Loss of mutual protection between human osteoclasts and chondrocytes in damaged joints initiates osteoclast-mediated cartilage degradation by MMPs.

Osteoclasts are multinucleated, bone-resorbing cells. However, they also digest cartilage during skeletal maintenance, development and in degradative conditions including osteoarthritis, rheumatoid arthritis and primary bone sarcoma. This study explores the mechanisms behind the osteoclast-cartilage interaction. Human osteoclasts differentiated on acellular human cartilage expressed osteoclast marker genes (e.g. CTSK, MMP9) and proteins (TRAP, VNR), visibly damaged the cartilage surface and released glycosaminoglycan in a contact-dependent manner. Direct co-culture with chondrocytes during differentiation increased large osteoclast formation (p < 0.0001) except when co-cultured on dentine, when osteoclast formation was inhibited (p = 0.0002). Osteoclasts cultured on dentine inhibited basal cartilage degradation (p = 0.012). RNA-seq identified MMP8 overexpression in osteoclasts differentiated on cartilage versus dentine (8.89-fold, p = 0.0133), while MMP9 was the most highly expressed MMP. Both MMP8 and MMP9 were produced by osteoclasts in osteosarcoma tissue. This study suggests that bone-resident osteoclasts and chondrocytes exert mutually protective effects on their 'native' tissue. However, when osteoclasts contact non-native cartilage they cause degradation via MMPs. Understanding the role of osteoclasts in cartilage maintenance and degradation might identify new therapeutic approaches for pathologies characterized by cartilage degeneration.

d-Mannose suppresses osteoarthritis development in vivo and delays IL-1ß-induced degeneration in vitro by enhancing autophagy activated via the AMPK pathway.

Osteoarthritis (OA) is a heterogeneous disease that is consistently difficult to treat due to the complexity of the regulatory network involved in OA pathogenesis, especially in terms of cartilage degeneration. As a C-2 epimer of glucose, d-mannose can alleviate bone loss and repress immunopathology by upregulating regulatory T cells; however, the role of d-mannose in OA-related cartilage degeneration remains unknown. In this study, we investigated the chondroprotective effect of d-mannose in vitro and in vivo on OA. We found that incubating interleukin (IL)-1ß-treated rat chondrocytes with d-mannose restrained OA degeneration by elevating cell proliferation, strongly activating autophagy, reducing apoptosis, and downregulating catabolism. Additionally, oral gavage administration of d-mannose to monosodium iodoacetate (MIA)-treated rats revealed that a median (1.25 g/kg/day) rather than high or low dose of d-mannose suppressed OA progression and attenuated OA development based on lower macroscopic scores for cartilage, decreased histological scores for cartilage and synovium, strongly activated autophagy, and downregulated catabolism. In terms of a downstream mechanism, we showed that d-mannose might attenuate OA degeneration by activating autophagy in IL-1ß-treated rat chondrocytes by promoting the phosphorylation of 5' AMP-activated protein kinase (AMPK). Our in vitro findings revealed that d-mannose delayed IL-1ß-induced OA degeneration in rat chondrocytes by enhancing autophagy activation through the AMPK pathway. Furthermore, the in vivo results indicated that a median dose of d-mannose suppressed MIA-induced OA development. These results suggested that d-mannose exhibits chondroprotective effects and represents a potential disease-modifying drug and novel therapeutic agent for OA.

Long noncoding RNA LINC00671 exacerbates osteoarthritis by promoting ONECUT2-mediated Smurf2 expression and extracellular matrix degradation.

Accumulating evidence has highlighted the remarkable role of long noncoding RNAs (lncRNAs) in the pathogenesis of various diseases including osteoarthritis (OA). Since current treatment available for OA has limited efficacy, it is urgent to elucidate the pathogenesis of OA. Therefore, we aimed at elucidating the specific regulatory role of LINC00671 in OA progression. Differentially expressed lncRNAs were initially screened using the OA profile. LINC00671, ONECUT2, and Smurf2 expression in OA cartilage tissues were determined, while their interaction was verified by RNA-pull down assay, ChIP, and dual-luciferase reporter gene assay. After chondrocytes were transfected with shRNA and overexpressed plasmids, the proliferation and apoptosis were determined. Meanwhile, extracellular matrix (ECM)-related proteins were detected by Western blot analysis. Establishment of the OA model was performed by surgical destabilization of the medial meniscus (DMM) surgery in mice. Upregulation of LINC00671, ONECUT2, and Smurf2 expression were detected in OA cartilage. LINC00671 was bound to ONECUT2 and ONECUT2 was conjugated to Smurf2. Overexpression of LINC00671 resulted in inhibited chondrocytes proliferation, enhanced apoptosis, and ECM degradation, which was readily reversed by silencing ONECUT2 or Smurf2. Furthermore, LINC00671 induced GSK-3ß ubiquitination and upregulated ß-catenin expression through Smurf2. In vivo experiment revealed that silencing of LINC00671 or GSK-3ß activator resulted in alleviated ECM degradation and ameliorated OA progression. Collectively, these data demonstrated that LINC00671 exacerbates OA progression through GSK-3ß ubiquitination by upregulating ONECUT2-mediated Smurf2.

PTH1-34 inhibited TNF-α expression and antagonized TNF-α-induced MMP13 expression in MIO mice.

This study aims to investigate the effects and mechanisms of parathyroid hormone [1-34] (PTH1-34) on TNF-α-stimulated mice chondrocytes, as well as cartilage from a meniscus injury induced osteoarthritis (MIO) mice model. The C57BL/6J mice received medial meniscectomy, and then administrated with PTH1-34. The results showed that PTH1-34 administration decreased secondary allodynia and the pain-related transcripts. The IHC, ELISA, Micro-CT imaging and histopathology analysis revealed the significantly improved subchondral plate thickness and bone porosity, the reduced pro-inflammatory cytokines in serum and joint fluid. In vitro, mice chondrocyte was treated with TNF-α or co-cultured with synovial cells. The results showed that TNF-α markedly upregulated the MMP13 expression, and the ERK1/2, NF-κB or PI3K signaling pathway inhibitors could reverse the induction effect of TNF-α on expression of MMP13 in chondrocytes. PTH1-34 alone has no effect on the expression of MMP13 and NF-κB signaling pathways, but the PTH1-34 could reverse the induction effect of TNF-α on MMP13 expression and NF-κB signaling pathway activation in chondrocytes. In addition, PTH1-34 administration inhibited the expression of TNF-α and MMP13, and chondrocyte viability, while the PKA repressor reversed the effect of PTH1-34 in chondrocytes co-cultured with synovial cells. In conclusion, PTH1-34 has an obvious analgesic and anti-inflammatory effect, inhibits the matrix synthesis and alleviates the progression of osteoarthritis. In vitro, PTH1-34 inhibited TNF-α expression and antagonized TNF-α-induced MMP13 expression via the PKA pathway and the NF-κB signaling pathways, respectively.

Acacetin Suppresses IL-1ß-Induced Expression of Matrix Metalloproteinases in Chondrocytes and Protects against Osteoarthritis in a Mouse Model by Inhibiting NF-κB Signaling Pathways.

Osteoarthritis (OA) is a very common chronic joint dysfunction, and there is currently a poor understanding of its etiology and pathogenesis. Therefore, there are no active disease-modifying drugs currently available for clinical treatment. Several natural compounds have been shown to play a role in inhibiting OA progression. The present study is aimed at investigating the curative effects of acacetin, a natural flavonoid compound, against OA. Our results demonstrated that MMP-1, MMP-3, and MMP-13 were highly expressed in OA specimens. Acacetin inhibited the interleukin-1ß- (IL-1ß-) induced expression of MMP-1, MMP-3, and MMP-13in chondrocytes by blocking nuclear factor-κB (NF-κB) signaling pathways. Furthermore, we found that acacetin suppressed OA progression and inhibited the expression of MMP-1, MMP-3, and MMP-13 in ACLT-induced OA mice. Taken together, our study revealed that acacetin may serve as a potential drug for treating OA.

Down-regulated ciRS-7/up-regulated miR-7 axis aggravated cartilage degradation and autophagy defection by PI3K/AKT/mTOR activation mediated by IL-17A in osteoarthritis.

Osteoarthritis (OA) is one of the most painful and widespread chronic degenerative joint diseases and is characterized by destructed articular cartilage and inflamed joints. Previously, our findings indicated that circular RNA ciRS-7 (ciRS-7)/microRNA 7 (miR-7) axis is abnormally expressed in OA, and regulates proliferation, inflammatory responses, and apoptosis of interleukin-1ß (IL-1ß)-stimulated chondrocytes. However, its underlying role in OA remains unknown. In this study, we first validated cartilage degradation and defection of autophagy in samples of OA patients. IL-1ß initially stimulated autophagy of chondrocytes, and ultimately significantly suppressed autophagy. Upregulated ciRS-7/down-regulated miR-7 aggravated IL-1ß-induced cartilage degradation, and restrained autophagy in vitro. Gene sequencing and bioinformatics analysis performed on a control group, IL-1ß group, and IL-1ß+miR-7-mimics group demonstrated that seven of the most significant mRNA candidates were enriched in the interleukin-17 (IL-17) signaling pathway. Increased IL-17A levels were also observed by qRT-PCR and ELISA. In addition, it was revealed that the ciRS-7/miR-7 axis ameliorated cartilage degradation and defection of autophagy by PI3K/AKT/mTOR activation in IL-1ß-induced chondrocytes. Furthermore, an OA model was established in rats with medial meniscus destabilization. miR-7-siRNA-expressing lentiviruses alleviated surgical resection-induced cartilage destruction of OA mice, whereas miR-7 mimics worsened the effects. Thus, these findings revealed that the mechanism of the ciRS-7/miR-7 axis involved regulating OA progression and provided valuable directions for OA treatment.

Inhibition of PI3K/Akt/NF-κB signaling by Aloin for ameliorating the progression of osteoarthritis: In vitro and in vivo studies.

Osteoarthritis (OA) is a progressive and degenerative joint disease. Aloin is a bitter and yellow-brown-coloured compound from the Aloe plant and is allowed for use in foods as a "natural flavour". In our study, we examined the protective effects of Aloin on the inhibition of OA development as well as its underlying mechanism in both in vitro and vivo experiments. In in-vitro experiments, the protective effect of aloin on the anabolism and catabolism of the extracellular matrix (ECM) induced by IL-1 ß in chondrocytes by inhibiting the expression of pro-inflammatory factors, including TNF-α (p = 0.016), IL-6 (p = 0.006), iNOS (p = 0.001) and COX-2 (p = 0.006). Mechanistically, Aloin suppressed the IL-1ß-induced activation of the PI3K/Akt/NF-κB signalling pathway cascades. Moreover, molecular docking studies demonstrated that Aloin bound strongly to PI3K. In vivo, Aloin ameliorated the OA process in the destabilization of the medial meniscus (DMM) model. In summary, our findings demonstrate that Aloin ameliorates the progression of OA via the PI3K/Akt/NF-κB signalling pathways, which supports Aloin as a promising therapeutic agent for the treatment of OA.

Chondroprotective Actions of Selective COX-2 Inhibitors In Vivo: A Systematic Review.

Knee osteoarthritis (OA) is a condition mainly characterized by cartilage degradation. Currently, no effective treatment exists to slow down the progression of OA-related cartilage damage. Selective COX-2 inhibitors may, next to their pain killing properties, act chondroprotective in vivo. To determine whether the route of administration is important for the efficacy of the chondroprotective properties of selective COX-2 inhibitors, a systematic review was performed according to the PRISMA guidelines. Studies investigating OA-related cartilage damage of selective COX-2 inhibitors in vivo were included. Nine of the fourteen preclinical studies demonstrated chondroprotective effects of selective COX-2 inhibitors using systemic administration. Five clinical studies were included and, although in general non-randomized, failed to demonstrate chondroprotective actions of oral selective COX-2 inhibitors. All of the four preclinical studies using bolus intra-articular injections demonstrated chondroprotective actions, while one of the three preclinical studies using a slow release system demonstrated chondroprotective actions. Despite the limited evidence in clinical studies that have used the oral administration route, there seems to be a preclinical basis for considering selective COX-2 inhibitors as disease modifying osteoarthritis drugs when used intra-articularly. Intra-articularly injected selective COX-2 inhibitors may hold the potential to provide chondroprotective effects in vivo in clinical studies.

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.

Long non-coding RNA XIST contributes to osteoarthritis progression via miR-149-5p/DNMT3A axis.

Long non-coding RNAs (lncRNAs) are largely involved in the development of osteoarthritis (OA), a chronic and degenerative joint disease. The objective of this paper is to research the functional role and molecular mechanism of lncRNA X inactive specific transcript (XIST) in OA. The levels of XIST, microRNA-149-5p (miR-149-5p), and DNA methyltransferase 3A (DNMT3A) were measured. Cell viability and apoptosis rate were determined. Associated protein levels were examined through Western blot. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were implemented for confirming the target relation. And the role of XIST on OA in vivo was investigated by a rat model. XIST was expressed at a high level in OA cartilage tissues and IL-1ß-treated chondrocytes. XIST knockdown promoted cell viability but restrained cell apoptosis and extracellular matrix (ECM) protein degradation in IL-1ß-treated chondrocytes. XIST directly targeted miR-149-5p and miR-149-5p down-regulation restored si-XIST-mediated pro-proliferative and anti-apoptotic or ECM degradative effects. DNMT3A was a target gene of miR-149-5p and DNMT3A overexpression ameliorated miR-149-5p-induced promotion of cell viability but repression of apoptosis and ECM degradation. Knockdown of XIST reduced DNMT3A level by motivating miR-149-5p expression. The inhibitory influence of XIST down-regulation on OA evolvement was also achieved by miR-149-5p/DNMT3A axis in vivo. In a word, knockdown of XIST can repress the development of OA by miR-149-5p/DNMT3A axis. This study discovers the XIST/miR-149-5p/DNMT3A axis in regulating OA evolution, which is beneficial for understanding the molecular pathomechanism and can lay a good foundation for targeted therapy of OA treatment.

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.

miR-122/SIRT1 axis regulates chondrocyte extracellular matrix degradation in osteoarthritis.

BACKGROUND/AIMS: MicroRNAs (miRNAs) are involved in the pathogenesis of osteoarthritis (OA). The present study aimed to investigate the potential function of miR-122 in the development of OA and its potential molecular mechanisms. METHODS: The expression of miR-122, silent information regulator 1 (SIRT1), collagen II, aggrecan, matrix metalloproteinase (MMP) 13 (MMP13) and ADAMTS4 in OA cartilage was detected by RT-qPCR. Target gene prediction and screening, luciferase reporter assay were used to verify downstream target genes of miR-122. RESULTS: Compared with osteonecrosis, the expression of miR-122 was significantly increased in OA cartilage, while the expression of SIRT1 was significantly decreased. Overexpression of miR-122 increased the expression of extracellular matrix (ECM) catabolic factors, for example disintegrins, MMPs and metalloproteinases with platelet reaction protein motifs, and inhibited the expression of synthetic metabolic genes such as collagen II and aggregating proteoglycan. Inhibition of miR-122 expression had the opposite effect. Furthermore, SIRT1 was identified as a direct target of miR-122. SIRT1 was significantly inhibited by miR-122 overexpression. Knockdown of SIRT1 reversed the degradation of chondrocyte ECM by miR-122 inhibitors. CONCLUSION: The miR-122/SIRT1 axis can regulate the degradation of ECM in OA, thus providing new insights into the treatment of OA.

miR-137 targets the inhibition of TCF4 to reverse the progression of osteoarthritis through the AMPK/NF-κB signaling pathway.

PURPOSE: To explore the regulatory mechanism of miR-137 and transcription factor 4 (TCF4) in the progression of osteoarthritis (OA). PATIENTS AND METHODS: The expressions of miR-137 and TCF4 were detected in OA cartilage tissue, chondrocytes and OA rat cartilage tissue. miR-137 and TCF4 were up-regulated or down-regulated and transfected into chondrocytes and OA rat cartilage tissue. The gene expression, protein level, cell proliferation, apoptosis and inflammatory factors were detected, respectively. LPS and anterior cruciate ligament transection (ACLT) on the right knee were used to induce chondrocyte inflammation and establish rat OA model, respectively. RESULTS: miR-137 was low expressed in cartilage tissue of OA group, while TCF4 expression and protein level were significantly higher, showing significant negative correlation. In LPS group, chondrocyte activity was significantly inhibited, cell apoptosis ability was significantly enhanced, and the levels of inflammatory factors TNF-α, IL-1ß, IL-6 were significantly increased. However, the above results were significantly improved after the up-regulation of miR-137 or down-regulation of TCF4. Double luciferase report revealed that miR-137 and TCF4 had targeted relationship. LPS induced activation of AMPK/NF-κB pathway and higher level of apoptosis. AMPK/NF-κB pathway inhibitor C could inhibit activation of this pathway, and up-regulation of miR-137 or down-regulation of TCF4 could significantly weaken the regulation of LPS on the pathway and apoptosis. Analysis of OA rat model showed that over-expression of miR-137 could inhibit up-regulation of inflammatory factors and activation of AMPK/NF-κB pathway. CONCLUSION: miR-137 targets the inhibition of TCF4 to reverse the progression of OA through the AMPK/NF-κB signaling pathway.

Per2 attenuates LPS-induced chondrocyte injury through the PTEN/PI3K/Akt signalling pathway.

This research aimed to explore the role of period circadian clock 2 (Per2) in the evolution of osteoarthritis (OA) and the relevant mechanisms. Per2 messenger RNA (mRNA) and protein levels were markedly reduced in NHAC-kn cells treated with 5 µg/ml lipopolysaccharide (LPS) for 12 h. Then, pcDNA3.1-Per2 and si-Per2 were recruited to boost and reduce the expression of Per2, respectively. MTT assay, apoptosis analysis and enzyme-linked immunosorbent assay (ELISA) results showed that Per2 increased cell proliferation, while inhibited apoptosis and inflammation. Furthermore, the PTEN/PI3K/Akt signalling pathway was activated by Per2 overexpression; the CO-IP data confirmed that Per2 specifically bound to PTEN. Through employing IGF-1, a PI3K activator, we determined that Per2-mediated inflammation response in LPS-stimulated NHAC-kn cells through the PTEN/PI3K/Akt signalling pathway. In summary, the present study indicates that Per2 may serve as a novel therapeutic target through activating the PTEN/PI3K/Akt signalling pathway.

GCN5 acetylation is required for craniofacial chondrocyte maturation.

Development of the craniofacial structures requires the precise differentiation of cranial neural crest cells into osteoblasts or chondrocytes. Here, we explore the epigenetic and non-epigenetic mechanisms that are required for the development of craniofacial chondrocytes. We previously demonstrated that the acetyltransferase activity of the highly conserved acetyltransferase GCN5, or KAT2A, is required for murine craniofacial development. We show that Gcn5 is required cell autonomously in the cranial neural crest. Moreover, GCN5 is required for chondrocyte development following the arrival of the cranial neural crest within the pharyngeal arches. Using a combination of in vivo and in vitro inhibition of GCN5 acetyltransferase activity, we demonstrate that GCN5 is a potent activator of chondrocyte maturation, acting to control chondrocyte maturation and size increase during pre-hypertrophic maturation to hypertrophic chondrocytes. Rather than acting as an epigenetic regulator of histone H3K9 acetylation, our findings suggest GCN5 primarily acts as a non-histone acetyltransferase to regulate chondrocyte development. Here, we investigate the contribution of GCN5 acetylation to the activity of the mTORC1 pathway. Our findings indicate that GCN5 acetylation is required for activation of this pathway, either via direct activation of mTORC1 or through indirect mechanisms. We also investigate one possibility of how mTORC1 activity is regulated through RAPTOR acetylation, which is hypothesized to enhance mTORC1 downstream phosphorylation. This study contributes to our understanding of the specificity of acetyltransferases, and the cell type specific roles in which these enzymes function.

Prematurely aging mitochondrial DNA mutator mice display subchondral osteopenia and chondrocyte hypertrophy without further osteoarthritis features.

Mitochondrial mutations and dysfunction have been demonstrated in several age-related disorders including osteoarthritis, yet its relative contribution to pathogenesis remains unknown. Here we evaluated whether premature aging caused by accumulation of mitochondrial DNA mutations in PolgD275A mice predisposes to the development of knee osteoarthritis. Compared with wild type animals, homozygous PolgD275A mice displayed a specific bone phenotype characterized by osteopenia of epiphyseal trabecular bone and subchondral cortical plate. Trabecular thickness was significantly associated with osteocyte apoptosis rates and osteoclasts numbers were increased in subchondral bone tissues. While chondrocyte apoptosis rates in articular and growth plate cartilage were similar between groups, homozygous mitochondrial DNA mutator mice displayed elevated numbers of hypertrophic chondrocytes in articular calcified cartilage. Low grade cartilage degeneration, predominantly loss of proteoglycans, was present in all genotypes and the development of osteoarthritis features was not found accelerated in premature aging. Somatically acquired mitochondrial DNA mutations predispose to elevated subchondral bone turnover and hypertrophy in calcified cartilage, yet additional mechanical or metabolic stimuli would seem required for induction and accelerated progression of aging-associated osteoarthritis.

Up-regulation of P21-activated kinase 1 in osteoarthritis chondrocytes is responsible for osteoarthritic cartilage destruction.

Osteoarthritis is mainly caused by a degenerative joint disorder, which is characterized by the gradual degradation of articular cartilage and synovial inflammation. The chondrocyte, the unique resident cell type of articular cartilage, is crucial for the development of osteoarthritis. Previous studies revealed that P21-activated kinase-1 (PAK1) was responsible for the initiation of inflammation. The purpose of the present study was to determine the potential role of PAK1 in osteoarthritis. The level of PAK1 expression was measured by Western blot and quantitative real-time PCR in articular cartilage from osteoarthritis model rats and patients with osteoarthritis. In addition, the functional role of aberrant PAK1 expression was detected in the chondrocytes. We found that the expression of PAK1 was significantly increased in chondrocytes treated with osteoarthritis-related factors. Increased expression of PAK1 was also observed in knee articular cartilage samples from patients with osteoarthritis and osteoarthritis model rats. PAK1 was found to inhibit chondrocytes proliferation and to promote the production of inflammatory cytokines in cartilages chondrocytes. Furthermore, we found that PAK1 modulated the production of extracellular matrix and cartilage degrading enzymes in chondrocytes. Results of the present studies demonstrated that PAK1 might play an important role in the pathogenesis of osteoarthritis.

4-Phenylbutyric Acid Reduces Endoplasmic Reticulum Stress in Chondrocytes That Is Caused by Loss of the Protein Disulfide Isomerase ERp57.

OBJECTIVE: The integrity of cartilage depends on the correct synthesis of extracellular matrix (ECM) components. In case of insufficient folding of proteins in the endoplasmic reticulum (ER) of chondrocytes, ECM proteins aggregate, ER stress evolves, and the unfolded protein response (UPR) is initiated. By this mechanism, chondrocytes relieve the stress condition or initiate cell death by apoptosis. Especially persistent ER stress has emerged as a pathogenic mechanism in cartilage diseases, such as chondrodysplasias and osteoarthritis. As pharmacological intervention is not available yet, it is of great interest to understand cartilage ER stress in detail and to develop therapeutics to intervene. METHODS: ERp57-deficient chondrocytes were generated by CRISPR/Cas9-induced KO. ER stress and autophagy were studied on mRNA and protein level as well as by transmission electron microscopy (TEM) in chondrocyte micromass or cartilage explant cultures of ERp57 KO mice. Thapsigargin (Tg), an inhibitor of the ER-residing Ca2+-ATPase, and 4-Phenylbutyric acid (4-PBA), a small molecular chemical chaperone, were applied to induce or inhibit ER stress. RESULTS: Our data reveal that the loss of the protein disulfide isomerase ERp57 is sufficient to induce ER stress in chondrocytes. 4-PBA efficiently diffuses into cartilage explant cultures and diminishes excessive ER stress in chondrocytes dose dependently, no matter if it is induced by ERp57 KO or stimulation with Tg. CONCLUSION: ER-stress-related diseases have different sources; therefore, various targets for therapeutic treatment exist. In the future, 4-PBA may be used alone or in combination with other drugs for the treatment of ER-stress-related skeletal disorders in patients.

Effect of luteolin on apoptosis, MAPK and JNK signaling pathways in guinea pig chondrocyte with osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease usually seen in the elderly, which incidence increases with age. Its pathogenesis and underlying mechanism are still unclear. The disease severely affects the physical health and life quality of patients, thereby constituting a huge economic burden to family and society. Luteolin (LUT) is a natural flavonoid with multiple pharmacological properties. Many plants containing LUT have been applied in the treatment of several inflammation-related diseases due the relatively strong anti-inflammatory effects of LUT. The present study investigated the influence of LUT on cell apoptosis and inflammatory reactions in cartilage of OA guinea pigs, and its underlying mechanism. It was found that LUT effectively inhibited proliferation of OA cartilage cells, down-regulated the expressions of JNK and p38MAPK in cartilage cells of OA, and downregulated NO, TNF-α and IL-6. Thus, it alleviated inflammatory reactions, protected cartilage cells, and delayed cartilage degeneration.

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.