Expression Of Catabolic Factors Research Articles

Schisandrol A Suppresses Catabolic Factor Expression by Blocking NF-κB Signaling in Osteoarthritis.

Schisandrol A possesses pharmacological properties and is used to treat various diseases; however, its effects on osteoarthritis (OA) progression remain unclear. Here, we investigated Schisandrol A as a potential therapeutic agent for OA. In vitro, Schisandrol A effects were confirmed based on the levels of expression of catabolic factors (MMPs, ADAMTS5, and Cox2) induced by IL-1β or Schisandrol A treatment in chondrocytes. In vivo, experimental OA in mice was induced using a destabilized medial meniscus (DMM) surgical model or oral gavage of Schisandrol A in a dose-dependent manner, and demonstrated using histological analysis. In vitro and in vivo analyses demonstrated that Schisandrol A inhibition attenuated osteoarthritic cartilage destruction via the regulation of Mmp3, Mmp13, Adamts5, and Cox2 expression. In the NF-κB signaling pathway, Schisandrol A suppressed the degradation of IκB and the phosphorylation of p65 induced by IL-1β. Overall, and Schisandrol A reduced the expression of catabolic factors by blocking NF-κB signaling and prevented cartilage destruction. Therefore, Schisandrol A attenuated OA progression, and can be used to develop novel OA drug therapies.

Therapeutic Single Compounds for Osteoarthritis Treatment

Osteoarthritis (OA) is an age-related degenerative disease for which an effective disease-modifying therapy is not available. Natural compounds derived from plants have been traditionally used in the clinic to treat OA. Over the years, many studies have explored the treatment of OA using natural extracts. Although various active natural extracts with broad application prospects have been discovered, single compounds are more important for clinical trials than total natural extracts. Moreover, although natural extracts exhibit minimal safety issues, the cytotoxicity and function of all single compounds in a total extract remain unclear. Therefore, understanding single compounds with the ability to inhibit catabolic factor expression is essential for developing therapeutic agents for OA. This review describes effective single compounds recently obtained from natural extracts and the possibility of developing therapeutic agents against OA using these compounds.

Antarctic Krill Oil Ameliorates Monosodium Iodoacetate-Induced Irregularities in Articular Cartilage and Inflammatory Response in the Rat Models of Osteoarthritis.

The aim of this study was to examine the effects of Antarctic krill oil (FJH-KO) in a rat model of monosodium iodoacetate (MIA) induced osteoarthritis. The effect of FJH-KO on the development and severity of MIA-induced osteoarthritis was assessed using hematoxylin and eosin (H&E) staining and micro-CT. The expression of PGE2, pro-inflammatory cytokines (IL-1β, TNF-α), and arthritics related genes in osteoarthritic rats in response to FJH-KO supplementation was investigated using real time PCR. FJH-KO supplementation in the arthritic rat model reduced tissue damage, cartilage degeneration, and reduced the MIA-induced irregularities in articular cartilage surface. Serum PGE2, IL-1β, IL-6, and TNF-α levels were higher in MIA treated animals, but these levels decreased upon FJH-KO supplementation. When FJH-KO was provided at a dose of 150 mg/kg b.w to MIA-treated animals, it significantly increased the mRNA expression of anabolic factors. The mRNA expression of catabolic factors was significantly decreased MIA-treated animals that were provided FJH-KO at a dose of 100 and 150 mg/kg b.w. Moreover, the mRNA expression of inflammatory mediators was significantly decreased MIA-treated animals supplemented with FJH-KO. These results suggest supplementation with FJH-KO ameliorates the irregularities in articular cartilage surface and improves the inflammatory response in the osteoarthritis. Thus, FJH-KO could serve as a potential therapeutic agent for osteoarthritis treatment.

Interleukin-1-Interleukin-17 Signaling Axis Induces Cartilage Destruction and Promotes Experimental Osteoarthritis.

Osteoarthritis (OA), which is the most common degenerative joint disorder, has been considered a non-inflammatory disease with abnormal mechanics. Interleukin (IL)-17 is a pleiotropic cytokine involved in inflammatory diseases and their production is driven by the cytokine including IL-1 and IL-23. However, little is known about the mechanism of IL-17 in the development of OA. Here, we investigated the role of IL-17 in the pathogenesis of OA using monosodium iodoacetate (MIA)-injected IL-17 and IL-1 receptor antagonist (IL-1Ra) double-deficient mice. In MIA-injected IL-1Ra KO mice, nociceptive properties, degree of cartilage damage, and the level of inflammatory factors in articular cartilage were increased compared to MIA-injected wild-type mice. Interestingly, the intestinal architecture was impaired in IL-1Ra KO mice compared to wild-type mice and the damage was further exacerbated by MIA injection. Deficiency of IL-17 reduced nociceptive properties and cartilage destruction, as well as inflammation-related factors in MIA-injected IL-1Ra KO mice compared to MIA-injected wild-type mice. Furthermore, IL-17-treated chondrocytes from OA patients showed enhanced expression of catabolic factors that are involved in the destruction of cartilage in OA. IL-17 accelerates the destruction of cartilage and small intestine via regulation of several inflammatory mediators in an OA murine model. These results suggest that IL-17 plays a critical role in the development of OA.

Hypoxia-inducible factor-1 alpha maintains mouse articular cartilage through suppression of NF-\u03baB signaling

HIF-1α, an essential transcription factor under hypoxic condition, is indispensable for chondrocytes during skeletal development but its expression and roles in articular chondrocytes are yet to be revealed. We examined HIF-1α protein expression and the hypoxic condition during mouse osteoarthritis (OA) development using state of the art hypoxic probes and found that its expression decreased as OA progressed, coinciding with the change in hypoxic conditions in articular cartilage. Gain- and loss-of-function of HIF-1α in cell culture experiments showed that HIF-1α suppressed catabolic genes such as Mmp13 and Hif2a. We confirmed these anticatabolic effects by measuring glycosaminoglycan release from wild type and conditional knock-out mice femoral heads cultured ex vivo. We went on to surgically induce OA in mice with chondrocyte-specific deletion of Hif1a and found that the development of OA was exacerbated. Increased expression of catabolic factors and activation of NF-κB signalling was clearly evident in the knock-out mice. By microarray analysis, C1qtnf3 was identified as a downstream molecule of HIF-1α, and experiments showed it exerted anti-catabolic effects through suppression of NF-κB. We conclude that HIF-1α has an anti-catabolic function in the maintenance of articular cartilage through suppression of NF-κB signalling.

Chondroprotective Effect of Cynaroside in IL-1β-Induced Primary Rat Chondrocytes and Organ Explants via NF-κB and MAPK Signaling Inhibition.

Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage degradation and inflammation. Interleukin-1β is the key player in the pathogenesis of OA, which induces the expression of various catabolic factors that contribute to cartilage degradation. Cynaroside (luteolin-7-O-glucoside or luteoloside) is a flavonoid that has various pharmacological properties, such as antitumor, anti-inflammatory, and antioxidant activities. In this study, we investigated the chondroprotective effects of cynaroside on IL-1β-stimulated chondrocytes and organ explants. The production of nitrite, PGE2, collagen type II, and aggrecan was measured by a Griess reagent and ELISAs, and the production of ROS was measured by H2DCF-DA fluorescence. The protein levels of iNOS, Cox-2, MMP-1, MMP-3, MMP-13, ADAMTS-4, MAPKs, and the NF-κB p65 subunit were measured by western blot. Proteoglycan analysis was performed by Alcian Blue staining (in vitro) and Safranin O staining (ex vivo). Cynaroside inhibited IL-1β-induced expression of catabolic factors (nitrite, iNOS, ROS, PGE2, Cox-2, MMP-1, MMP-3, MMP-13, and ADAMTS-4) and degradation of anabolic factors (collagen type II and aggrecan). Furthermore, cynaroside suppressed IL-1β-induced phosphorylation of MAPKs and translocation of the NF-κB p65 subunit into the nucleus. Collectively, these results suggest that cynaroside may be a potential candidate for the development of new therapeutic drugs for the alleviation of OA progression.

Pulsed electromagnetic fields reduce acute inflammation in the injured rat-tail intervertebral disc.

Pro‐inflammatory cytokines are recognized contributors to intervertebral disc (IVD) degeneration and discogenic pain. We have recently reported the anti‐inflammatory effect of pulsed electromagnetic fields (PEMF) on IVD cells in vitro. Whether these potentially therapeutic effects are sufficiently potent to influence disc health in vivo has not been demonstrated. We report here the effect of PEMF on acute inflammation arising from a rat‐tail IVD injury model. Disc degeneration was induced by percutaneously stabbing the Co6‐7, Co7‐8, and Co8‐9 levels using a 20‐gauge needle. Seventy‐two (72) rats were divided into three groups: sham control, needle stab, needle stab+PEMF. Treated rats were exposed to PEMF immediately following surgery and for either 4 or 7 days (4 hr/d). Stab and PEMF effects were evaluated by measuring inflammatory cytokine gene expression (RT‐PCR) and protein levels (ELISA assay), anabolic and catabolic gene expression (RT‐PCR), and histologic changes. We observed in untreated animals that at day 7 after injury, inflammatory cytokines (interleukin [IL]‐6, tumor necrosis factor α, and IL‐1β) were significantly increased at both gene and protein levels (P < .05). Similarly, catabolic factors (MMP [metalloproteinases]‐2, MMP‐13 and the transcriptional factor NF‐kβ gene expression) were significantly increased (P < .05). At day 7, PEMF treatment significantly inhibited inflammatory cytokine gene and protein expression induced by needle stab injury (P < .05). At day 4, PEMF downregulated FGF‐1 and upregulated MMP‐2 compared to the stab‐only group. These data demonstrate that previously reported anti‐inflammatory effects of PEMF on disc cells carry over to the in vivo situation, suggesting potential therapeutic benefits. Though we observed an inhibitory effect of PEMF on acute inflammatory cytokine expression, a consistent effect was not observed for acute changes in disc histology and anabolic and catabolic factor expression. Therefore, these findings should be further investigated in studies of longer duration following needle‐stab injury.

Depletion of glycosphingolipids induces excessive response of chondrocytes under mechanical stress

Glycosphingolipids (GSLs) are ubiquitous membrane components that play an indispensable role in maintaining chondrocyte homeostasis. To gain better insight into roles of GSLs, we studied the effects of GSL-deletion on the physiological responses of chondrocytes to mechanical stress. Mice lacking Ugcg gene (Ugcg-/-) were genetically generated to obtain GSL-deficient mice, and their chondrocytes from the joints were used for functional analyses in vitro culture experiments. The cells were seeded in a three-dimensional collagen gel and subjected to 5%, 10% or 16% cyclic tensile strain for either 3 or 24 h. The gene expressions of chondrocyte anabolic and catabolic factors, and the induction of Ca2+ signaling were analyzed. Our results revealed that chondrocytes derived from GSL-deficient mice exhibited an elevation in the expression of catabolic factors (ADAMTS-5, MMP-13) following the exposure to strain with amplitudes of 10%. Likewise, applying cyclic tensile strain with these amplitudes resulted in an increased Ca2+ oscillation ratio in chondrocytes from GSL-deficient as compared to the ratio from control mice. These results demonstrated that deletion of GSL stimulated the catabolic responses of chondrocytes to mechanical stress via the augmentation of the sensitivity to mechanical stress that may lead to the cartilage deterioration. These findings suggest that the regulation of the physiological responses of chondrocytes by GSLs could be a potential target in a therapeutic intervention in osteoarthritis.

Elevated expression of hypoxia-inducible factor-2alpha regulated catabolic factors during intervertebral disc degeneration

Headings aimsThe present study determined whether nucleus pulposus (NP) cells express hypoxia-inducible factor-2alpha (HIF-2α) and assessed its role in regulating the expression of catabolic factors during intervertebral disc degeneration. Materials and methodsHuman degenerated NP tissues were acquired to examine the HIF-2α expression levels using immunohistochemistry, western blotting, and Real-time PCR. Human NP cells were cultivated under normoxic or hypoxic conditions, and the HIF-2α expression was determined. Then, human NP cells were treated with HIF-2α plasmids, HIF-2α siRNA, and tumor necrosis factor-alpha (TNF-α) to evaluate the role of HIF-2α in regulating matrix metalloproteinase (MMP) and aggrecanase expression. An in vivo rabbit disc degeneration model was established to demonstrate that HIF-2α plays a critical role in disc degeneration. Key findingsWe found that HIF-2α had a markedly elevated expression in human degenerated discs in the Grade III stage. HIF-2α protein and gene transcript levels in vitro were relatively higher under hypoxic conditions. The expression of MMP-13, ADAMTS-4 was decreased significantly in HIF-2α silencing condition, while the over-expression resulted in significantly increased levels of MMP-13 and ADAMTS-4. When cytokine TNF-α was added, HIF-2α was induced by nuclear factor-κB (NF-κB). The in vivo experiments showed that the HIF-2α controlled the catabolic factors MMP-13 and ADAMTS-4 that regulated the collagen II and aggrecan metabolism in disc degeneration. SignificanceHIF-2α is a catabolic regulator in disc degeneration and directly controls the catabolic genes. The suppression of HIF-2α expression leads to decelerates extracellular matrix degradation that might represent a therapeutic target for the degenerative disc.

Induction of pro-inflammatory cytokines by 29-kDa FN-f via cGAS/STING pathway

The cGAS-STING pathway plays an important role in pathogen-induced activation of the innate immune response. The 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) found predominantly in the synovial fluid of osteoarthritis (OA) patients increases the expression of catabolic factors via the toll-like receptor-2 (TLR-2) signaling pathway. In this study, we investigated whether 29-kDa FN-f induces inflammatory responses via the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon gene (STING) pathway in human primary chondrocytes. The levels of cGAS and STING were elevated in OA cartilage compared with normal cartilage. Long-term treatment of chondrocytes with 29-kDa FN-f activated the cGAS/STING pathway together with the increased level of gamma-H2AX, a marker of DNA breaks. In addition, the expression of pro-inflammatory cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF/CSF-2), granulocyte colony-stimulating factor (G-CSF/CSF-3), and type I interferon (IFN-α), was increased more than 100-fold in 29-kDa FN-f-treated chondrocytes. However, knockdown of cGAS and STING suppressed 29-kDa FN-f-induced expression of GM-CSF, G-CSF, and IFN-α together with the decreased activation of TANK-binding kinase 1 (TBK1), interferon regulatory factor 3 (IRF3), and inhibitor protein κBα (IκBα). Furthermore, NOD2 or TLR-2 knockdown suppressed the expression of GM-CSF, G-CSF, and IFN-α as well as decreased the activation of the cGAS/STING pathway in 29-kDa FN-f-treated chondrocytes. These data demonstrate that the cGAS/STING/TBK1/IRF3 pathway plays a critical role in 29-kDa FN-f-induced expression of pro-inflammatory cytokines.

Cirsium japonicum var. maackii and apigenin block Hif-2α-induced osteoarthritic cartilage destruction.

Although Hif‐2α is a master regulator of catabolic factor expression in osteoarthritis development, Hif‐2α inhibitors remain undeveloped. The aim of this study was to determine whether Cirsium japonicum var. maackii (CJM) extract and one of its constituents, apigenin, could attenuate the Hif‐2α‐induced cartilage destruction implicated in osteoarthritis progression. In vitro and in vivo studies demonstrated that CJM reduced the IL‐1β‐, IL‐6, IL‐17‐ and TNF‐α‐induced up‐regulation of MMP3, MMP13, ADAMTS4, ADAMTS5 and COX‐2 and blocked osteoarthritis development in a destabilization of the medial meniscus mouse model. Activation of Hif‐2α, which directly up‐regulates MMP3, MMP13, ADAMTS4, IL‐6 and COX‐2 expression, is inhibited by CJM extract. Although cirsimarin, cirsimaritin and apigenin are components of CJM and can reduce inflammation, only apigenin effectively reduced Hif‐2α expression and inhibited Hif‐2α‐induced MMP3, MMP13, ADAMTS4, IL‐6 and COX‐2 expression in articular chondrocytes. IL‐1β induction of JNK phosphorylation and IκB degradation, representing a critical pathway for Hif‐2α expression, was completely blocked by apigenin in a concentration‐dependent manner. Collectively, these effects indicate that CJM and one of its most potent constituents, apigenin, can lead to the development of therapeutic agents for blocking osteoarthritis development as novel Hif‐2α inhibitors.

Expression of the RANK/RANKL/OPG system in the human intervertebral disc: implication for the pathogenesis of intervertebral disc degeneration

BackgroundThe expression of the receptor activator of nuclear factor kappa B (RANK) /RANK ligand (RANKL) /osteoprotegerin (OPG) system and its association with the progression of intervertebral disc (IVD) degeneration has recently been reported in a human IVD. However, the effect of the RANK/RANKL/OPG system on the matrix metabolism of human IVD cells, especially on the expression of catabolic factors relevant to IVD degeneration, remains unknown. The purpose of this study was to examine the expression of the RANK/RANKL/OPG system, and then to evaluate the effect of this system on the expression of catabolic factors by human IVD cells.MethodsAnnulus fibrosus (AF) and nucleus pulposus (NP) cells isolated by sequential enzyme digestion from human IVD tissues obtained during spine surgeries were monolayer cultured. The expression of the RANK/RANKL/OPG system was determined using immunohistochemical methods and real-time polymerase chain reaction (PCR). To evaluate the influence of interleukin-1 beta (IL-1β) stimulation on the mRNA expression of RANK, RANKL, and OPG, recombinant human IL-1β (rhIL-1β) was administered in the culture media of IVD cells. To examine the influence of RANKL signaling on the expression of matrix metalloprotease-3 (MMP-3), MMP-13, and IL-1β, the cells were cultured with exogenous recombinant human RANKL (rhRANKL), recombinant human OPG (rhOPG) or anti-human RANKL mouse monoclonal antibody (ahRANKL-mAB) with or without rhIL-1β.ResultsImmunoreactivity to RANK/RANKL/OPG and the mRNA expression of the three genes were obviously identified in both AF and NP cells. rhIL-1β stimulation significantly upregulated the mRNA expression level of RANK/RANKL/OPG. The mRNA expression of catabolic factors was significantly upregulated by stimulation of rhRANKL in the presence of rhIL-1β. On the other hand, the administration of either rhOPG or ahRANKL-mAB significantly suppressed the mRNA expression of catabolic factors that had been upregulated by rhIL-1β stimulation. The suppressive effect of ahRANKL-mAB against rhIL-1β stimulation was also confirmed by the protein expression of MMP-3.ConclusionsThe present study showed that the RANK/RANKL/OPG system may be involved in the progression of IVD degeneration. This study also suggested the potential use of anti-RANKL monoclonal antibody and OPG as therapeutic agents to suppress the progression of IVD degeneration.

Theaflavins prevent cartilage degeneration via AKT/FOXO3 signaling invitro.

Theaflavins (TFs) are the main bioactive polyphenols in tea and contribute to protection against oxidative stress. Excessive reactive oxygen species (ROS) accumulation can lead to the disruption of cartilage homeostasis. The present study examined the potential effects of TFs on H2O2-induced cartilage degeneration in vitro. Cell Counting kit (CCK-8) was used to determine cell viability, and flow cytometric analysis was used to detect ROS, apoptosis and DNA damage. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to detect the expression levels of target factors. The present study revealed that TFs effectively reduced the expression of catabolic factors, including matrix metalloproteinase-13, interleukin-1 and cartilage glycoprotein 39. TFs inhibited ROS generation in cartilage degeneration, and suppressed apoptosis and DNA damage caused by oxidative stress. TFs also downregulated the expression levels of cleaved caspase-3 and B-cell lymphoma 2-associated X protein, and the DNA damage-related genes, ATR serine/threonine kinase and ATM serine/threonine kinase. Furthermore, TFs enhanced the activity of glutathione peroxidase 1 and catalase, but reduced the expression levels of phosphorylated (p)-AKT serine/threonine kinase (AKT) and p-Forkhead box O3 (FOXO3)a. Conversely, the effects of TFs on apoptosis and DNA damage were reversed by persistent activation of AKT. In conclusion, TFs prevented cartilage degeneration via AKT/FOXO3 signaling in vitro. The present study suggested that TFs may be a potential candidate drug for the prevention of cartilage degeneration.

The protective effect of sophocarpine in osteoarthritis: An in vitro and in vivo study

BackgroundOsteoarthritis (OA) is a type of degenerative joint disease affecting millions of individuals worldwide. However, there are currently no great inflammatory treatments available for it. Sophocarpine (SPC), one of the key bioactive compounds derived from Sophora flavescens, has shown remarkable anti-inflammatory effects. MethodsIn this study, we evaluated the effect of SPC on preventing the progression of OA and investigated its molecular target involved. In brief, rat chondrocytes were pretreated with SPC and subsequently stimulated with IL-1β. We found that SPC reduced the production of pro-inflammatory cytokines, such as nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6). SPC also inhibited the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) at both the gene and protein level. Moreover, SPC promoted the expression of anabolic factors Sox-9 and aggrecan, while inhibiting the expression of catabolic factors, such as matrix metalloproteinases 13 (MMP-13) and thrombospondin motifs 5 (ADAMTS-5) in rat chondrocytes. Mechanistically, we found that SPC inhibited nuclear factor kappa B (NF-κB) via the phosphatidylinositol 3 kinase (PI3K)/AKT pathway. The beneficial effects of SPC were also observed in vivo using a rat OA model. ConclusionsOur findings indicate that SPC may be a potential novel therapeutic in the treatment of OA.

Effect of hypoxia/reoxygenation on the biological effect of IGF system and the inflammatory mediators in cultured synoviocytes

Hypoxia/reoxygenation (H/R) plays an important role in the pathogenesis of osteoarthritis. Fibroblast-like synoviocytes (FLS), which are highly sensitive to H/R, are thought to be associated with cartilage degradation during osteoarthritis development. In this study, we investigated the biological effects of insulin-like growth factor (IGF) system and the expression of inflammatory mediators in FLS. We also pretreated FLS with tumor necrosis factor-α (TNF-α) before H/R in order to observe the response of FLS with the background of inflammatory cytokines. H/R increased the levels of TNF-α-induced C-C chemokine ligand 5 (CCL5), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in cell-free culture supernatants; H/R also increased the expression of TNF-α-induced insulin-like growth factor binding protein 3 (IGFBP-3), downregulated the expression of insulin-like growth factor 1 (IGF-1), promoted the loss of mitochondrial membrane potential (MMP), the openness of mitochondrial permeability transition pore (MPTP), the release of intracellular reactive oxygen species (ROS), and mitochondrial matrix swelling, outer membrane rupture and decrease in cristae. Furthermore, H/R induced the expression of catabolic factors and activated the NF-κB signaling pathway in FLS. We therefore concluded that H/R may play a role in inducing inflammation and increase the TNF-α-induced inflammatory effect in FLS, contributing to osteoarthritis pathogenesis.

3'-Sialyllactose as an inhibitor of p65 phosphorylation ameliorates the progression of experimental rheumatoid arthritis.

Background and Purpose3′‐Sialyllactose (3′‐SL) is a safe compound that is present in high levels in human milk. Although it has anti‐inflammatory properties and supports immune homeostasis, its effect on collagen‐induced arthritis (CIA) is unknown. In this study, we investigated the prophylactic and therapeutic effect of 3′‐SL on the progression of rheumatoid arthritis (RA) in in vitro and in vivo models.Experimental ApproachThe anti‐arthritic effect of 3′‐SL was analysed with fibroblast‐like synoviocytes in vitro and an in vivo mouse model of CIA. RT‐PCR, Western blotting and ELISA were performed to evaluate its effects in vitro. Histological analysis of ankle and knee joints of mice with CIA was performed using immunohistochemistry, as well as safranin‐O and haematoxylin staining.Key Results3′‐SL markedly alleviated the severity of CIA in the mice by reducing paw swelling, clinical scores, incidence rate, serum levels of inflammatory cytokines and autoantibody production. Moreover, 3′‐SL reduced synovitis and pannus formation and suppressed cartilage destruction by blocking secretion of chemokines, pro‐inflammatory cytokines, https://en.wikipedia.org/wiki/Matrix_metalloproteinases and osteoclastogenesis via NF‐κB signalling. Notably, phosphorylation of p65, which is a key protein in the NF‐κB signalling pathway, was totally blocked by 3′‐SL in the RA models.Conclusions and Implications3′‐SL ameliorated pathogenesis of CIA by suppressing catabolic factor expression, proliferation of inflammatory immune cells and osteoclastogenesis. These effects were mediated via blockade of the NF‐κB signalling pathway. Therefore, 3′‐SL exerted prophylactic and therapeutic effects and could be a novel therapeutic agent for the treatment of RA.

Alleviation of Murine Osteoarthritis by Cartilage-Specific Deletion of IκBζ.

IκBζ, an atypical IκB family member, regulates gene expression in the nucleus as a transcriptional cofactor. Although IκBζ has been extensively studied in the immune system, its specific roles in osteoarthritis (OA) are currently unknown. The objective of this study was to investigate the potential role of IκBζ in chondrocyte catabolism and OA pathogenesis. We also determined the molecular mechanism underlying its relationship to the transcription factor NF-κB. We determined expression levels of IκBζ in mouse chondrocytes treated with interleukin-1β (IL-1β), in human OA cartilage, and in mouse experimental OA cartilage. Adenovirus-mediated overexpression and small interfering RNA knockdown of IκBζ were performed to determine the impact of IκBζ on catabolic gene expression in vitro. Cartilage-specific IκBζ-transgenic and -knockout mice were generated and used for in vivo studies. Experimental and spontaneous OA were induced by surgical destabilization of the medial meniscus and by aging, respectively. Coimmunoprecipitation assay was used to examine the association between IκBζ and NF-κB subunits. IκBζ was highly up-regulated in chondrocytes in response to IL-1β and in OA cartilage of human and mouse knee joints. Overexpression of IκBζ in chondrocytes promoted spontaneous OA development by activating chondrocyte catabolism. Genetic ablation of IκBζ in chondrocytes abolished catabolic gene induction by IL-1β and protected against the development of experimental OA. IκBζ formed complexes with NF-κB members to regulate catabolic factor expression. These findings demonstrate a critical role for IκBζ in OA pathogenesis. Inhibition of IκBζ function might be an effective therapeutic approach for OA treatment.

Sauchinone inhibits IL-1β induced catabolism and hypertrophy in mouse chondrocytes to attenuate osteoarthritis via Nrf2/HO-1 and NF-κB pathways

BackgroundOsteoarthritis (OA) is a common degenerative joint disease for which currently no anti-inflammatory therapy is available. Sauchinone (SAU), a key bioactive compound derived from Saururus chinensis, which has shown remarkable anti-inflammatory effects. MethodsTo evaluate the effect of SAU on OA progression, mouse chondrocytes were pretreated with SAU and subsequently stimulated with interleukin (IL)-1β. We found that SAU reduced the production of pro-inflammatory cytokines, such as nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α), and IL-6. SAU also inhibited the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) at both the gene and protein level. Moreover, SAU promoted the expression of aggrecan, while inhibiting the expression of catabolic factors, such as matrix metalloproteinases (MMPs) and thrombospondin motifs 5 (ADAMTS-5) in mouse chondrocytes. Col X, vascular endothelial growth factor-A (VEGF)-A, and Runx2, major markers of hypertrophic chondrocytes, were markedly elevated following IL-1β stimulation, and were reduced by SAU treatment while having the opposite effect on Col II. Mechanistically, we found that SAU inhibited nuclear factor kappa B (NF-κB) and activated the Nrf2/HO-1 pathway. The beneficial effects of SAU were also observed in vivo using a mouse OA model. ConclusionsOur findings indicate that SAU may be a potential novel therapeutic for the treatment of OA.

Downregulation of aquaporin 9 decreases catabolic factor expression through nuclear factor‑κB signaling in chondrocytes.

Aquaporins (AQPs) are small integral membrane proteins that are essential for water transport across membranes. AQP9, one of the 13 mammalian AQPs (including AQP0 to 12), has been reported to be highly expressed in hydrarthrosis and synovitis patients. Given that several studies have identified signal transduction as an additional function of AQPs, it is hypothesized that AQP9 may modulate inflammatory signal transduction in chondrocytes. Therefore, the present study used a model of interleukin (IL)‑1β‑induced inflammation to determine the mechanisms associated with AQP9 functions in chondrocytes. Osteoarthritis (OA) and normal cartilage samples were subjected to immunohistological analysis. In addition, matrix metalloproteinase (MMP)3, MMP13 and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS‑5) mRNA and protein analysis was conducted in normal human articular chondrocytes from the knee (NHAC‑Kn) stimulated with IL‑1β by reverse transcription‑polymerase chain reaction (RT‑qPCR) and western blotting, respectively. AQP9 knockdown was also performed by transfection of AQP9‑specific small interfering RNA using Lipofectamine. AQP1, 3, 7, 9 and 11 mRNA expression levels were detected in OA human chondrocytes and in IL‑1β‑treated normal human chondrocytes. The levels of AQP9, MMP‑3, MMP‑13 and ADAMTS‑5 mRNA were increased by treatment with 10ng/ml IL‑1β in a time‑dependent manner, while knockdown of AQP9 expression significantly decreased the mRNA levels of the MMP3, MMP13 and ADAMTS‑5 genes, as well as the phosphorylation of IκB kinase (IKK). Treatment with a specific IKK inhibitor also significantly decreased the expression levels of MMP‑3, MMP‑13 and ADAMTS‑5 in response to IL‑1β stimulation. Furthermore, immunohistochemical analysis demonstrated that AQP9 and inflammatory markers were highly expressed in OA cartilage. In addition, the downregulation of AQP9 in cultured chondrocytes decreased the catabolic gene expression in response to IL‑1β stimulation through nuclear factor‑κB signaling. Therefore, AQP9 may be a promising target for the treatment of OA.

Depletion of aquaporin 1 decreased ADAMTS‑4 expression in human chondrocytes.

Inflammation serves an important role in the progression of osteoarthritis (OA), and IL-1β may act as a catabolic factor on cartilage, reducing the synthesis of primary cartilage components type II collagen and aggrecan. Aquaporin 1 (AQP1) is a 28-kDa water channel formed of six transmembrane domains on the cell membrane. AQP1 is highly expressed in the anus, gallbladder and liver, and is moderately expressed in the hippocampus, ependymal cells of the central nervous system and articular cartilage. It was hypothesized that AQP1 may be highly expressed in OA cartilage and that it may increase the expression of catabolic factors during inflammatory OA progression. Therefore, the present study evaluated AQP1 functions in human OA articular chondrocytes. Primary chondrocytes were isolated from human hip and knee cartilage tissues, cultured and transfected with AQP1-specific small interfering RNA with or without subsequent IL-1β treatment. In vitro explant culture from hip cartilages were also prepared. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to assess the expression of AQP genes in human articular cartilage, AQP1 immunohistochemistry of the cartilages and explant culture, as well as RT-quantitative PCR, western blotting and immunocytochemistry/immunofluorescence of OA chondrocytes to evaluate the expression of AQP1, and catabolic and anabolic factors. RT-PCR results demonstrated that AQP0, 1, 3, 7, 9, and 11 were expressed in OA chondrocytes. Immunohistochemistry revealed that AQP1 was highly expressed in the superficial to middle zones of OA articular cartilages. Additionally, AQP1 mRNA was significantly higher in OA cartilage and IL-1β treatment significantly increased AQP1 expression in hip explant cartilage. Furthermore, AQP1 downregulation decreased a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS)-4 expression in OA chondrocytes, though it did not affect other associated genes. Immunofluorescence showed that AQP1 and ADAMTS-4 were co-localized. These findings indicated that AQP1 depletion may decrease ADAMTS-4 expression in human OA chondrocytes. Therefore, regulating AQP1 expression may be a strategy to suppress catabolic factors during OA progression.