated that DHA significantly ameliorated LPS-induced AKI as evidenced by improvement of renal function (serum creatinine and blood urea nitrogen), amelioration of renal pathological injury, and inhibition of tubular cell apoptosis. Meanwhile, DHA also strikingly attenuated inflammatory response, suppressed NF-κB signaling pathway activation, and inhibited oxidative stress in LPS-challenged mice. In conclusion, DHA could protect against LPS-induced AKI possibly by anti-inflammatory and antioxidant activities.
... Furthermore, immunostaining for VCAM-1, a vascular inflammation marker, showed that LPS-AKI kidneys exhibited higher VCAM-1 expression in the cortex compared to the medulla (Figure 3e). Prussian blue-positive iron also co-localized with dilated proximal tubules as shown on Periodic acid-Schiff (PAS) staining (Figure 3f), with the tubular damage being a hallmark of the LPS-AKI model (Zhang et al., 2017). In contrast, kidneys from control mice injected with either magneto-EVs or SPIO-His particles exhibited fewer hypointense areas, and those hypointense areas on T 2 *w images (Figure 3g) correlated well with the negative Prussian blue staining (Figure 3h). ...
Human stem-cell-derived extracellular vesicles (EVs) are currently being investigated for cell-free therapy in regenerative medicine applications, but their biodistribution and tropic properties for homing to injured tissues are largely unknown. Here, we labeled EVs with magnetic nanoparticles to create magneto-EVs that can be tracked by magnetic resonance imaging (MRI). Superparamagnetic iron oxide (SPIO) nanoparticles were coated with polyhistidine tags, which enabled purification of labeled EVs by efficiently removing unencapsulated SPIO particles in the solution. The biodistribution of systemically injected human induced pluripotent stem cell (iPSC)-derived magneto-EV was assessed in three different animal models of kidney injury and myocardial ischemia. Magneto-EVs were found to selectively home to the injury sites and conferred substantial protection in a kidney injury model. In vivo MRI tracking of magnetically labeled EVs represents a new powerful method to assess and quantify their whole-body distribution, which may help optimize further development of EV-based cell-free therapy.
... The NF-κB signaling pathway is central to the inflammation and immune responses [14]. Shi and coworkers found that expression of the inflammatory cytokines TNF-α and IL-1β were greatly elevated in rats with acute kidney injuries [15]. ...
Manganese (Mn) can have adverse effects on organisms as a result of heavy or chronic exposure, including neurological damage. This study examined the effects of chronic exposure to manganese chloride (MnCl2) on various biochemical indices of inflammatory cytokines, antioxidant enzymes, and heat shock proteins (HSPs) in the kidneys of Hy-line cocks. The exposures were carried out using 600, 900, or 1800 mg/kg doses of MnCl2 administered for periods of 30, 60, and 90 days. The exposure experiments indicated that Mn concentration in the kidneys increased over time and that Mn exposure potentially caused ultrastructural changes to the cells. Treatment with Mn was seen to increase the levels of various biomarkers, including protein carbonyl group content; DNA-protein cross-links (DPCs) and the mRNA expression of inflammatory factors such as tumor necrosis factor-α (TNF-α), nuclear factor-κB p50 (NF-κB p50), cyclooxygenase-2 (COX-2), and prostaglandin E synthase (PGES). The levels of other biomarkers were found to decrease as a result of Mn exposure, including the mRNA expression of oxidation indexes such as copper-zinc superoxide dismutase (CuZn–SOD), manganese superoxide dismutase (Mn-SOD), glutathione peroxidase (GSH-Px), and catalase (CAT). Accompanying the above changes, Mn exposure was seen to result in the relative mRNA and protein expression of HSPs 90, 70, 60, 40, and 27 increasing significantly. Thus, in cock kidneys, HSPs attenuated the biological changes caused by toxic exposure to Mn. This mechanism needs further exploration.
... In addition, nerolidol has several pharmacological effects, such as anti-inflammatory and antioxidant activities. In a previous study, nerolidol at concentrations of 100 and 200 mg/kg has been shown to suppress LPS-induced acute kidney inflammation in rat models [21]. Moreover, nerolidol reduces the generation of proinflammatory mediators in LPS-activated peritoneal macrophages [22]. ...
Acute lung injury (ALI) is a life-threatening disease that is characterised by the rapid onset of inflammatory responses. Lipopolysaccharide (LPS) is an endotoxin that plays an important role in triggering ALI via pneumonia and sepsis. However, no effective therapeutic strategies are currently available to treat ALI. Nerolidol is an aliphatic sesquiterpene alcohol that is found in the essential oils of many flowers as well as floral plants. It has been shown to exhibit anti-inflammatory, antioxidant, and anticancer properties. Herein, we show that nerolidol pretreatment counteracted the histopathological hallmarks in LPS-induced ALI mice. Indeed, nerolidol pretreatment inhibited LPS-induced alveolar-capillary barrier disruption, lung edema, and lipid peroxidation. Moreover, nerolidol pretreatment prevented the LPS from decreasing the enzymatic activities of superoxide dismutase, catalase, and glutathione peroxidase. Importantly, nerolidol treatment enhanced phosphorylation of AMP-activated protein kinase (AMPK) and expression of nuclear factor erythroid-derived 2-related factor 2 (Nrf-2) and heme oxygenase-1 (HO-1). Taken together, our study reveals the novel protective effects of nerolidol in LPS-induced ALI via the induction of antioxidant responses and activation of the AMPK/Nrf-2/HO-1 signalling pathway.
... The NF-B signal pathway lies in the center of inflammatory and immune response [32,33]. ROS has been reported to activate NF-B through the classical IKK-dependent pathway and induces a positive feedback mechanism associated with inflammation and kidney injury [34][35][36]. ...
Acute kidney injury (AKI) is a critical care syndrome, which is usually associated with sepsis-related endotoxemia. Evodiamine (EVO) is an active ingredient of many traditional medicinal formulations that possess a battery of biological activities. In the study, we aimed to evaluate the potential protective effect of EVO against lipopolysaccharide- (LPS-) induced AKI and cytotoxicity. LPS-resulted pathological injuries were significantly ameliorated by the administration of EVO. EVO reduced the levels of blood urea nitrogen (BUN) and creatinine in LPS-treated rats. EVO also inhibited LPS-induced reduction of cell viability in NRK-52E cells. LPS-resulting increase of TNF α and IL-1 β in both serum and kidney of rats and NRK-52E cells was inhibited by EVO. LPS-induced increase of P65 NF- κ B expression was markedly inhibited by EVO. EVO-induced reduction of TNF α and IL-1 β expression in LPS-treated cells was blocked by overexpression of P65 NF- κ B. Moreover, the increase of cell viability in LPS-treated cells induced by EVO was remarkably suppressed by overexpression of P65 NF- κ B. LPS-resulting increase of reactive oxygen species (ROS) production was suppressed by EVO. H 2 O 2 suppressed EVO-induced decrease of P65 NF- κ B expression and increase of cell viability in LPS-treated NRK-52E cells. Moreover, the antioxidant NAC significantly promoted EVO-induced decrease of P65 NF- κ B expression and increase of cell viability in LPS-treated NRK-52E cells. In conclusion, EVO had crucial protective effects against LPS-induced AKI and cytotoxicity through the antioxidant activities and thus the inhibition of inflammation. Our data highlight EVO as a potential candidate for the development of new strategies for the treatment of AKI.
... To demonstrate the effects of ISL on AKI induced by sepsis, we established murine AKI model by LPS injection which used in many researches [18][19][20]. In pathologic process of LPS-induced AKI, vacuolization could be found in renal tubular cells, indicating the moderate/ severe renal injury. ...
Septic acute kidney injury (AKI) characterized as acute infection and renal inflammation, still lacks of effective therapies. Isoliquiritigenin (ISL) as a small molecular from licorice, is able to inhibit the expression of HMGB1. However, the role and mechanism of ISL in septic AKI has not been investigated. In this study, we used LPS injection to induce murine septic AKI. One hour before LPS injection, 50 mg/kg ISL was once orally given to the mice. For the in vitro study, HK2 human tubular cells were respectively treated with 50 μM and 100 μM ISL 5 hrs before 2 μg/ml LPS stimulation. Then we observed that ISL ameliorated renal dysfunction and attenuated renal tubular injury. ISL inhibited the phosphorylation of IκB-α and NF-κB p65 after LPS induction both in vivo and in vitro. ISL also inhibited NF-κB p65 translocation from cytoplasm to the nucleus upon LPS stimulation. Further, NF-κB p65 translocation could trigger macrophage polarization, neutrophil activation and pro-inflammatory cytokines secretion in LPS-induced inflammation. These results showed that ISL could alleviate LPS-induced AKI by suppressing NF-κB p65 translocation and inhibiting inflammatory responses, indicating protective effects of ISL in LPS-induced acute renal inflammation. This study might be useful for designing potential clinical trials to prevent and treat sepsis induced AKI in patients with serious illness.
... In addition, consistent with our findings on anti-oxidative effect of Inflammation is another pivotal factor that is strongly involved in pathogenesis of AKI [5,6]. After LPS challenge, over-production or over-activity and subsequent release of inflammatory mediators such as NF-jB and TLR4 [46], IL-1b and TNF-a [47], IL-6 [39], and Cox2 [48] are observed. Since comparable data were also noted in our study, this evidently showed the development of an inflammatory process in kidney tissue after LPS injection. ...
Context: Acute kidney injury (AKI) is considered a major public health concern in today’s world. Sepsis‐induced AKI is large as a result of exposure to lipopolysaccharide (LPS) that is the major outer membrane component of Gram‐negative bacteria. Sesamin is the main lignan of sesame seeds with multiple protective effects.
Objective: In this research, we tried to demonstrate the protective effect of sesamin pretreatment in LPS-induced mouse model of AKI.
Methods: LPS was injected at a single dose of 10 mg/kg (i.p.) and sesamin was given p.o. at doses of 25, 50, or 100 mg/kg, one hour prior to LPS.
Results: Treatment of LPS-challenged mice with sesamin reduced serum level of creatinine and blood urea nitrogen (BUN) and returned back renal oxidative stress-related parameters including glutathione (GSH), malondialdehyde (MDA), and activity of catalase and superoxide dismutase (SOD). Moreover, sesamin alleviated inappropriate changes of renal nuclear factor-kappaB (NF-κB), toll-like receptor 4 (TLR4), cyclooxygenase-2 (COX2), tumor necrosis factor α (TNFα), interleukin-6, DNA fragmentation (an apoptotic index), and nuclear factor (erythroid-derived 2)-like 2 (Nrf2). In addition, sesamin diminished magnitude of kidney tissue damage due to LPS.
Conclusion: In summary, sesamin could dose-dependently abrogate LPS-induced AKI via attenuation of renal oxidative stress, inflammation, and apoptosis.
... TLR4 plays a critical role in the LPS-induced inflammatory response [22]. In this study, the expression of TLR4 was measured by Western blot. ...
Background
Nuciferine, a major bioactive component from the lotus leaf, has been reported to have notable anti-inflammatory activities such as renal inflammation and acute lung injury in previous studies. Mastitis is one of the most prevalent diseases in the dairy cattle, which causes large economic losses for the dairy industry. However, the effects of nuciferine on lipopolysaccharide (LPS)-induced mastitis have not been reported.
Methods and results
Here, we investigated the anti-inflammatory effects of nuciferine on LPS-induced mastitis in mice and illuminated its potential mechanism on the TLR4-mediated signaling pathway in mouse mammary epithelial cells (mMECs). Histopathological changes and myeloperoxidase (MPO) activity assay showed that nuciferine treatment significantly alleviated the LPS-induced injury of mammary gland flocculus, inflammatory cells infiltration. qPCR and ELISA assays indicated that nuciferine dose-dependently reduced the levels of TNF-α and IL-1β, which indicated that nuciferine might have therapeutic effects on mastitis. Furthermore, nuciferine treatment significantly decreased the expression of TLR4 in a dose-dependent manner. Besides, nuciferine was also found to suppress LPS-induced NF-κB activation.
Conclusion
These findings indicate that nuciferine potently ameliorates LPS-induced mastitis by inhibition of the TLR4-NF-κB signaling pathway.
... In addition, the treatment was able to prevent oxidative stress and alterations in Na þ , K þ -ATPase and AChE activities caused by the infection. Using the same model for the mouse liver, heart and blood, the authors demonstrated that the use of NOH and NOH-loaded nanospheres resulted in similar effects, ameliorating the oxidative stress and inflammation via the inhibition of TNFa, IL-1b and TLR4 [250]. According to high throughput data, NOH inhibited LPS-induced production of TNF-a (with an activity value of 2.0 mM) and IL-12p40 (7.6 mM) (https://pubchem.ncbi.nlm.nih.gov/compound/ ...
Cannabaceae plants Cannabis sativa L. and Humulus lupulus L. are rich in terpenes-both are typically comprised of terpenes as up to 3-5% of the dry-mass of the female inflorescence. Terpenes of cannabis and hops are typically simple mono-and sesquiterpenes derived from two and three isoprene units, respectively. Some terpenes are relatively well known for their potential in biomedicine and have been used in traditional medicine for centuries, while others are yet to be studied in detail. The current, comprehensive review presents terpenes found in cannabis and hops. Terpenes' medicinal properties are supported by numerous in vitro, animal and clinical trials and show anti-inflammatory, antioxidant, analgesic, anticonvulsive, antidepressant, anxiolytic, anticancer, antitumor, neuroprotective, anti-mutagenic, anti-allergic, antibiotic and anti-diabetic attributes, among others. Because of the very low toxicity, these terpenes are already widely used as food additives and in cosmetic products. Thus, they have been proven safe and well-tolerated.
Background
Diabetic retinopathy (DR) is the foremost microvascular problem that causes drastic visual impairment in diabetes patients. Hyperglycemia-triggered reaction cascade of inflammation and oxidative stress constitute the DR pathogenesis. The existing treatment options are not completely satisfactory.
Materials and Methods
We investigated the cell viability by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, inflammatory mediators, lactate dehydrogenase (LDH), superoxide dismutase, glutathione, and malonaldehyde (MDA) levels by ELISA and qRT-PCR assay, protein expression of Nrf2 and heme oxygenase-1 (HO-1) by western blotting assay were analyzed.
Results
According to our research, nerolidol (NRD) increases the proliferation and antioxidant activity of human retinal endothelial cells (HRECs) by inducing Nrf2/HO-1 signaling, while attenuating MDA, an oxidative stress marker, LDH, and inflammatory mediators. These outcomes suggest that a substantial reaction of inflammation and oxidative stress injury happened in DR, which might be correlated to the instigation of the signaling Nrf2/HO-1.
Conclusion
NRD effectively suppresses oxidative stress and inflammation in HG-induced HRECs. The primary mechanism of NRD on DR may be linked to the activation of the Nrf2/HO-1 pathway and may give a useful medicine for DR treatment.
Despite the observation of diabetes‐induced brain tissue damage and impaired learning and memory, the underlying mechanism of damage remains elusive, and effective, targeted therapeutics are lacking. Notably, the NLRP3 inflammasome is highly expressed in the hippocampus of diabetic individuals. Nerolidol, a naturally occurring compound with anti‐inflammatory and antioxidant properties, has been identified as a potential therapeutic option for metabolic disorders. However, the ameliorative capacity of nerolidol on diabetic hippocampal injury and its underlying mechanism remain unclear. Network pharmacology and molecular docking was used to predict the signaling pathways and therapeutic targets of nerolidol for the treatment of diabetes. Then established a diabetic rat model using streptozotocin (STZ) combined with a high‐fat diet and nerolidol was administered. Morris water maze to assess spatial learning memory capacity. Hematoxylin and eosin and Nissl staining was used to detect neuronal damage in the diabetic hippocampus. Transmission electron microscopy was used to detect the extent of damage to mitochondria, endoplasmic reticulum (ER) and synapses. Immunofluorescence was used to detect GFAP, IBA1, and NLRP3 expression in the hippocampus. Western blot was used to detect apoptosis (Bcl‐2, BAX, and Cleaved‐Caspase‐3); synapses (postsynaptic densifying protein 95, SYN1, and Synaptophysin); mitochondria (DRP1, OPA1, MFN1, and MFN2); ER (GRP78, ATF6, CHOP, and caspase‐12); NLRP3 inflammasome (NLRP3, ASC, and caspase‐1); inflammatory cytokines (IL‐18, IL‐1β, and TNF‐α); AKT (P‐AKT); and mitogen‐activated protein kinase (MAPK) pathway (P‐ERK, P‐p38, and P‐JNK) related protein expression. Network pharmacology showed that nerolidol's possible mechanisms for treating diabetes are the MAPK/AKT pathway and anti‐inflammatory effects. Animal experiments demonstrated that nerolidol could improve blood glucose, blood lipids, and hippocampal neuronal damage in diabetic rats. Furthermore, nerolidol could improve synaptic, mitochondrial, and ER damage in the hippocampal ultrastructure of diabetic rats by potentially affecting synaptic, mitochondrial, and ER‐related proteins. Further studies revealed that nerolidol decreased neuroinflammation, NLRP3 and inflammatory factor expression in hippocampal tissue while also decreasing MAPK pathway expression and enhancing AKT pathway expression. However, nerolidol improves hippocampal damage in diabetic rats cannot be shown to improve cognitive function. In conclusion, our study reveals for the first time that nerolidol can ameliorate hippocampal damage, neuroinflammation, synaptic, ER, and mitochondrial damage in diabetic rats. Furthermore, we suggest that nerolidol may inhibit NLRP3 inflammasome and affected the expression of MAPK and AKT. These findings provide a new experimental basis for the use of nerolidol to ameliorate diabetes‐induced brain tissue damage and the associated disease.
Ethnopharmacological relevance: Hedychium coccineum rhizome is an anti-inflammatory ethnomedicine used to remedy inflammation-related swelling and bronchial asthma.
Aim of the study: The study aimed to analyze the phytochemical constituents of H. coccineum rhizome essential oil (EO) and evaluate its in vitro and in vivo anti-inflammatory effects and underlying mechanisms.
Materials and methods: Phytochemical constituents of H. coccineum rhizome EO were analyzed using GC-FID/MS. In RAW264.7 macrophages induced by LPS, blockade of PGE2, NO, IL-1β, IL-6, and TNF-α secretion by H. coccineum rhizome EO was measured, and then western blot, qRT-PCR, and immunofluorescent staining were used to evaluate its underlying mechanisms. Moreover, we used the xylene-induced ear edema model for testing anti-inflammatory potential in vivo and examined auricular swelling as well as tissue and serum contents of IL-1β, IL-6, and TNF-α.
Results: EO's main components were E-nerolidol (40.5%), borneol acetate (24.8%), spathulenol (4.5%), linalool (3.8%), elemol (3.5%), and borneol (3.4%). In RAW264.7 cells stimulated by LPS, EO downregulated the expression of pro-inflammatory enzyme (iNOS and COX-2) genes and proteins, thereby suppressing pro-inflammatory mediators (NO and PGE2) secretion. Simultaneously, it reduced TNF-α, IL-1β, and IL-6 release by downregulating their mRNA expression. Besides, H. coccineum EO attenuated LPS-stimulated activation of NF-κB (by reducing IκBα phosphorylation and degradation to inhibit NF-κB nuclear translocation) and MAPK (by downregulating JNK, p38, and ERK phosphorylation). In xylene-induced mouse ear edema, EO relieved auricular swelling and lowered serum and tissue levels of TNF-α, IL-1β, and IL-6.
Conclusions: H. coccineum EO had powerful in vivo and in vitro anti-inflammatory effects by inhibiting MAPK and NF-κB activation. Hence, H. coccineum EO should have great potential for application in the pharmaceutical field as a novel anti-inflammatory agent.
Asthma is a common respiratory disease associated with airway inflammation. Nerolidol is an acyclic sesquiterpenoid with anti‐inflammatory properties. BALB/C mice were sensitized with ovalbumin (OVA) to induce asthma symptoms and given different doses of Nerolidol. We found that Nerolidol reduced OVA‐induced inflammatory cell infiltration, the number of goblet cells and collagen deposition in lung tissue. Nerolidol reduced the OVA‐specific IgE levels in serum and alveolar lavage fluid in an asthma model. Immunohistochemical staining of α‐SMA (the marker of airway smooth muscle) showed that Nerolidol caused bronchial basement membrane thinning in asthmatic mice. The hyperplasia of airway smooth muscle cells (ASMCs) is an important feature of airway remodeling in asthma. ASMCs were treated with 10 ng/mL TGF‐β to simulate the pathological environment of asthma in vitro and then treated with different doses of Nerolidol. Nerolidol inhibited the activity of TGF‐β/Smad signaling pathway both in the lung tissue of OVA‐induced mouse and TGF‐β‐stimulated ASMCs. 16s rRNA sequencing was performed on feces of normal mice, the changes of intestinal flora in OVA‐induced asthmatic mice and Nerolidol‐treated asthmatic mice were studied. The results showed that Nerolidol reversed the reduced gut microbial alpha diversity in asthmatic mice. Nerolidol changed the relative abundance of gut bacteria at different taxonomic levels. At the phylum level, the dominant bacteria were Bacteroidota, Firmicutes, and Proteobacteria. At the genus level, the dominant bacteria were Lactobacillus, Muribaculaceae, Bacteroides , and Lachnospiraceae . We conclude that Nerolidol attenuates OVA‐induced airway inflammation and alters gut microbes in mice with asthma via TGF‐β/Smad signaling.
Torularhodin is a carotenoid that exerts beneficial effects on chronic kidney disease (CKD), which is strongly linked to a high-fat diet (HFD), however, its functional properties have not yet been fully determined. In this study, torularhodin bilosomes, which feature good processing properties and physical stability, were chosen for delivery. We evaluated their effect on mice with CKD induced by HFD and determined the relevant mechanisms. In HFD-fed mice, both structural and functional renal injury was observed, including significant increases in BUN, SCr, urinary protein levels, capillary basement membrane thickening, and inflammatory cell infiltration. The results showed that SOD content, GSH-px content, and CAT activity were all significantly higher, while the MDA level was significantly lower in the tor-emu and tor-bilo groups, indicating that torularhodin effectively alleviated oxidative stress. In addition, the expression of the inflammatory factors TNF-a, IL-6, and IL-1β was reduced in the torularhodin group compared with the model group, and both the transcription and expression of the TLR4, MyD88, TIRAP, TRIF, and NF-κB proteins related to the TLR4/NF-κB pathway were reduced. Mechanistically, torularhodin reduced the renal inflammatory response by decreasing TLR4 protein expression and the signaling of proteins such as MyD88, as well as preventing NF-κB from undergoing dissociation, reducing its transcriptional activity, and decreasing the release of downstream pro-inflammatory mediators. In conclusion, torularhodin bilosomes can alleviate high-fat diet-induced CKD in mice by modulating the TLR4/NF-κB pathway. This result further deepens the understanding of torularhodin activity and confirms the role of torularhodin as an effective health supplement.
Asiatic acid (AA), a triterpenoid compound isolated from Centella asiatica, has anti-inflammatory, antioxidant and anticancer biological characteristics. To explore the effect of AA on LPS-induced acute kidney injury (AKI) in broilers, a total of 60 one-day-old broilers were randomly divided into 6 groups, including the normal group, AKI model group, AKI + AA 15 mg/kg group, AKI + AA 30 mg/kg group, AKI + AA 60 mg/kg group and normal + AA 60 mg/kg group. Hematoxylin-eosin staining was used to observe the histopathology in kidney tissue, and the mRNA and protein expressions related to oxidative stress and ferroptosis were tested by qPCR and western blotting respectively. AA mitigated vacuolar degeneration and enlarged glomerular space caused by LPS in kidney tissue. Additionally, AA significantly increased the mRNA levels of Nrf2, HO-1, NQO1, GCLC, GCLM, GPX4, SLC7A11 and FTH1, and decreased the mRNA levels of Keap1 and PTGS2 in LPS-induced AKI. Likewise, AA significantly upregulated the protein expressions of Nrf2, HO-1, NQO1, GPX4, SLC7A11 and FTH1, and downregulated the protein expressions of Keap1 and PTGS2 in LPS-induced AKI. These results suggested that AA alleviated LPS-induced AKI by inhibiting oxidative stress and ferroptosis through targeting regulation of the Nrf2 pathway in broilers.
The aim of the present study was to determine the cell proliferation, apoptotic pathway analysis through protein, mRNA and cell cycle arrest mechanism in nerolidol induced osteosarcoma MG‐63 cells. The osteosarcoma MG‐63 cells were treated with various doses of nerolidol (15 and 20 μM/ml) for 24 h. Cell proliferation was examined using assist method of MTT assay, fixed the IC50 value of nerolidol 15 μM/ml. Reactive oxygen species (ROS) generation was analyzed by DCFH‐DA dye, mitochondrial potential detected by Rh‐123 dye, apoptotic morphological changes identified by AO/EtBr, PI, DAPI staining, and cell adhesion were detected by using fluorescence microscope. Cell proliferation, and apoptotic molecular protein and mRNA expressions such as ERK, P38, p‐PI3K, p‐JNK, Bcl‐2, JNK, p‐P38, cyclin‐D1, and Bax were analyzed in osteosarcoma MG‐63 cells. Nerolidol significantly suppressed the osteosarcoma cells progression in a dose dependent manner (p < .05) evident in the oxidative stress induction and apoptotic morphological changes. Nerolidol also regulated the protein PI3K/AKT mechanistically via induction of apoptosis Nerolidol suppresses osteosarcoma MG‐63 cells by PI3K/AKT by cell cycle arrest at early phase of G0/G1. To sum up, nerolidol suppressed the growth of bone cancer cells and can be finally targeted as a potent drug for analyzing its chemotherapeutic effects in future.
The objective of this study is to examine the chemopreventive effects of Nerolidol (NER) on hamster buccal pouch carcinogenesis (HBC) induced by 7,12‐dimethylbenz(a)anthracene (DMBA) in male golden Syrian hamsters. In this study, oral squamous cell carcinoma was developed in the buccal pouch of an oral painted hamster with 0.5% DMBA in liquid paraffin three times weekly for 12 weeks. To assess DMBA‐induced hamster buccal tissue carcinogenesis, biochemical endpoints such as Phase I and II detoxification enzymes, antioxidants, lipid peroxidation (LPO) by‐products, and renal function markers, as well as histopathological examinations, were used. Furthermore, the immunohistochemical studies of interleukin‐6 were investigated to find the inflammatory link in the HBC carcinogenesis. In our results, DMBA alone exposed hamsters showed 100% tumor growth, altered levels of antioxidants, detoxification agents, LPO, and renal function identifiers as compared to the control hamsters. The outcome in present biochemical, histopathological, and immunohistochemistry studies has been found a reverse in NER‐treated hamsters against the tumor. This study concluded that NER modulated the biochemical profiles (antioxidants, detoxification, LPO, and renal function markers) and inhibited tumor development in DMBA induced oral carcinogenesis.
Objective
To explore the effects of miR-494 inhibition through the NF-κB signaling pathway on lipopolysaccharide (LPS)-induced acute kidney injury (AKI) mouse model.
Methods
The AKI mice induced by LPS were treated with miR-494 antagomir, and the kidney parameters and indicators of oxidative stress were detected. HE and TUNEL staining were performed to observe the kidney histopathology and the apoptosis in renal tubular epithelial cells (RTECs), respectively. The ROS level was measured using dihydroethidium (DHE) staining. In addition, qRT-PCR, western blotting, immunohistochemistry (IHC), and ELISA were also used to detect gene or protein expression.
Results
LPS-induced AKI mice injected with the miR-494 antagomir showed reduced blood urea nitrogen (BUN) and serum creatinine (Cr) with improved kidney histopathology. The expression levels of p-IKKα/β, p-IκBα and p65 NF-κB in the nucleus were increased in kidney tissues from the LPS-induced AKI mice, and they were decreased by the miR-494 antagomir. Moreover, the results of IHC showed that the miR-494 antagomir downregulated p65 NF-κB in kidney tissues from the LPS-induced AKI mice, accompanied by decreased levels of TNF-α, IL-1β, IL-6, MDA, NO, and ROS but increased levels of SOD and GSH. In addition, the LPS-induced AKI mice had increased apoptosis in RTECs, as well as increased Caspase-3 and Bax and decreased Bcl-2, which were reversed by the miR-494 antagomir.
Conclusions
The inhibition of miR-494 could reduce inflammatory responses and improve oxidative stress in kidney tissues from LPS-induced AKI mice by blocking the NF-κB pathway accompanying by reduced apoptosis in RTECs.
Acute kidney injury (AKI) is a progressive renal complication which significantly affects the patient's life with huge economic burden. Untreated acute kidney injury eventually progresses to a chronic form and end-stage renal disease. Although significant breakthroughs have been made in recent years, there are still no effective pharmacological therapies for the treatment of acute kidney injury. Toll-like receptor 4 (TLR4) is a well-characterized pattern recognition receptor, and increasing evidence has shown that TLR4 mediated inflammatory response plays a pivotal role in the pathogenesis of acute kidney injury. The expression of TLR4 has been seen in resident renal cells, including podocytes, mesangial cells, tubular epithelial cells and endothelial cells. Activation of TLR4 signaling regulates the transcription of numerous pro-inflammatory cytokines and chemokines, resulting in renal inflammation. Therefore, targeting TLR4 and its downstream effectors could serve as an effective therapeutic intervention to prevent renal inflammation and subsequent kidney damage. For the first time, this review summarizes the literature on acute kidney injury from the perspective of TLR4 from year 2010 to 2020. In the current review, the role of TLR4 signaling pathway in AKI with preclinical evidence is discussed. Furthermore, we have highlighted several compounds of natural and synthetic origin, which have the potential to avert the renal TLR4 signaling in preclinical AKI models and have shown protection against AKI. This scientific review provides new ideas for targeting TLR4 in the treatment of AKI and provides strategies for the drug development against AKI.
Exposure to high levels of manganese (Mn) leads to brain Mn accumulation, and a disease referred to as manganism. Activation of microglia plays an important role in Mn-induced neuroinflammation. Sodium p-aminosalicylic acid (PAS-Na) is a non-steroidal anti-inflammatory drug that inhibits Mn-induced neuroinflammation. The aim of the current study was to explore the role of NF-κB in the protective mechanism of PAS-Na on Mn-induced neuroinflammation in BV2 microglial experimental model. We treated BV2 microglia with 200 μM Mn for 24 h followed by 48 h treatment with graded concentrations of PAS-Na, using an NF-kB inhibitor, JSH-23, as a positive control. MTT results established that 200 and 400 μM PAS-Na treatment increased the Mn-induced cell viability reduction. NF-κB (P65) mRNA expression and the phosphorylation of p65 were increased in Mn-treated BV2 cell, and suppressed by PAS-Na, analogous to the effect of JSH-23 pretreatment. Furthermore, PAS-Na significantly reduced the contents of the inflammatory cytokine TNF-α and IL-1β, both of which were increased by Mn treatment. The current results show that PAS-Na attenuated Mn-induced inflammation by abrogating the activation of the NF-κB signaling pathways and reduced the release of pro-inflammatory cytokines.
Ethnopharmacological relevance
Baccharis dracunculifolia (Asteraceae) is a commonly used plant in traditional medicine known as "alecrim-do-campo". Popularly it has been used as an immunostimulant, antibiotic, anti-inflammatory among other applications. So far, only a few studies have investigated the B. dracunculifolia anti-inflammatory effect and none has investigated the effectiveness of essential oil on skin diseases.
Aim of the study
The study aimed at evaluating the topical anti-inflammatory activity of B. dracunculifolia essential oil (BdEO) in mice models of acute and chronic skin inflammation.
Materials and methods
BdEO was obtained from leaves and it was analyzed with Gas Chromatograph. Topical anti-inflammatory activity of BdEO (0.1, 0.3 and 1.0 mg/ear) was evaluated in Arachidonic Acid or TPA-induced acute and chronic skin inflammation in mice. Parameters such edema, cell migration and keratinocytes proliferation were evaluated. In addition, safety and a possible mechanism of action for BdEO essential oil were also investigated.
Results
Our results indicate that mainly terpenoids compounds compose BdEO. In addition, topical treatment with BdEO inhibited inflammatory parameters in both acute and chronic models of skin inflammation. This protective effect was associated with reduced edema formation, smaller cellular influx into the inflamed tissue and reduction of keratinocytes hyperproliferation. Although BdEO appears to exert its anti-inflammatory effect through a corticosteroid pathway, no local or systemic side effects were observed.
Conclusion
Taken together, the present results showed that the essential oil obtained by hydrodistillation from B. dracunculifolia leaf samples exhibit remarkable topical anti-inflammatory properties. Therefore, our study demonstrated evidence for BdEO topical anti-inflammatory efficacy and safety, suggesting that it could be considered for developing of a new phytotherapeutic formulation as treatment for skin diseases.
Purpose:
Nerolidol, a naturally occurring sesquiterpene has both anti-microbial and anti-inflammatory properties. The current study aims to investigate the antifungal and the anti-inflammatory effects of nerolidol against mouse Aspergillus fumigatus (A. fumigatus) keratitis.
Methods:
The minimum inhibitory concentration (MIC) and cytotoxicity tests were used to study the antifungal ability. For in vivo and in vitro studies, the mouse corneas and the human corneal epithelial cells (HCECs) infected with A. fumigatus spores were intervented with nerolidol or phosphate buffer saline (PBS). Thereafter, the effect of the nerolidol on the response against inflammation was analyzed using the following parameters: recruitment of the neutrophils or macrophages and the expression of the lectin-type oxidized low density lipoprotein receptor-1 (LOX-1) and interleukin 1β (IL-1β). Techniques used were the slit lamp, immunofluorescence, myeloperoxidase (MPO) detection, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot.
Results:
Nerolidol directly inhibits the growth of A. fumigatus. The administration of nerolidol reduced the severity of fungal keratitis with infiltration of fewer inflammatory cells and reduced levels of the LOX-1, as well the anti-inflammatory cytokines such as IL-1β were reduced compared with the PBS group. Additionally, in vitro studies showed that treatment with nerolidol inhibited the production of the LOX-1 / IL-1β levels in A. fumigatus stimulated HCECs.
Conclusion:
Nerolidol attenuated the A. fumigatus keratitis inflammatory response by inhibiting the growth of A. fumigatus, reducing the recruitment of the neutrophils and the macrophages, and inhibiting the LOX-1/ IL-1β signaling.
Objective:
To investigate the protective effect of quercetin against lipopolysaccharide (LPS)- induced acute kidney injury (AKI) in mice and explore its mechanism.
Methods:
Forty male BALB/c mice were randomly divided into control group (with saline treatment), 15 mg/kg LPS group, and quercetin-treated groups with intragastric quercetin treatment (once daily for 3 consecutive days) at low (25 mg/kg) and high (50 mg/kg) dose prior to 15 mg/kg LPS injection. LPS was administered by intraperitoneally injection 1 after the last gavage of quercetin. The mice were sacrificed 24 h after LPS injection for analysis of kidney pathologies, blood urea nitrogen (BUN) and creatinine levels; serum levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 were detected by ELISA, and the expressions of Toll-like receptor-4 (TLR4), MyD88, TRAF-6 and NF-κBp65 in the kidney were detected by Western blotting.
Results:
Quercetin significantly lessened renal pathologies, lowered BUN and creatinine levels (P < 0.05) and inhibited TNF-α, IL-1β, and IL-6 production in mice with LPS-induced AKI (P < 0.05). Pretreatment with quercetin also significantly inhibited TLR4, MyD88, and TRAF-6 expressions and NF-κBp65 activation in the kidneys of the rats with LPS challenge (P < 0.05).
Conclusions:
Quercetin pretreatment can protect mice against LPSinduced AKI by inhibiting TLR4/NF-κB signaling pathway.
Acute kidney injury (AKI) is a clinical syndrome characterized by a rapid loss of renal function, which may further develop into chronic kidney damage (CKD) or even end-stage renal disease (ESRD). AKI is a global health problem associated with high morbidity and costly treatments, and there is no specific or effective strategy to treat AKI. In recent years, Traditional Chinese Medicine (TCM) has attracted more attention, with lines of evidence showing that application of TCM improved AKI, and the mechanisms of action for some TCMs have been well illustrated. However, reviews summarizing the progress in this field are still lacking. In this paper, we reviewed TCM preparations and TCM monomers in the treatment of AKI over the last 10 years, describing their renal protective effects and mechanisms of action, including alleviating inflammation, programmed cell death, necrosis, and reactive oxygen species. By focusing on the mechanisms of TCMs to improve renal function, we provide effective complementary evidence to promote the development of TCMs to treat AKI. Moreover, we also summarized TCMs with nephrotoxicity, which provides a more comprehensive understanding of TCMs in the treatment of AKI. This review may provide a theoretical basis for the clinical application of TCMs in the future.
Acute kidney injury (AKI) is a clinical syndrome that results in severe tubular damage with high morbidity and mortality. However, there is a lack of effective therapy strategies. Therefore, it is critical to develop effective drugs for AKI. Dexmedetomidine (DEX), a highly selective α2-adrenoreceptor agonist, has neuroprotective, anti-inflammatory and sympatholytic properties. The present study aimed to investigate the effect DEX on attenuating the inflammatory reaction and apoptosis in the kidney tissues of septic mice and to explore its underlying mechanisms. Sepsis-induced AKI mice models were generated via intraperitoneal injection of lipopolysaccaride (LPS). DEX reduced LPS-induced local inflammation and tubular apoptosis, which was aggravated in the pathogenesis of renal dysfunction. Reverse transcription-quantitative polymerase chain reaction and western blot analysis results revealed that the expression of pro-apoptotic genes and inflammatory factors were markedly reduced by DEX pretreatment. Furthermore, the protective role of DEX was markedly inhibited by the α7 nicotinic acetylcholine receptor (nAChR) antagonist α-bungarotoxin. These findings provided novel evidence for the anti-apoptotic and anti-inflammatory effects of DEX in LPS-induced AKI mice through an α7 nAChR-dependent signaling pathway.
Schistosomiasis is a neglected tropical disease that affects hundreds of millions of people worldwide. Since the treatment of this disease currently relies on a single drug, praziquantel, new and safe schistosomicidal agents are urgently required. Nerolidol, a sesquiterpene present in the essential oils of several plants, is found in many foods and was approved by the U.S. Food and Drug Administration. In this study we analysed the in vitro antiparasitic effect of nerolidol on Schistosoma mansoni adult worms. Nerolidol at concentrations of 31.2 and 62.5 μM reduced the worm motor activity and caused the death of all male and female schistosomes, respectively. In addition, confocal laser scanning microscopy revealed morphological alterations on the tegument of worms such as disintegration, sloughing and erosion of the surface, and a correlation between viability and tegumental damage was observed. In conclusion, nerolidol may be a promising lead compound for the development of antischistosomal natural agents.
Acute kidney injury (AKI) is a frequent condition in hospitalised patients undergoing major surgery or the critically ill and is associated with increased mortality. Based on the volume of the published literature addressing this condition, reporting both supporting as well as conflicting molecular evidence, it is apparent that a comprehensive analysis strategy is required to understand and fully delineate molecular events and pathways which can be used to describe disease induction and progression as well as lead to a more targeted approach in intervention therapies.
We used a Systems Biology approach coupled with a de-novo high-resolution proteomic analysis of kidney cortex samples from a mouse model of folic acid-induced AKI (12 animals in total) and show comprehensive mapping of signalling cascades, gene activation events and metabolite interference by mapping high-resolution proteomic datasets onto a de-novo hypothesis-free dataspace. The findings support the involvement of the glutamatergic signalling system in AKI, induced by over-activation of the N-methyl-D-aspartate (NMDA)-receptor leading to apoptosis and necrosis by Ca2+-influx, calpain and caspase activation, and co-occurring reactive oxygen species (ROS) production to DNA fragmentation and NAD-rundown. The specific over-activation of the NMDA receptor may be triggered by the p53-induced protein kinase Dapk1, which is a known non-reversible cell death inducer in a neurological context. The pathway mapping is consistent with the involvement of the Renin-Angiotensin Aldosterone System (RAAS), corticoid and TNFalpha signalling, leading to ROS production and gene activation through NFkappaB, PPARgamma, SMAD and HIF1alpha trans-activation, as well as p53 signalling cascade activation. Key elements of the RAAS-glutamatergic axis were assembled as a novel hypothetical pathway and validated by immunohistochemistry.
This study shows to our knowledge for the first time in a molecular signal transduction pathway map how AKI is induced, progresses through specific signalling cascades that may lead to end-effects such as apoptosis and necrosis by uncoupling of the NMDA receptor. Our results can potentially pave the way for a targeted pharmacological intervention in disease progression or induction.
Reactive oxygen species (ROS) and oxidative stress are thought to play a central role in the etiology of cell dysfunction and tissue damage in sepsis. However, there is limited and controversial evidence from in vivo studies that ROS mediate cell signaling processes that elicit acute inflammatory responses during sepsis. Because NADPH oxidase is one of the main cellular sources of ROS, we investigated the role of this enzyme in lipopolysaccharide (LPS)-induced acute inflammation in vivo, utilizing mice deficient in the gp91phox or p47phox subunits of NADPH oxidase. Age-and body weight-matched C57BL/6J wild-type (WT) and gp91phox−/− and p47phox−/− mice were injected ip with 50 μg LPS or saline vehicle and sacrificed at various time points up to 24 h. We found that LPS-induced acute inflammatory responses in serum and tissues were not significantly diminished in gp91phox−/− and p47phox−/− mice compared to WT mice. Rather, genetic deficiency of NADPH oxidase was associated with enhanced gene expression of inflammatory mediators and increased neutrophil recruitment to lung and heart. Furthermore, no protection from LPS-induced septic death was observed in either knockout strain. Our findings suggest that NADPH oxidase-mediated ROS production and cellular redox signaling do not promote, but instead limit, LPS-induced acute inflammatory responses in vivo.
The pathophysiology of sepsis involves complex cytokine and inflammatory mediator networks, a mechanism to which NF-κB activation is central. Downregulation of endothelial nitric oxide synthase (eNOS) contributes to sepsis-induced endothelial dysfunction. Erythropoietin (EPO) has emerged as a major tissue-protective cytokine in the setting of stress. We investigated the role of EPO in sepsis-related acute kidney injury using a cecal ligation and puncture (CLP) model. Wistar rats were divided into three primary groups: control (sham-operated); CLP; and CLP+EPO. EPO (4,000 IU/kg body wt ip) was administered 24 and 1 h before CLP. Another group of rats received N-nitro-l-arginine methyl ester (l-NAME) simultaneously with EPO administration (CLP+EPO+l-NAME). A fifth group (CLP+EPOtreat) received EPO at 1 and 4 h after CLP. At 48 h postprocedure, CLP+EPO rats presented significantly higher inulin clearance than did CLP and CLP+EPO+l-NAME rats; hematocrit levels, mean arterial pressure, and metabolic balance remained unchanged in the CLP+EPO rats; and inulin clearance was significantly higher in CLP+EPOtreat rats than in CLP rats. At 48 h after CLP, creatinine clearance was significantly higher in the CLP+EPO rats than in the CLP rats. In renal tissue, pre-CLP EPO administration prevented the sepsis-induced increase in macrophage infiltration, as well as preserving eNOS expression, EPO receptor (EpoR) expression, IKK-α activation, NF-κB activation, and inflammatory cytokine levels, thereby increasing survival. We conclude that this protection, which appears to be dependent on EpoR activation and on eNOS expression, is attributable, in part, to inhibition of the inflammatory response via NF-κB downregulation.
The rhizomes of Hedychium coronarium have been used for the treatment of inflammation, skin diseases, headache, and sharp pain due to rheumatism in traditional medicine. From this plant, three new labdane-type diterpenes 1-3, named coronarins G-I as well as seven known 4-10, coronarin D, coronarin D methyl ether, hedyforrestin C, (E)-nerolidol, β-sitosterol, daucosterol, and stigmasterol were isolated. Their chemical structures were elucidated by mass, 1D- and 2D-nuclear magnetic resonance spectroscopy. They were evaluated for inhibitory effects on lipopolysaccharide-stimulated production of pro-inflammatory cytokines in bone marrow-derived dendritic cells. Among of them, compounds 1, 2, and 6 were significant inhibitors of LPS-stimulated TNF-α, IL-6, and IL-12 p40 productions with IC(50) ranging from 0.19±0.11 to 10.38±2.34 μM. The remains of compounds showed inactivity or due to cytotoxicity. These results warrant further studies concerning the potential anti-inflammatory benefits of labdane-type diterpenes from H. coronarium.
To assess renoprotective effects of a blueberry-enriched diet in a rat model of hypertension. Oxidative stress (OS) appears to be involved in the development of hypertension and related renal injury. Pharmacological antioxidants can attenuate hypertension and hypertension-induced renal injury; however, attention has shifted recently to the therapeutic potential of natural products as antioxidants. Blueberries (BB) have among the highest antioxidant capacities of fruits and vegetables.
Male spontaneously hypertensive rats received a BB-enriched diet (2% w/w) or an isocaloric control diet for 6 or 12 weeks or 2 days. Compared to controls, rats fed BB-enriched diet for 6 or 12 weeks exhibited lower blood pressure, improved glomerular filtration rate, and decreased renovascular resistance. As measured by electron paramagnetic resonance spectroscopy, significant decreases in total reactive oxygen species (ROS), peroxynitrite, and superoxide production rates were observed in kidney tissues in rats on long-term dietary treatment, consistent with reduced pathology and improved function. Additionally, measures of antioxidant status improved; specifically, renal glutathione and catalase activities increased markedly. Contrasted to these observations indicating reduced OS in the BB group after long-term feeding, similar measurements made in rats fed the same diet for only 2 days yielded evidence of increased OS; specifically, significant increases in total ROS, peroxynitrite, and superoxide production rates in all tissues (kidney, brain, and liver) assayed in BB-fed rats. These results were evidence of "hormesis" during brief exposure, which dissipated with time as indicated by enhanced levels of catalase in heart and liver of BB group.
Long-term feeding of BB-enriched diet lowered blood pressure, preserved renal hemodynamics, and improved redox status in kidneys of hypertensive rats and concomitantly demonstrated the potential to delay or attenuate development of hypertension-induced renal injury, and these effects appear to be mediated by a short-term hormetic response.
Acute kidney injury (AKI) is a frequent complication in critically ill patients and sepsis is the most common contributing factor. We aimed to determine the risk factors associated with AKI development in patients with septic shock.
Observational cohort study consisted of consecutive adults with septic shock admitted to a medical intensive care unit (ICU) of a tertiary care academic hospital from July 2005 to September 2007. AKI was defined according to RIFLE criteria (urine output and creatinine criteria). Demographic, clinical, and treatment variables were reviewed. Main outcomes measured were AKI occurrence, all-cause hospital mortality, and hospital and ICU length of stay.
Three hundred ninety patients met inclusion criteria, of which 237 (61%) developed AKI. AKI development was independently associated with delay to initiation of adequate antibiotics, intra-abdominal sepsis, blood product transfusion, use of angiotensin-converting enzyme inhibitor/angiotensin-receptor blocker, and body mass index (kg/m²). Higher baseline GFR and successful early goal directed resuscitation were associated with a decreased risk of AKI. Hospital mortality was significantly greater in patients who developed AKI (49 versus 34%).
In a contemporary cohort of patients with septic shock, both patient and health care delivery risk factors seemed to be important for AKI development.
Sepsis is a severe and dysregulated inflammatory response to infection characterized by end-organ dysfunction distant from the primary site of infection. Development of acute kidney injury (AKI) during sepsis increases patient morbidity, predicts higher mortality, has a significant effect on multiple organ functions, is associated with an increased length of stay in the intensive care unit, and hence consumes considerable healthcare resources. When compared with AKI of nonseptic origin, septic AKI is characterized by a distinct pathophysiology and therefore requires a different approach. Despite impressive advances in several fields of medicine, the pathophysiology, diagnostic procedures, and appropriate therapeutic interventions in sepsis are still highly debatable. Numerous immunomodulatory agents showing promise in preclinical studies fail to reduce the overwhelmingly high mortality rate of sepsis and provoke AKI when compared with other critically ill patients. Major impediments to progress in understanding, early diagnosis, and application of appropriate therapeutic modalities in sepsis-induced AKI include limited histopathologic information, few animal models that closely mimic human sepsis, and a relative shortage of specific diagnostic tools. Here we discuss the most recent advances in understanding the fundamental mechanisms of sepsis-induced AKI, characteristics of relevant animal models available, and potential therapies.
Analyses of essential oils obtained from fresh and dried leaves and inflorescences of Piper clausenianum were performed using GC-FID, GC-MS and NMR techniques. Forty compounds were detected for these four oils with the total of identified constituents ranging from 88.7% for the dried inflorescences to 97.7% for the dried leaves. Sesquiterpenes were the main constituents in the volatile fraction from leaves with a high percentage of (E)-nerolidol (up to 83%). However, monoterpenes were identified in greater amount in the inflorescences, with linalool percentages from 50% up. The essential oils from fresh leaves and inflorescences were submitted to anti-parasitic activity against a strain of Leishmania amazonensis. Both samples showed biological activity, but the essential oil from P. claussenianum fresh leaves, which was rich in (E)-nerolidol, showed effective growth inhibition of L. amazonensis due to the high percentage of this metabolite in the mixture.
Morbidity and mortality are markedly elevated in chronic kidney disease (CKD) patients as consequence of cardiovascular risk factors clustering. Non-traditional risk factors such as inflammation are far more prevalent in this population and contribute significantly to atherosclerosis and cardiovascular disease (CVD). CKD results in a chronic, low-grade inflammatory process that becomes evident even in the early stages of the disease. C-reactive protein (CRP) and interleukin-6 (IL-6) are the most extensively studied inflammatory biomarkers in CVD. Circulating levels of both of these factors are elevated in CKD patients and increase with renal function deterioration. In end-stage renal disease (ESRD), elevated CRP levels are a strong predictor of all-cause and cardiovascular mortality. Recent studies showed IL-6 to predict more reliably CVD and mortality in ESRD patients. However, the issue of the ideal inflammatory marker remains open. Several factors are involved in triggering the inflammatory process including patient-related factors, such as underlying disease, comorbidity, oxidative stress, infectious, genetic or immunologic factors and uremia per se, as well as those arising from dialysis treatment itself, mainly membrane and dialysate biocompatibility. This inflammatory state is associated with adverse outcomes, such as malnutrition, anemia and erythropoietin hyporesponsiveness, high rate of CVD, decreased quality of life, as well as increased mortality and hospitalization in CKD patients. There is currently no consensus on how to manage the inflammatory syndrome in this population. However, adequate knowledge of its causes and their potential prevention or treatment may improve poor clinical outcome in CKD patients.
The nuclear factor-kappa B (NF-kappaB) transcription factor plays a critical role in diverse cellular processes associated with proliferation, cell death, development, as well as innate and adaptive immune responses. NF-kappaB is normally sequestered in the cytoplasm by a family of inhibitory proteins known as inhibitors of NF-kappaB (IkappaBs). The signal pathways leading to the liberation and nuclear accumulation of NF-kappaB, which can be activated by a wide variety of stimuli, have been extensively studied in the past two decades. After gaining access to the nucleus, NF-kappaB must be actively regulated to execute its fundamental function as a transcription factor. Recent studies have highlighted the importance of nuclear signaling in the regulation of NF-kappaB transcriptional activity. A non-Rel subunit of NF-kappaB, ribosomal protein S3 (RPS3), and numerous other nuclear regulators of NF-kappaB, including Akirin, Nurr1, SIRT6, and others, have recently been identified, unveiling novel and exciting layers of regulatory specificity for NF-kappaB in the nucleus. Further insights into the nuclear events that govern NF-kappaB function will deepen our understanding of the elegant control of its transcriptional activity and better inform the potential rational design of therapeutics for NF-kappaB-associated diseases.
In renal tissue injury, activation of the transcription factor NF-kappaB has a central role in the induction of proinflammatory gene expression, which are involved in the development of progressive renal inflammatory disease. The function of NF-kappaB during the switch from the inflammatory process toward resolution, however, is largely unknown. Therefore, we assessed the time-dependent activation and function of NF-kappaB in two different models of acute nephritis. Our experiments demonstrate a biphasic activation of NF-kappaB in the anti-Thy-1 model of glomerulonephritis in rats and the LPS-induced nephritis in mice, with a first peak during the induction phase and a second peak during the resolution period. After induction of glomerular immune injury in rats, predominantly NF-kappaB p65/p50 heterodimer complexes are shifted to the nucleus whereas during the resolution phase predominantly p50 homodimers could be demonstrated in the nuclear compartment. In addition, we could demonstrate that p50 protein plays a pivotal role in the resolution of LPS-induced renal inflammation since NF-kappaB p50 knockout mice demonstrate significantly higher chemokine expression, prolonged renal inflammatory cell infiltration with consecutive tissue injury, and reduced survival. In conclusion, our studies indicate that NF-kappaB subunit p50 proteins have critical in vivo functions in immunologically mediated renal disease by downregulating inflammation during the resolution period.
In this study, we found 985 genes that change expression in the cortex and the medulla of the kidney with age. Some of the genes whose transcripts increase in abundance with age are known to be specifically expressed in immune cells, suggesting that immune surveillance or inflammation increases with age. The age-regulated genes show a similar aging profile in the cortex and the medulla, suggesting a common underlying mechanism for aging. Expression profiles of these age-regulated genes mark not only age, but also the relative health and physiology of the kidney in older individuals. Finally, the set of aging-regulated kidney genes suggests specific mechanisms and pathways that may play a role in kidney degeneration with age.
The activity of nerolidol, a sesquiterpene used as a food-flavoring agent and currently under testing as a skin penetration
enhancer for the transdermal delivery of therapeutic drugs, was evaluated against Leishmania species. Nerolidol inhibited the growth of Leishmania amazonensis, L. braziliensis, and L. chagasi promastigotes and L. amazonensis amastigotes with in vitro 50% inhibitory concentrations of 85, 74, 75, and 67 μM, respectively. The treatment of L. amazonensis-infected macrophages with 100 μM nerolidol resulted in 95% reduction in infection rates. Inhibition of isoprenoid biosynthesis,
as shown by reduced incorporation of [2-14C]mevalonic acid (MVA) or [1-14C]acetic acid precursors into dolichol, ergosterol, and ubiquinone, was observed in nerolidol-treated promastigotes. This
drug effect can be attributed to the blockage of an early step in the mevalonate pathway, since incorporation of the precursor
[1(n)-3H]farnesyl pyrophosphate in polyisoprenoids is not inhibited by nerolidol. L. amazonensis-infected BALB/c mice were treated with intraperitoneal doses of 100 mg/kg/day for 12 days or topically with 5 or 10% ointments
for 4 weeks. Significant reduction of lesion sizes in nerolidol treated mice was observed for both treatment routes. However,
long-term follow up indicated that the disease was not cured in this highly susceptible animal model. Nonetheless, the in
vitro activity of nerolidol against these parasites may prove a useful tool for the development of new drugs for the treatment
of leishmaniasis. In addition, biosynthesis of dolichols with 11 and 12 isoprene units was identified in Leishmania, as described for other trypanosomatids and Apicomplexa.
Although acute renal failure (ARF) is believed to be common in the setting of critical illness and is associated with a high risk of death, little is known about its epidemiology and outcome or how these vary in different regions of the world.
To determine the period prevalence of ARF in intensive care unit (ICU) patients in multiple countries; to characterize differences in etiology, illness severity, and clinical practice; and to determine the impact of these differences on patient outcomes.
Prospective observational study of ICU patients who either were treated with renal replacement therapy (RRT) or fulfilled at least 1 of the predefined criteria for ARF from September 2000 to December 2001 at 54 hospitals in 23 countries.
Occurrence of ARF, factors contributing to etiology, illness severity, treatment, need for renal support after hospital discharge, and hospital mortality.
Of 29 269 critically ill patients admitted during the study period, 1738 (5.7%; 95% confidence interval [CI], 5.5%-6.0%) had ARF during their ICU stay, including 1260 who were treated with RRT. The most common contributing factor to ARF was septic shock (47.5%; 95% CI, 45.2%-49.5%). Approximately 30% of patients had preadmission renal dysfunction. Overall hospital mortality was 60.3% (95% CI, 58.0%-62.6%). Dialysis dependence at hospital discharge was 13.8% (95% CI, 11.2%-16.3%) for survivors. Independent risk factors for hospital mortality included use of vasopressors (odds ratio [OR], 1.95; 95% CI, 1.50-2.55; P<.001), mechanical ventilation (OR, 2.11; 95% CI, 1.58-2.82; P<.001), septic shock (OR, 1.36; 95% CI, 1.03-1.79; P = .03), cardiogenic shock (OR, 1.41; 95% CI, 1.05-1.90; P = .02), and hepatorenal syndrome (OR, 1.87; 95% CI, 1.07-3.28; P = .03).
In this multinational study, the period prevalence of ARF requiring RRT in the ICU was between 5% and 6% and was associated with a high hospital mortality rate.
Acute kidney injury (AKI) complicates about 3–50% of hospital admissions [1] depending on patient comorbidities and on medical procedures performed during the stay. The greatest incidence of AKI is observed in the intensive care unit (ICU): indeed, critically ill patients with hospital-acquired AKI have a greater in-hospital mortality (30–70%) and a more than double risk of severe adverse outcomes even 5 years after discharge, including an increased incidence of end-stage chronic kidney disease (CKD), than patients without AKI [2]. The most common cause of inhospital AKI is sepsis, the systemic inflammatory response to infection that is often complicated by multiple organ failure and death [3]. In recent years, experimental and clinical studies have provided new insights into the pathogenic mechanisms of sepsis-associated AKI, also explaining how the loss of renal function may impair the immune system and lead to the subsequent development of sepsis. In this chapter, we review the recent findings on the detrimental cross-talk between sepsis and AKI and the identification of new, early biomarkers and targeted therapies aimed at improving the outcome of these critically ill patients.
Nerolidol, an acyclic sesquiterpene found as a major constituent of several essential oils, has several pharmacological activities, but its action in pain processes has never been studied. The purpose of our research was to evaluate the antinociceptive and anti-inflammatory activities of nerolidol, as well as possible mechanisms of action, in experimental mouse models of pain. Antinociceptive activity was evaluated using the acetic acid-induced writhing test, the formalin test, and the hot-plate test. The nerolidol-treated group showed lesser acetic acid-induced abdominal contractions than the control group in all of the three doses tested (200, 300, and 400 mg/kg, p.o.). The formalin test doses of 300 and 400 mg/kg p.o. inhibited licking time, in both the first phase and the second phase. In the hot-plate test, nerolidol did not alter latency at any of the observed time points. Motor coordination, evaluated through the rotarod test, was not hindered in animals treated with nerolidol. Regarding the mechanism of action, the antinociceptive activity of nerolidol is related to the GABAergic system, and not to the opioidergic or ATP-sensitive K+ channels. Treatment with nerolidol reduced carrageenan-induced paw edema. In the model of carrageenan-induced peritonitis, nerolidol decreased the influx of polymorphonuclear cells and also reduced levels of tumor necrosis factor (TNF-α) in peritoneal lavage. Nerolidol reduced production of interleukin 1 beta (IL-1β) in LPS-stimulated, peritoneal macrophages. Thus, these results showed that nerolidol has antinociceptive activity with possible involvement of the GABAergic system, and anti-inflammatory activity, attributed to the suppression of TNF-α and IL-1β proinflammatory cytokines. © 2015 Société Française de Pharmacologie et de Thérapeutique.
Ethnopharmacological relevance:
Ribes diacanthum Pall. (Saxifragaceae), a Mongolian folk medicinal plant, was used to treat urinary system diseases. The present work aims to investigate the protective effects of Ribes diacanthum Pall (RDP) against cisplatin-induced nephrotoxicity.
Methods:
The renal injury was modeled by intraperitoneal injection of cisplatin for 5 consecutive days (5mg/kg). Nephroprotection of RDP was investigated by oral administration of RDP aqueous extract at a daily dose of 40mg/kg for 14 consecutive days, starting 7 days prior to cisplatin administration.
Results:
We demonstrated that pretreatment with RDP aqueous extract protected the mice from death induced by cisplatin administration. RDP treatment also significantly reduced blood urea nitrogen (BUN) and serum creatinine (Cr) levels observed in cisplatin-administrated mice. Histopathological analysis demonstrated that RDP administration protected cisplatin-induced renal tubular cell apoptosis. Further western blotting analysis revealed that RDP significantly reversed cisplatin-increased expression levels of cleaved-Caspase-3, Bax and cisplatin-decreased expression level of Bcl-2 in renal tissue. Finally, RDP markedly enhanced enzyme activities of reduced superoxide dismutase (SOD), Heme oxygenase 1 (HO-1) and catalase (CAT), suppressed lipid peroxidation as well as reactive oxygen species (ROS) production. Conclusion We concluded that RDP displayed nephroprotective effects against cisplatin-induced renal tubular cell apoptosis, possibly associated with both enhanced antioxidase activity and suppressed ROS generation. Given the major nephrotoxicity of cisplatin cancer chemotherapy, RDP might be a potential candidate for neoadjuvant chemotherapy.
The analgesic and anti-inflammatory properties of Citrus aurantium L. blossoms essential oil (neroli) were investigated in mice and rats. The analgesic activity of neroli was assessed by acetic acid-induced writhing and Eddy's hot plate methods, while acute and chronic anti-inflammatory effects were investigated by inflammatory paw edema in rat and the cotton pellet-induced granuloma tissue model, respectively. Mechanistic studies were conducted using L-nitro arginine methyl ester (L-NAME), an inhibitor of NO synthase. Neroli significantly decreased the number of acetic acid-induced writhes in mice compared to animals that received vehicle only. Also, it exhibited a central analgesic effect, as evidenced by a significant increase in reaction time in the hot plate method. The oil also significantly reduced carrageenan-induced paw edema in rats. The inhibitory activity of neroli (especially at 40 mg/kg) was found to be very close to the standard drug, diclofenac sodium (50 mg/kg). In cotton pellet-induced granuloma, neroli was effective regarding the transudate and granuloma formation amount. Neroli was analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) and twenty-three constituents, representing 91.0 % of the oil, were identified. The major components of neroli were characterized as linalool (28.5 %), linalyl acetate (19.6 %), nerolidol (9.1 %), E,E-farnesol (9.1 %), α-terpineol (4.9 %), and limonene (4.6 %), which might be responsible for these observed activities. The results suggest that neroli possesses biologically active constituent(s) that have significant activity against acute and especially chronic inflammation, and have central and peripheral antinociceptive effects which support the ethnomedicinal claims of the use of the plant in the management of pain and inflammation.
Objective
To optimize the extraction yields of essential oil from Fagraea fragrans Roxb. flowers in hydro-distillation using a central composite design (CCD) and to evaluate its biological activities for perfumery and cosmetic applications.MethodsCCD was applied to study the influences of operational parameters [water to flower weight (X1) and distillation time (X2)] on the yields of essential oil (Y). Chemical compositions of the essential oil extracted from the optimized condition were identified by gas chromatography-mass spectrometry. Antioxidant activities of the essential oil were determined against ABTS•+ and DPPH• radicals and the cytotoxic effects were assessed on human embryonic kidney (HEK293) cells by the use of the MTT assay. Also, the aromatic properties of the essential oil were evaluated by 5 healthy trained volunteers.ResultsThe best conditions to obtain the maximum essential oil yield were 7.5 L g-1 (X1) and 215 min (X2). The experimental yield of the essential oil (0.35±0.02% v/w) was close to the value predicted by a mathematical model (0.35±0.01% v/w). 3-Octadecyne, Z,Z,Z-7,10,13-hexadecatrienal, E-nerolidol, pentadecanal, and linalool were the major constituents of the essential oil. The essential oil showed moderate antioxidant capacities with no toxic effects on HEK293 cells at 1-250 μg mL-1. Also, the essential oil exhibited a very strong aroma and was classified to be top to middle notes.Conclusion
The results offer the effectively operational conditions in the extraction of essential oil from F. fragrans using hydro-distillation. The essential oil could be used as a natural fragrance, having antioxidant activity with slight cytotoxicity, for perfumery and cosmetic applications.This article is protected by copyright. All rights reserved.
Approximately 18% of patients undergoing cardiac surgery experience AKI (on the basis of modern standardized definitions of AKI), and approximately 2%-6% will require hemodialysis. The development of AKI after cardiac surgery portends poor short- and long-term prognoses, with those developing RIFLE failure or AKI Network stage III having an almost 2-fold increase in the risk of death. AKI is caused by a variety of factors, including nephrotoxins, hypoxia, mechanical trauma, inflammation, cardiopulmonary bypass, and hemodynamic instability, and it may be affected by the clinician's choice of fluids and vasoactive agents as well as the transfusion strategy used. The risk of AKI may be ameliorated by avoidance of nephrotoxins, achievement of adequate glucose control preoperatively, and use of goal-directed therapy hemodynamic strategies. Remote ischemic preconditioning is an exciting future strategy, but more work is needed before widespread implementation. Unfortunately, there are no pharmacologic agents known to reduce the risk of AKI or treat established AKI.
Acute kidney injury (AKI) is an abrupt loss of kidney function. Severe AKI requiring renal replacement therapy and has high mortality. Leonurine (LEO), an alkaloid isolated from Leonurus cardiaca, has shown biological effects such as antioxidant, anticoagulant, anti-apoptosis. We have examined the effect of LEO on lipopolysaccharide (LPS) - induced AKI in mice and further studied the mechanism involved. Blood urea nitrogen (BUN), creatinine and cytokine were estimated in the serum or tissue. Kidney tissue specimens were used for biochemical estimations of lipid peroxides (LPO), reduced glutathione (GSH), reactive oxygen species (ROS). The effects of LEO on LPS-induced renal tissue damage were detected by Hematoxylin and eosin (HE) stain and electron microscopy. The production of cytokines in tissue and blood were measured by Elisa. Protein phosphorylation and protein subcellular localization were tested by Western blot. LEO protected against LPS-induced AKI, improved animal survival and maintained the redox balance. The beneficial effects of LEO were accompanied by the down-regulation of TNF-α, IL-1, IL-6, IL-8, KIM-1 expression and inhibition the phosphorylation of IκBα and p65 translocalization. These results suggest that LEO may suppress NF-κB activation and inhibit pro-inflammatory cytokine production via decreasing cellular ROS production. Accumulating studies have demonstrated that LEO reduces kidney injury and protects renal functions from LPS-induced kidney injury.
Protein overload activates proximal tubule epithelial cells (PTECs) to release chemokines. Bone morphogenetic protein-7 (BMP-7) reduces infiltrating cells and tissue damage in acute and chronic renal injuries. The present study examines the inhibitory effect and related molecular mechanism of BMP-7 on chemokine and adhesion molecule synthesis by PTECs activated with human serum albumin (HSA). The expression profiles of chemokines and adhesion molecules in cultured human PTECs were screened by PCR array. Expression of CXCL1, CXCL2 and vascular cell adhesion protein 1 (VCAM-1) by PTECs was significantly upregulated by HSA and reduced by BMP-7. HSA activated both the canonical and noncanonical nuclear factor (NF)-κB pathways in PTECs, as indicated by the increased nuclear translocation of NF-κB p50 and p52 subunits. The nuclear translocation of NF-κB p52 was completely abrogated by BMP-7, whereas NF-κB p50 activation was only partially repressed. BMP-7 increased the expression of cellular inhibitor of apoptosis 1 (cIAP1), tumor necrosis factor receptor-associated factor (TRAF)2 and TRAF3, but not of NF-κB-inducing kinase (NIK) that was significantly upregulated by HSA. Silencing NIK recapitulated the partial inhibitory effect on HSA-induced chemokine synthesis by BMP-7. Complete abolishment of the chemokine synthesis was only achieved by including additional blockade of the NF-κB p65 translocation on top of NIK silencing. Our data suggest that BMP-7 represses the NIK-dependent chemokine synthesis in PTECs activated with HSA through blocking the noncanonical NF-κB pathway and partially interfering with the canonical NF-κB pathway.Immunology and Cell Biology advance online publication, 14 January 2014; doi:10.1038/icb.2013.106.
OBJECTIVE:
Acute kidney injury in the critically ill is an independent risk factor for adverse outcome. The magnitude of the impact of acute kidney injury on outcome, however, is still unclear. This study aimed to estimate the excess mortality attributable to acute kidney injury.
DESIGN:
We performed a sequentially matched analysis according to the day of acute kidney injury diagnosis after ICU admission. Patients with acute kidney injury and those without acute kidney injury were matched according to age, sex, ICU admission diagnosis, Simplified Acute Physiology Score II without renal and age components, and the propensity to develop acute kidney injury at each of the four matching time points.
SETTING:
Cohort of 16 participating ICUs from the prospective Finnish Acute Kidney Injury study.
PATIENTS:
Cohort of 2,719 consecutive patients with either emergency admission or elective postsurgical patients with an expected ICU stay greater than 24 hours.
INTERVENTIONS:
None.
MEASUREMENTS AND MAIN RESULTS:
Of the 2,719 patients included in the study, acute kidney injury developed in 1,081 patients (39.8%) according to the Kidney Disease: Improving Global Outcomes-definition during ICU treatment on days 1-5. Of these, 477 patients were successfully matched to 477 patients who did not develop acute kidney injury. The 90-day mortality of the matched patients with acute kidney injury was 125 of 477 (26.2%) compared with 84 of 477 (17.6%) for their matched controls without acute kidney injury. Thus, the absolute excess 90-day mortality attributable to acute kidney injury was estimated at 8.6 percentage points (95% CI, 2.6-17.6 percentage points). The population attributable risk (95% CI) of 90-day mortality associated with acute kidney injury was 19.6% (10.3-34.1%).
CONCLUSIONS:
In general ICU patients, the absolute excess 90-day mortality statistically attributable to acute kidney injury is substantial (8.6%), and the population attributable risk was nearly 20%. Our findings are useful in planning suitably powered future clinical trials to prevent and treat acute kidney injury in critically ill patients.
Sickness behaviours are host defence adaptations that arise from integrated autonomic outputs in response to activation of the innate immune system. These behaviours include fever, anorexia, and hyperalgesia intended to promote survival of the host when encountering pathogens. Cannabinoid (CB) receptor activation can induce hypothermia and attenuate lipopolysaccharide (LPS)-evoked fever. The aim of the present study was to examine the role of CB1 receptors in the LPS-evoked febrile response. CB1 receptor-deficient (CB1(-/-)) mice did not display LPS-evoked fever; likewise pharmacological blockade of CB1 receptors in wild-type mice blocked LPS-evoked fever. This unresponsiveness is not limited to thermogenesis, as the animals were not hyperalgesic after LPS administration. A toll-like receptor (TLR)3 agonist and viral mimetic polyinosinic:polycytidylic acid evoked a robust fever in CB1(-/-) mice suggesting TLR3-mediated responses are functional. LPS-evoked c-Fos activation in areas of the brain associated with the febrile response was evident in wild-type mice but not in CB1(-/-) mice. Liver and spleen TLR4 mRNA were significantly lower in CB1(-/-) mice compared to wild-type, and peritoneal macrophages from CB1(-/-) mice did not release pro-inflammatory cytokines in response to LPS. These data indicate that CB1 receptors play a critical role in LPS-induced febrile responses through inhibiting TLR4-mediated cytokine production.
World Kidney Day will be celebrated on March 14 2013, and this year, aims to increase awareness of the global increase in acute kidney injury (AKI). An urgent need exists for a global health strategy to reduce the burden of AKI; efforts focused on preventing AKI should be coupled with early detection, treatment, and follow-up strategies.
Acute kidney injury (AKI) is a common and serious problem affecting millions and causing death and disability for many. In 2012, Kidney Disease: Improving Global Outcomes completed the first ever international multidisciplinary clinical practice guideline for AKI. The guideline is based on evidence review and appraisal, and covers AKI definition, risk assessment, evaluation, prevention, and treatment. Two topics, contrast-induced AKI and management of renal replacement therapy, deserve special attention because of the frequency in which they are encountered and the availability of evidence. Recommendations are based on systematic reviews of relevant trials. Appraisal of the quality of the evidence and the strength of recommendations followed the Grading of Recommendations Assessment, Development and Evaluation approach. Limitations of the evidence are discussed and a detailed rationale for each recommendation is provided. This review is an abridged version of the guideline and provides additional rationale and commentary for those recommendation statements that most directly impact the practice of critical care.