Molecular Targets of Natural Products for Chondroprotection in Destructive Joint Diseases.

Osteoarthritis (OA) is the most common type of arthritis that occurs in an aged population. It affects any joints in the body and degenerates the articular cartilage and the subchondral bone. Despite the pathophysiology of OA being different, cartilage resorption is still a symbol of osteoarthritis. Matrix metalloproteinases (MMPs) are important proteolytic enzymes that degrade extra-cellular matrix proteins (ECM) in the body. MMPs contribute to the turnover of cartilage and its break down; their levels have increased in the joint tissues of OA patients. Application of chondroprotective drugs neutralize the activities of MMPs. Natural products derived from herbs and plants developed as traditional medicine have been paid attention to, due to their potential biological effects. The therapeutic value of natural products in OA has increased in reputation due to their clinical impact and insignificant side effects. Several MMPs inhibitor have been used as therapeutic drugs, for a long time. Recently, different types of compounds were reviewed for their biological activities. In this review, we summarize numerous natural products for the development of MMPs inhibitors in arthritic diseases and describe the major signaling targets that were involved for the treatments of these destructive joint diseases.

Gumiganghwal-tang ameliorates cartilage destruction via inhibition of matrix metalloproteinase.

ETHNOPHARMACOLOGICAL RELEVANCE: Kyung-Bang Gumiganghwal-tang tablet (GMGHT) is a standardized Korean Medicine that could treat a cold, headache, arthralgia and fever. Although GMGHT has been used for arthritis-related diseases including a sprain, arthralgia, unspecified arthritis and knee arthritis, there is no pre-clinical evidence to treat osteoarthritis (OA). This study determined the drug dosage and the mechanisms of GMGHT for OA. METHODS: OA was induced by intra-articular monoiodoacetic acid (MIA) injection in Sprague-Dawley rats. As calculated from the human equivalent dose formula, GMGHT was orally administered at the doses of 9.86, 98.6 and 986 mg/kg for 4 weeks. The arthritis score was performed by a blind test, and histological changes in articular cartilage were indicated by hematoxylin and eosin, Safranin O and toluidine blue staining. SW1353 chondrocytes were stimulated by interleukin (IL)-1ß recombinant to analyze the expressions of Type II collagen, matrix metalloproteinases (MMPs) and nuclear factor (NF)-κB. RESULTS: Rough and punctate surfaces of the femoral condyle induced by MIA, were recovered by the GMGHT treatment. The arthritis score was significantly improved in the 968 mg/kg of GMGHT-treated cartilage. Loss of chondrocytes and proteoglycan were ameliorated at the deep zone of the subchondral bone plate by the GMGHT administration in OA rats. The expression of Type II collagen was increased, while MMP-1, -3 and -13 levels were decreased in the GMGHT-treated SW1353 chondrocytes. In addition, the GMGHT treatment regulated NF-κB activation along with IL-6, transforming growth factor-ß and IL-12 production. CONCLUSIONS: GMGHT promoted the recovery of articular cartilage damage by inhibiting MMPs, accompanied with its anti-inflammatory effects in OA. GMGHT might be an alternative therapeutic treatment for OA.

Acute Changes in NADPH Oxidase 4 in Early Post-Traumatic Osteoarthritis.

Knee injuries cause structural damage and acute inflammation that initiates the development of post-traumatic osteoarthritis (PTOA). NADPH oxidase 4 (Nox4), a member of a family of enzymes that generates reactive oxygen species (ROS), plays a pivotal role in normal development of the musculoskeletal system, but may increase ROS production to harmful levels after joint injury. The role of ROS in both normal joint homeostasis and injury is poorly understood, but inhibition of excessive ROS production by Nox4 after joint injury could be protective to the joint, decreasing oxidative stress, and initiation of PTOA. Knee injuries were simulated using inflammatory cytokines in cultured primary human chondrocytes and a non-invasive mouse model of PTOA in C57BL/6N and Nox4 knockout mice. There is an acute decrease in Nox4 activity within 24 h after injury in both systems, followed by a subsequent sustained low-level increase, a novel finding not seen in any other system. Inhibition of Nox4 activity by GKT137831 was protective against early structural changes after non-invasive knee injury in a mouse model. Nox4 knockout mice had significant differences in structural and mechanical properties of bone, providing further evidence for the role of Nox4 in development of joint tissues and biochemical response after joint injury. Nox4 plays a significant role in the acute phase after joint injury, and targeted inhibition of inflammation caused by Nox4 may be protective against early joint changes in the pathogenesis of PTOA. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2429-2436, 2019.

Ligustilide attenuates nitric oxide-induced apoptosis in rat chondrocytes and cartilage degradation via inhibiting JNK and p38 MAPK pathways.

Ligustilide (LIG) is the main lipophilic component of the Umbelliferae family of pharmaceutical plants, including Radix angelicae sinensis and Ligusticum chuanxiong. LIG shows various pharmacological properties associated with anti-inflammation and anti-apoptosis in several kinds of cell lines. However, the therapeutic effects of LIG on chondrocyte apoptosis remain unknown. In this study, we investigated whether LIG had an anti-apoptotic activity in sodium nitroprusside (SNP)-stimulated chondrocyte apoptosis and could delay cartilage degeneration in a surgically induced rat OA model, and elucidated the potential mechanisms. In vitro studies revealed that LIG significantly suppressed chondrocyte apoptosis and cytoskeletal remodelling, which maintained the nuclear morphology and increased the mitochondrial membrane potential. In terms of SNP, LIG treatment considerably reduced the expression levels of cleaved caspase-3, Bax and inducible nitric oxide synthase and increased the expression level of Bcl-2 in a dose-dependent manner. The LIG-treated groups presented a significantly suppressed expression of activating transcription factor 2 and phosphorylation of Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). The inhibitory effect of LIG was enhanced by the p38 MAPK inhibitor SB203580 or the JNK inhibitor SP600125 and offset by the agonist anisomycin. In vivo studies demonstrated that LIG attenuated osteoarthritic cartilage destruction by inhibiting the cartilage chondrocyte apoptosis and suppressing the phosphorylation levels of activating transcription factor 2, JNK and p38 MAPK. This result was confirmed by histological analyses, micro-CT, TUNEL assay and immunohistochemical analyses. Collectively, our studies indicated that LIG protected chondrocytes against SNP-induced apoptosis and delayed articular cartilage degeneration by suppressing JNK and p38 MAPK pathways.

Critical role for arginase II in osteoarthritis pathogenesis.

OBJECTIVE: Osteoarthritis (OA) appears to be associated with various metabolic disorders, but the potential contribution of amino acid metabolism to OA pathogenesis has not been clearly elucidated. Here, we explored whether alterations in the amino acid metabolism of chondrocytes could regulate OA pathogenesis. METHODS: Expression profiles of amino acid metabolism-regulating genes in primary-culture passage 0 mouse chondrocytes were examined by microarray analysis, and selected genes were further characterised in mouse OA chondrocytes and OA cartilage of human and mouse models. Experimental OA in mice was induced by destabilisation of the medial meniscus (DMM) or intra-articular (IA) injection of adenoviruses expressing catabolic regulators. The functional consequences of arginase II (Arg-II) were examined in Arg2-/- mice and those subjected to IA injection of an adenovirus encoding Arg-II (Ad-Arg-II). RESULTS: The gene encoding Arg-II, an arginine-metabolising enzyme, was specifically upregulated in chondrocytes under various pathological conditions and in OA cartilage from human patients with OA and various mouse models. Adenovirus-mediated overexpression of Arg-II in mouse joint tissues caused OA pathogenesis, whereas genetic ablation of Arg2 in mice (Arg2-/-) abolished all manifestations of DMM-induced OA. Mechanistically, Arg-II appears to cause OA cartilage destruction at least partly by upregulating the expression of matrix-degrading enzymes (matrix metalloproteinase 3 [MMP3] and MMP13) in chondrocytes via the nuclear factor (NF)-κB pathway. CONCLUSIONS: Our results indicate that Arg-II is a crucial regulator of OA pathogenesis in mice. Although chondrocytes of human and mouse do not identically, but similarly, respond to Arg-II, our results suggest that Arg-II could be a therapeutic target of OA pathogenesis.

Zinc Protects Articular Chondrocytes through Changes in Nrf2-Mediated Antioxidants, Cytokines and Matrix Metalloproteinases.

Osteoarthritis (OA) is an age-related degenerative joint disease characterized by high oxidative stress, chondrocyte death and cartilage damage. Zinc has been implicated in the antioxidant capacity of the cell, and its deficiency might inhibit chondrocyte proliferation. The present study examined the potential of zinc as a preventive supplement against OA using the in vitro chondrosarcoma cell line SW1353 and an in vivo Wistar rat model to mimic OA progress induced by monosodium iodoacetate (MIA). The results demonstrated that, in SW1353 cells, 5 µM MIA exposure increased oxidative stress and decreased the expression of GPx1 and Mn-SOD but still increased GSH levels and HO-1 expression and enhanced the expression of interleukin (IL)-10, IL-1ß, and matrix metalloproteinase (MMP)-13. Zinc addition could block these changes. Besides, the expression of Nrf2 and phosphorylated (p)-Akt was dramatically increased, implicating the p-Akt/Nrf2 pathway in the effects of zinc on MIA-treated cells. A rat model achieved similar results as those of cell culture, and 1.6 mg/kg/day of zinc supplementation is sufficient to prevent OA progress, while 8.0 mg/kg/day of zinc supplementation does not have a better effect. These findings indicate that zinc supplementation exerts a preventive effect with respect to MIA-induced OA progress.

Synergistic effect of l-ascorbic acid and hyaluronic acid on the expressions of matrix metalloproteinase-3 and -9 in human chondrocytes.

Proinflammatory cytokines and reactive oxygen species (ROS) are known to be involved in the initiation and progression of osteoarthritis (OA). New evidence clarifying the correlation between ROS and inflammation has indicated that oxidative stress can up-regulate inflammatory cytokines. l-Ascorbic acid (AA), an antioxidant, has been shown to have anti-inflammatory effects and improve matrix deposition in chondrocytes. The purpose of this study was to examine the effects of hyaluronic acid (HA; 100 µg/mL) supplemented with AA (50 µg/mL) on human normal and interleukin-1 beta-stimulated (IL-1ß, 10 ng/mL) chondrocytes. HA, AA, and HA + AA treatment did not change cell morphology, viability, proliferation, and glycosaminoglycan production in normal chondrocytes. HA, AA, and HA + AA, by contrast, partially restored viability and morphology of hypertrophic chondrocytes, and HA and HA + AA further decreased the cytotoxicity of IL-1ß. Real-time PCR revealed that AA and HA + AA had no substantial effects on unstimulated chondrocytes, except for down-regulation of matrix metalloproteinase (MMP)-9 mRNA levels. For IL-1ß-stimulated chondrocytes, significant down-regulation of IL-1ß, tumor necrosis factor-alpha (TNF-α), MMP-3, and MMP-9 mRNA expression was found when cells were cultured in HA-supplemented media. Moreover, HA + AA supplementation further significantly decreased MMP-3 and MMP-9 mRNA expression. The protein production of MMP-3 was decreased, with a significant difference between the HA + AA group and HA group. The antioxidant capacity and superoxide dismutases activity were also partially restored in stimulated chondrocytes. HA supplemented with AA modulates MMPs expression and antioxidant fuction in chondrocytes. AA may enhance the anticatabolic effects of HA on OA chondrocytes. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1809-1817, 2018.

Oleanolic acid (OA) regulates inflammation and cellular dedifferentiation of chondrocytes via MAPK signaling pathways.

Oleanolic acid (OA) is a bioactive triterpenoid in medicinal plants. It possesses various pharmacological properties, including analgesic, anti-inflammatory, and antitumor effects. The effects of OA in chondrocytes, however, are not well characterized. Here, we used rabbit articular chondrocytes as a cellular model to investigate the effects and regulatory mechanisms of OA on dedifferentiation and pro-inflammation. OA promoted dedifferentiation of chondrocytes by inhibiting type II collagen and pro-inflammatory activity by increasing cyclooxygenase-2 (COX-2) expression. Furthermore increased phosphorylation of p38 kinases and down-regulated phosphorylation of ERK was observed. Inhibition of p38 with SB203580 in OA-treated cells rescued the expression of type II collagen and suppressed the expression of COX-2. However, ERK inhibition with PD98059 accelerated the OA-induced inflammatory responses. These results suggest that OA induces loss of type II collagen expression via the p38 pathway and induces inflammation through the p38 and ERK pathways in rabbit articular chondrocytes.

Glucose adsorption to chitosan membranes increases proliferation of human chondrocyte via mammalian target of rapamycin complex 1 and sterol regulatory element-binding protein-1 signaling.

Osteoarthritis (OA) is currently still an irreversible degenerative disease of the articular cartilage. Recent, dextrose (d-glucose) intraarticular injection prolotherapy for OA patients has been reported to benefit the chondrogenic stimulation of damaged cartilage. However, the detailed mechanism of glucose's effect on cartilage repair remains unclear. Chitosan, a naturally derived polysaccharide, has recently been investigated as a surgical or dental dressing to control breeding. Therefore, in this study, glucose was adsorbed to chitosan membranes (CTS-Glc), and the study aimed to investigate whether CTS-Glc complex membranes could regulate the proliferation of human OA chondrocytes and to explore the underlying mechanism. Human OA and SW1353 chondrocytes were used in this study. The experiments involving the transfection of cells used SW1353 chondrocytes. A specific inhibitor and siRNAs were used to investigate the mechanism underlying the CTS-Glc-regulated proliferation of human chondrocytes. We found that CTS-Glc significantly increased the proliferation of both human OA and SW1353 chondrocytes comparable to glucose- or chitosan-only stimulation. The role of mammalian target of rapamycin complex 1 (mTORC1) signaling, including mTOR, raptor, and S6k proteins, has been demonstrated in the regulation of CTS-Glc-increased human chondrocyte proliferation. mTORC1 signaling increased the expression levels of maturated SREBP-1 and FASN and then induced the expressions of cell cycle regulators, that is, cyclin D, cyclin-dependent kinase-4 and -6 in human chondrocytes. This study elucidates the detailed mechanism behind the effect of CTS-Glc complex membranes in promoting chondrocyte proliferation and proposes a possible clinical application of the CTS-Glc complex in the dextrose intraarticular injection of OA prolotherapy in the future to attenuate the pain and discomfort of OA patients.

Inhibitory effects of EGb761 on the expression of matrix metalloproteinases (MMPs) and cartilage matrix destruction.

Cytokines such as tumor necrosis factor alpha (TNF-α)-induced expression of matrix metalloproteinase (MMP) play a pivotal role in the destruction of articular cartilage in patients who are suffering from osteoarthritis (OA). Collagen type II, the basis for articular cartilage, can be degraded by MMP-1, MMP-3, and 13. EGb761, the standardized extract of Ginkgo biloba produced by Dr. Willar Schwabe Pharmaceuticals, has shown its anti-inflammatory capacity. This study aimed to determine a mechanism whereby EGb761 may inhibit cartilage degradation. Our results indicated that pretreatment with EGb761 abolishes MMP-1, MMP-3, and MMP-13 gene expression and protein expression induced by TNF-α in human chondrocyte monolayer. In addition, the reduction of the tissue inhibitor of metalloproteinase-1(TIMP-1) and metalloproteinase-2 gene expression induced by TNF-α was rescued by pretreatment with EGb761. Importantly, TNF-α-induced degradation of collagen type II was ameliorated by EGb761 in a dose-dependent manner. Mechanistically, our results indicated that EGb761 treatment attenuated TNF-α-induced NF-κB activation. These actions of EGb761 suggest a mechanism by which EGb761 may act to prevent cartilage breakdown in arthritis.

Inhibition of matrix metalloproteinase-13 expression in IL-1ß-treated articular chondrocytes by a steroidal saponin, spicatoside A, and its cellular mechanisms of action.

Matrix metalloproteinase-13 (MMP-13) plays a critical role in degrading major collagens in human cartilage under some pathological conditions such as osteoarthritis. To establish the therapeutic potential against cartilage degradation, the effects of 12 naturally-occurring triterpenoids and steroids on MMP-13 induction were examined in the human chondrocyte cell line, SW1353. They included coreanoside F1, suavissimoside R1, spicatoside A, 25(S)-ruscogenin, methyl protogracillin, hederagenin, loniceroside A, loniceroside B, loniceroside C, smilaxin A, smilaxin C, and ursolic acid. Among these, only spicatoside A and 25(S)-ruscogenin were found to inhibit MMP-13 expression in IL-1ß-treated SW1353 cells at a pharmacologically-relevant concentration of 10 µM. These effects were also supported by the finding that spicatoside A (20 µM) reduced glycosaminoglycan release from IL-1α-treated rabbit joint cartilage culture to some degree. When the cellular mechanisms of action of spicatoside A in MMP-13 inhibition were investigated, the blocking point was not found among the MMP-13 signaling molecules examined such as mitogen-activated protein kinases, activator protein-1, and nuclear transcription factor-κB. Instead, spicatoside A was found to reduce MMP-13 mRNA stability. All of these findings suggest that spicatoside A and 25(S)-ruscogenin have a therapeutic potential for protecting against cartilage breakdown in arthritic disorders.

Achyranthes bidentata polysaccharides activate the Wnt/ß-catenin signaling pathway to promote chondrocyte proliferation.

Achyranthes bidentata polysaccharides (ABPS) are the active components of Radix Achyranthis Bidentatae (AB), which has been extensively used in Traditional Chinese medicine (TCM) in the treatment of osteoarthritis (OA). Our previous study provided evidence that ABPS regulated the G1/S transition to promote chondrocyte proliferation. However, the precise mechanisms involved remain to be elucidated. In the present study, we aimed to investigate the effects of ABPS on the Wnt/ß­catenin signaling pathway in chondrocytes. Chondrocytes, obtained from the knee cartilage of Sprague-Dawley rats, were identified by type II collagen immunohistochemistry. ABPS upregulated the expression of Wnt-4, Frizzled-2, ß-catenin and cyclin D1, and downregulated the expression of glycogen synthase kinase 3ß (GSK-3ß), as shown by reverse transcription PCR (RT-PCR) and western blot analysis. Using immunofluorescence, we also found that ABPS induced ß-catenin nuclear translocation. Importantly, the expression of ß-catenin and cyclin D1 was partly inhibited by Dickkopf-1 (DKK-1), an inhibitor of the Wnt/ß-catenin signaling pathway. In addition, we found that ABPS increased the expression of type II collagen in chondrocytes. These results suggest that ABPS promote chondrocyte proliferation by activating the Wnt/ß-catenin signaling pathway.

Overexpression of microRNA-25 by withaferin A induces cyclooxygenase-2 expression in rabbit articular chondrocytes.

Increasing evidence supports the role of microRNAs (miRNA) in the regulation of inflammation in various human disorders. Several recent studies have demonstrated that microRNA-25 (miR-25) has multiple functions, and it affects the expression of inflammatory mediators. Withaferin A (WFA), a natural compound derived from the medicinal plant Withania somnifera, has shown the potential to be an effective drug for arthritis treatment in several preclinical and clinical studies. We investigated the role of miR-25 in the WFA-mediated up-regulation of cyclooxygenase-2 (COX-2) expression in rabbit articular chondrocytes. WFA induced COX-2 expression in a dose-dependent manner as analyzed by western blot analysis and immunofluorescence staining in rabbit articular chondrocytes. WFA up-regulated miR-25 expression as determined by real-time PCR. Overexpression of miR-25 in the presence of WFA increased the expression of COX-2 compared to that observed with just WFA treatment alone, as indicated by western blot analysis and Real-time PCR. Moreover, silencing of miR-25 by anti-miR25 inhibited COX-2 expression in a dose-dependent manner. Since miR-25 up-regulation by WFA treatment induced the expression of COX-2 in rabbit articular chondrocytes, these findings collectively suggest that miR-25 mediates the WFA-induced inflammatory responses in chondrocytes.

Zhuanggu Jianxi Decoction () limits interleukin-1 ß-induced degeneration chondrocytes via the caveolin-p38 MAPK signal pathway.

OBJECTIVE: To evaluate the effect of Zhuanggu Jianxi Decoction (, ZGJXD) on interleukin-1 ß (IL-1 ß)-induced degeneration of chondrocytes (CDs) as well as the activation of caveolin-p38 mitogen-activated protein kinase (MAPK) signal pathway, investigating the possible molecular mechanism that ZGJXD treats osteoarthritis. METHODS: Serum pharmacology was applied in the present study, where ZGJXD was orally administrated to New Zealand rabbits and then ZGJXD containing serum (ZGJXD-S) was collected for following in vitro experiments. CDs were isolated aseptically from New Zealand rabbits and then cultured in vitro. Upon IL-1 ß stimulation, the degeneration of CDs was verified by inverted microscope, toluidine blue stain and type II collagen immunocytochemistry. After IL-1 ß-stimulated CDs were intervened with blank control serum, ZGJXD-S, together with or without SB203580 (a specific inhibitor of p38 MAPK) for 48 h, caveolin-1 protein expression and the phosphorylation level of p38 were determined by Western blotting, and the mRNA expression of IL-1 ß, tumor necrosis factor α (TNF-α), matrix metalloproteinase 3 (MMP-3) and MMP-13 were examined by real-time polymerase chain reaction. RESULTS: IL-1 ß stimulation induced degeneration of CDs, increased caveolin-1 expression and p38 phosphorylation, up-regulated the mRNA level of IL-1 ß, TNF-α, MMP-3 and MMP-13. However, the IL-1 ß-induced activation of caveolin-p38 signaling and alteration in the expression of p38 downstream target genes were suppressed by ZGJXD-S and/or SB203580 in CDs. CONCLUSION: ZGJXD can prevent CDs degeneration via inhibition of caveolin-p38 MAPK signal pathway, which might be one of the mechanisms that ZGJXD treats osteoarthritis.

Homeopathic Rhus toxicodendron treatment increased the expression of cyclooxygenase-2 in primary cultured mouse chondrocytes.

BACKGROUND: Rhus toxicodendron (Rhus tox) is a homeopathic remedy with anti-inflammatory activities used for arthritis pain. METHODS: We studied the effects of 4×, 30×, 30c and 200c homeopathic dilutions of Rhus tox in primary cultured mouse chondrocytes. We examined the expression of collagen type II, a marker protein of chondrocytes, and cyclooxygenase-2 (COX-2), which is responsible for the biosynthesis of prostaglandin E2 (PGE2) and the regulation of the inflammatory response. We assessed the expression of collagen type II and COX-2 using biochemical and immunological methods, such as reverse transcription polymerase chain reaction (RT-PCR), quantitative (or real-time) RT-PCR (qRT-PCR) and immunoblot assays. RESULTS: Stimulation with different concentrations of Rhus tox increased the mRNA expression of COX-2, and stimulation with 30× Rhus tox showed the most prominent mRNA expression in both RT-PCR and qRT-PCR analyses. We also observed that homeopathic dilutions of 4×, 30× and 30c Rhus tox inhibited collagen type II expression, suggesting that Rhus tox induced the dedifferentiation of chondrocytes. In addition, treatment with 30× Rhus tox significantly increased PGE2 release compared with other homeopathic dilutions of Rhus tox. CONCLUSIONS: Taken together, these results suggest that homeopathic treatment with Rhus tox induced chondrocyte dedifferentiation and inflammatory responses, such as COX-2 expression and PGE2 production, in primary cultured chondrocytes.

Production of reactive oxygen species by withaferin A causes loss of type collagen expression and COX-2 expression through the PI3K/Akt, p38, and JNK pathways in rabbit articular chondrocytes.

Withaferin A (WFA) is a major chemical constituent of Withania somnifera, also known as Indian ginseng. Many recent reports have provided evidence of its anti-tumor, anti-inflammation, anti-oxidant, and immune modulatory activities. Although the compound appears to have a large number of effects, its defined mechanisms of action have not yet been determined. We investigated the effects of WFA on loss of type collagen expression and inflammation in rabbit articular chondrocytes. WFA increased the production of reactive oxygen species, suggesting the induction of oxidative stress, in a dose-dependent manner. Also, we confirmed that WFA causes loss of type collagen expression and inflammation as determined by a decrease of type II collagen expression and an increase of cyclooxygenase-2 (COX-2) expression via western blot analysis in a dose- and time- dependent manner. WFA also reduced the synthesis of sulfated proteoglycan via Alcian blue staining and caused the synthesis of prostaglandin E2 (PGE2) via assay kit in dose- and time-dependent manners. Treatment with N-acetyl-L-cysteine (NAC), an antioxidant, inhibited WFA-induced loss of type II collagen expression and increase in COX-2 expression, accompanied by inhibition of reactive oxygen species production. WFA increased phosphorylation of both Akt and p38. Inhibition of PI3K/Akt, p38, and JNK with LY294002 (LY), SB203580 (SB), or SP600125 (SP) in WFA-treated cells rescued the expression of type II collagen and suppressed the expression of COX-2. These results demonstrate that WFA induces loss of type collagen expression and inflammation via PI3K/Akt, p38, and JNK by generating reactive oxygen species in rabbit articular chondrocytes.

Treatment with SiMiaoFang, an anti-arthritis chinese herbal formula, inhibits cartilage matrix degradation in osteoarthritis rat model.

A Chinese herbal preparation, SiMiaoFang (SMF), has been used clinically for treating arthralgia by virtue of its anti-inflammatory and pain-relieving activities. However, no evidence base links SMF to anti-osteoarthritis (OA), particularly its link to inhibiting cartilage matrix degradation. In this study, we undertook a characterization of anti-OA activity of SMF using an in vivo rat model induced by anterior cruciate ligament transection and medial meniscus resection (ACLT+MMx) together with in vitro studies with chondrocytes for further molecular characterization. ACLT+MMx rats were treated with SMF at doses of 0.63, 1.25, and 2.5 grams/kg per day for 6 weeks. SMF treatments significantly inhibited cartilage matrix degradation, as indicated by increasing proteoglycan and collagen content, particularly type II collagen expression in articular cartilage, decreasing CTX-II (collagen type II degradation marker), and increasing CPII (collagen type II synthesis marker) in circulation. Moreover, SMF suppressed synovial inflammation and inhibited release of interleukin-1ß (IL-1ß) and tumor necrosis factor-α in serum. The levels of serum prostaglandin E2 and nitric oxide productions were decreased via suppression of the production of cyclooxygenase-2 and inducible nitric oxide synthase, respectively. Importantly, SMF interfered with OA-augmented expression of matrix metalloproteinases (MMPs) -3 and -13 and aggrecanases (ADAMTS) -4 and -5, which are considered to be key enzymes in cartilage matrix degradation, and simultaneously augmented OA-reduced tissue inhibitors of metalloproteinases (TIMPs) -1 and -3 expression in the joints. The largest changes in these parameters were found at the highest dose. Meanwhile, SMF significantly decreased MMP-3 and -13 and increased TIMP-1 and -3 at mRNA and protein levels in IL-1ß-induced chondrocytes. These findings provide the first evidence that SMF effectively treats OA by inhibiting cartilage matrix degradation.

Salubrinal reduces expression and activity of MMP13 in chondrocytes.

OBJECTIVE: Stress to the endoplasmic reticulum (ER) and inflammatory cytokines induce expression and activity of matrix metalloproteinase 13 (MMP13). Since a synthetic agent, salubrinal, is known to alleviate ER stress and attenuate nuclear factor kappa B (NFκB) signaling, we addressed a question whether upregulation of MMP13 by ER stress and cytokines is suppressed by administration of salubrinal. METHODS: Using C28/I2 human chondrocytes, we applied ER stress with tunicamycin and inflammatory distress with tumor necrosis factor α (TNFα) and interleukin 1ß (IL1ß). RNA interference with siRNA specific to NFκB p65 (RelA) was employed to examine a potential involvement of NFκB signaling in salubrinal's action in regulation of MMP13. We also employed primary human chondrocytes and evaluated MMP13 activity. RESULTS: The result showed that tunicamycin activated p38 mitogen-activated protein kinase (MAPK), while inflammatory cytokines activated p38 MAPK and NFκB. In both cases, salubrinal significantly reduced expression and activity of MMP13. Silencing NFκB reduced inflammatory cytokine-driven upregulation of MMP13 activity. CONCLUSIONS: The results demonstrate that salubrinal downregulates expression and activity MMP13 through p38 and NFκB signaling, suggesting its potential usage to treat degenerative diseases such as osteoarthritis.

Increase of TNFα-stimulated osteoarthritic chondrocytes apoptosis and decrease of matrix metalloproteinases 9 by NF-κB inhibition.

OBJECTIVE: To investigate the in vitro effect of caffeic acid phenethyl ester (CAPE), a NF-κB inhibitor, on the apoptosis of osteoarthritic (OA) chondrocytes and on the regulation of the gelatinases matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9). METHODS: Annexin V-FITC/propidium iodide (PI) labeling and western blotting were used to observe and determine the apoptosis in TNFα-stimulated primary cultured osteoarthritic chondrocytes. Also, gelatin zymography was applied to examine MMP-2 and MMP-9 activities in supernatants. RESULTS: It was confirmed by both flow cytometry and western blotting that chondrocytes from OA patients have an apoptotic background. Use of CAPE in combination with 10 ng/mL of TNFα for 24 h facilitated the apoptosis. MMP-9 in the supernatant could be autoactivated (from proMMP-9 to active MMP-9), and the physiologic calcium concentration (2.5 mmol/L) could delay the autoactivation of MMP-9. The activities of MMP-2 and MMP-9 in the fresh supernatant increased significantly in response to stimulation by 10 ng/mL of TNFα for 24 h. The stimulatory effect of TNFα just on proMMP-9 was counteracted significantly by CAPE. CONCLUSION: NF-κB could prevent chondrocytes apoptosis though its activation was attributed to the increase of proMMP-9 activity induced by TNFα (a pro-apoptotic factor). Therefore, therapeutic NF-κB inhibitor was a 'double-edged swords' to the apoptosis of chondrocytes and the secretion of MMP-9.

Protective effects of ginsenoside Rg3 on human osteoarthritic chondrocytes.

OBJECTIVES: To explore whether Rg3, a major and especially potent ginsenoside, modulates human osteoarthritic (OA) chondrocyte senescence. METHODS: Isolated chondrocytes were cultured in medium containing interleukin-1 beta (IL-1ß) with or without Rg3. The expression levels of mRNAs encoding aggrecan (ACAN), a major structural proteoglycan, type II collagen (COL2A1), and metalloproteinases (MMP) -1, -3, and -13, respectively, were determined using real-time PCR. Cellular senescence was detected by measuring senescence-associated ß-galactosidase (SA-ß-Gal) activity. Chondrocyte telomerase activity also served as a senescence marker. RESULTS: Chondrocytes stimulated by IL-1ß showed increased MMP-1, MMP-3, and MMP-13 levels, whereas the expression of COL2A1 and ACAN decreased. However, in cells co-treated with IL-1ß and Rg3, the levels of MMP-1 and MMP-13 were lower than in cells treated with IL-1ß alone, and COL2A1 and ACAN expression levels recovered from the low values seen when cultured only in the presence of IL-1ß. Also, compared to vehicle-treated controls, IL-1ß stimulation alone resulted in an increased number of SA-ß-Gal-positive cells, while co-incubation with IL-1ß and Rg3 significantly suppressed the expression of this senescence marker. Chondrocytes cultured with Rg3 showed significantly higher proliferative and telomerase activities than did control cells. CONCLUSIONS: These findings indicate that Rg3 protects the cell against the development of chondrocyte senescence in osteoarthritis.