ATDC5 Chondrocytes Research Articles

Downregulation of heat shock protein 47 caused lysosomal dysfunction leading to excessive chondrocyte apoptosis

Heat shock protein 47 (HSP47) is a collagen-specific chaperone present in several regions of the endoplasmic reticulum and cytoplasm. Elevated HSP47 expression in cells causes various cancers and fibrotic disorders. However, the consequences of HSP47 downregulation leading to chondrocyte death, as well as the underlying pathways, remain largely unclear. This study presents the first experimental evidence of the localization of HSP47 on lysosomes. Additionally, it successfully designed and generated shRNA HSP47 target sequences to suppress the expression of HSP47 in ATDC5 chondrocytes using lentiviral vectors. By employing a chondrocyte model that has undergone stable downregulation of HSP47, we observed that HSP47 downregulation in chondrocytes, disturbs the acidic homeostatic environment of chondrocyte lysosomes, causes hydrolytic enzyme activity dysregulation, impairs the lysosome-mediated autophagy-lysosome pathway, and causes abnormal expression of lysosomal morphology, number, and functional effector proteins. This implies the significance of the presence of HSP47 in maintaining proper lysosomal function. Significantly, the inhibitor CA-074 Me, which can restore the dysfunction of lysosomes, successfully reversed the negative effects of HSP47 on the autophagy-lysosomal pathway and partially reduced the occurrence of excessive cell death in chondrocytes. This suggests that maintaining proper lysosomal function is crucial for preventing HSP47-induced apoptosis in chondrocytes. The existence of HSP47 is crucial for preserving optimal lysosomal function and autophagic flux, while also inhibiting excessive apoptosis in ATDC5 chondrocytes.

Syringic Acid Attenuates the IL-1β-Induced Akt Pathway in Chondrocyte ATDC5 Cells.

Osteoarthritis (OA) is a chronic progressive joint ailment that is largely predominant worldwide. However, it typically gets worse over time, occurs more frequently, and becomes more crippling. Syringic acid (SA) is a well-known phenolic compound reported to suppress inflammation, cell proliferation, and apoptosis of various cancer cells. Since the role of SA in OA remains unknown, there is a need to hypothesize the anti-inflammatory activities of SA on IL- 1β-induced ATDC5 chondrocyte‑like cells and to elucidate its protective action against OA. The cytotoxicity, inflammatory mediators, mRNA expression of MMPs, ADAMTS, COX-2, and Akt/NF-κB protein expression of SA activity on ATDC5 cells were examined through CCK-8 assay, ELISA, RT-qPCR, and western blot. It was found that SA (10, 20, and 30 μM) did not show any inhibitory effects on the viability of the ATDC5 cells in a concentrationdependent manner. SA markedly reduced the inflammatory mediators, cytokines, PGE2, MMPs, COX-2, and ADAMTS in a concentration-dependent manner. Likewise, SA expressively attenuated IL- 1β-stimulated Akt phosphorylation and NF-κB activation as well as IL-1β- induced ATDC5 chondrocytes. This study revealed that SA is a novel candidate applicable for the treatment of OA.

Catalpol protects mouse ATDC5 chondrocytes against interleukin-1β-induced catabolism.

Catalpol is a natural product with promising anti-inflammatory effects, however, its effects on chondrocytes and osteoarthritis (OA) have not been well investigated. OA is a painful and debilitating joint disease that affects people worldwide. Traditional Chinese Medicine has been sought to treat OA, including the Rehmannia extract, Catalpol. Here, we examined the effects of Catalpol, a plant derivative used in traditional Chinese medicine, on ATDC5 chondrocytes originating from mouse teratocarcinoma cells stimulated with interleukin-1β (IL-1β) to mimic the OA cellular environment. Catalpol significantly reduced matrix metalloproteinase-1, -3, -13 (MMP-1, -3, -13), a disintegrin and metalloproteinase with thrombospondin motifs -4, -5 (ADAMTS-4, -5) against IL-1β, demonstrating a likely anti-cartilage degradation activity. We also found that Catalpol exerted a significant anti-oxidative stress effect by downregulating the production of inducible nitric oxide synthase (iNOS), nitric oxide (NO), reactive oxygen species (ROS), and malondialdehyde (MDA). Catalpol treatment significantly reduced the levels of several key inflammatory factors, including Prostaglandin E₂ (PGE₂), cyclooxygenase-2 (COX-2), interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1). We further demonstrate that the effects of Catalpol were mediated by the nuclear factor -κB (NF-κB) pathway via downregulation of the phosphorylation of inhibitor of nuclear factor κB-α (IκBα). This was confirmed by measuring p38 and p65 protein levels as well as the luciferase activity of NF-κB. Altogether, we demonstrate the potential of Catalpol as a novel treatment agent against cartilage matrix degradation, oxidative stress, and inflammation in OA.

General Control Non-derepressible 2 Alleviates Cartilage Degeneration and Inhibits NLRP3 Inflammasome Activation in Osteoarthritis.

This study aimed to evaluate the effects of general control non-derepressible 2 (GCN2) on osteoarthritis (OA) in vivo and in vitro. First, anterior cruciate ligament transection (ACLT)-induced rat model and interleukin (IL)-1β-induced ATDC5 chondrocyte were established. Hematoxylin and eosin staining and safranin O/fast green staining were employed for analyzing the histological changes in the rat cartilage. In addition, immunohistochemistry, quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, western blot, and immunofluorescence staining were employed for examining cartilage degeneration-, inflammation-, autophagy-, and NLR family pyrin domain containing 3 (NLRP3) inflammasome-associated genes expression. Moreover, 2,7-dichlorodihydrofluorescein acetoacetic acid probe was utilized for examining the intracellular reactive oxygen species. In addition, 5-ethynyl-2'-deoxyuridine assay and flow cytometry were applied for detecting chondrocyte proliferation and apoptosis IL-1β-treated ATDC5 chondrocytes. GCN2 overexpression ameliorated articular cartilage degeneration and inflammation but promoted chondrocyte autophagy in ACLT-induced OA rats. Similarly, we demonstrated that the upregulation of GCN2 could promote chondrocyte proliferation, suppress chondrocyte apoptosis, attenuate chondrocyte inflammation and extracellular matrix degradation, and promote chondrocyte autophagy. Moreover, GCN2 overexpression could inhibit the activation of NLRP3 inflammasome in IL-1β-induced ATDC5 chondrocyte. Furthermore, 3-methyladenine neutralized the protective and autophagy-promoting effects of GCN2 overexpression on ATDC5 chondrocytes. GCN2 could attenuate inflammation and cartilage degeneration, promote chondrocyte autophagy, and inhibit NLRP3 inflammasome activation in OA.

Resatorvid alleviates experimental inflammatory TMJOA by restraining chondrocyte pyroptosis and synovial inflammation

ObjectivesInnate immunity plays a significant role in the pathogenesis of temporomandibular joint osteoarthritis (TMJOA), which is characterized by synovial inflammation and condylar cartilage degradation. We are urged to investigate the impact of Resatorvid, a preventative drug that inhibits Toll-like receptor 4 (TLR4), on experimental inflammatory TMJOA pathology.MethodsAn intra-articular injection of complete Freund’s adjuvant (CFA) was used to induce an experimental inflammatory mouse TMJOA model, and TLR4 expression was identified by immunofluorescent labeling. Intraperitoneal injections of Resatorvid were administered to CFA-induced TMJOA mice, and the pathology of TMJOA animals with and without Resatorvid treatment was examined by H&E, Safranin-O/Fast Green, and TRAP staining, as well as micro-CT, immunohistochemistry, and immunofluorescence. The impact of Resatorvid on chondrocyte pyroptosis and macrophage inflammation was further investigated using ATDC5 chondrocytes and RAW264.7 macrophages pretreated with relevant antagonists.ResultsCFA-induced TMJOA mice revealed remarkable synovial inflammation, together with a time course of cartilage degradation and bone destruction, with TLR4 elevated in the synovium and condylar cartilage. Prophylactic treatment with Resatorvid mitigated synovial inflammation, cartilage degeneration, and bone destruction in CFA-induced TMJOA mice and downregulated MyD88/NF-κB expression. Ex vivo studies demonstrated that Resatorvid treatment alleviated NOD-like receptor protein 3 (NLRP3)-mediated chondrocyte pyroptosis and degeneration and relieved macrophage inflammation by preventing reactive oxygen species (ROS) production through NLRP3 signaling.ConclusionProphylactic treatment with Resatorvid alleviates TMJOA pathology by inhibiting chondrocyte pyroptosis and degeneration, as well as ROS-induced macrophage inflammation, through TLR4/MyD88/NF-κB/NLRP3.

Pyroptosis of chondrocytes activated by synovial inflammation accelerates TMJ osteoarthritis cartilage degeneration via ROS/NLRP3 signaling

ObjectivesSynovial inflammation and chondrocyte death have been widely acknowledged as key contributors to the pathological progression of temporomandibular joint osteoarthritis (TMJ-OA), a degenerative joint disease currently lacking definitive treatments. This study aims to understand the regulatory role of chondrocyte pyroptosis in condylar cartilage degradation during TMJ-OA. MethodsThe levels of cytokines, cartilage degeneration markers, and pyroptotic biomarkers in the synovium and synovial fluid of temporomandibular disorders (TMD) patients were examined. The synovitis, cartilage degradation, and chondrocyte pyroptosis in wild-type and alpha-kinase 1 (ALPK1)-deficient TMJ-OA mice were then compared following monosodium iodoacetate (MIA) induction. Subsequently, we investigated the downstream mechanisms of cytokines- or macrophage supernatants-induced metabolic disorders and pyroptosis in chondrocytes using primary TMJ chondrocytes and ATDC5 chondrocyte cultures. ResultsWe found a positive correlation between pyroptotic biomarkers and cartilage degradation mediators and cytokines in the synovial fluid of TMD patients. MIA-induced TMJ-OA mice demonstrated significant synovitis, cartilage degradation, and chondrocyte pyroptosis, which were mitigated in ALPK1-deficient TMJ-OA mice, inflammation-restrained mice. Ex-vivo study revealed the contribution of reactive oxygen species (ROS) to inflammation-irritated macrophage supernatants-induced pyroptosis and metabolic disorders in chondrocytes. Targeting NOD-like receptor protein 3 (NLRP3) alleviated cytokines- or ROS-induced pyroptosis and metabolic disorders in chondrocytes by inhibiting caspase-1 activation and interleukin-1β (IL-1β) secretion. ConclusionOur findings offer novel insight into the role of synovial inflammation-induced chondrocyte pyroptosis in promoting cartilage degradation during TMJ-OA via the ROS and NLRP3 signaling pathway.

Tanshinone IIA alleviates chondrocyte apoptosis and extracellular matrix degeneration by inhibiting ferroptosis.

Articular cartilage degeneration caused by chondrocyte damage is the primary pathological mechanism of osteoarthritis (OA). Oxidative stress is correlated with chondrocyte injury by potentiating ferroptosis, a newly identified form of cell death. Given the effects of Tanshinone IIA (Tan IIA) on alleviating oxidative stress, we further explored whether Tan IIA inhibited chondrocyte death and cartilage degeneration by decreasing ferroptosis. ATDC5 chondrocytes were treated with lipopolysaccharides (LPS) and Tan IIA, and cell viability was assessed using cell counting kit-8 (CCK-8) assays. Matrix metalloproteinase-13 (MMP13), a disintegrin and metalloproteinase with thrombospondin motif-5 (ADAMTS5), and type II collagen (Col II) levels were measured using quantitative real-time polymerase chain reaction (qRT‒PCR), western blotting, and immunofluorescence (IF) analysis. We demonstrated that Tan IIA treatment prominently increased ATDC5 cell viability and decreased cell apoptosis in the presence of LPS-induced stress. MMP13 and ADAMTS5 expression was increased, and Col II expression was decreased in ATDC5 cells after LPS stimulation, whereas these changes were reversed by Tan IIA. Mechanistically, Tan IIA inhibited LPS-induced ferroptosis in ATDC5 cells, as indicated by decreased levels of iron, reactive oxygen species, and malondialdehyde and increased GSH levels. Importantly, a ferroptosis agonist partially abrogated the effect of Tan IIA on alleviating chondrocyte damage and death. Taken together, these results suggest that Tan IIA ameliorates chondrocyte apoptosis and cartilage degeneration by inhibiting ferroptosis and may be a potential therapeutic agent for OA.

FOXO 1 deletion in chondrocytes rescues diabetes-impaired fracture healing by restoring angiogenesis and reducing apoptosis.

Diabetes mellitus is associated with higher risks of long bone and jaw fractures. It is also associated with a higher incidence of delayed union or non-union. Our previous investigations concluded that a dominant mechanism was the premature loss of cartilage during endochondral bone formation associated with increased osteoclastic activities. We tested the hypothesis that FOXO1 plays a key role in diabetes-impaired angiogenesis and chondrocyte apoptosis. Closed fractures of the femur were induced in mice with lineage-specific FOXO1 deletion in chondrocytes. The control group consisted of mice with the FOXO1 gene present. Mice in the diabetic group were rendered diabetic by multiple streptozotocin injections, while mice in the normoglycemic group received vehicle. Specimens were collected 16 days post fracture. The samples were fixed, decalcified, and embedded in paraffin blocks for immunostaining utilizing anti cleaved caspase-3 or CD31 specific antibodies compared with matched control IgG antibody, and apoptosis by the TUNEL assay. Additionally, ATDC5 chondrocytes were examined in vitro by RT-PCR, luciferase reporter and chromatin immunoprecipitation assays. Diabetic mice had ~ 50% fewer blood vessels compared to normoglycemic mice FOXO1 deletion in diabetic mice partially rescued the low number of blood vessels (p < 0.05). Additionally, diabetes increased caspase-3 positive and apoptotic chondrocytes by 50%. FOXO1 deletion in diabetic animals blocked the increase in both to levels comparable to normoglycemic animals (p < 0.05). High glucose (HG) and high advanced glycation end products (AGE) levels stimulated FOXO1 association with the caspase-3 promoter in vitro, and overexpression of FOXO1 increased caspase-3 promoter activity in luciferase reporter assays. Furthermore, we review previous mechanistic studies demonstrating that tumor necrosis factor (TNF) inhibition reverses impaired angiogenesis and reverses high levels of chondrocyte apoptosis that occur in fracture healing. New results presented here, in combination with recent studies, provide a comprehensive overview of how diabetes, through high glucose levels, AGEs, and increased inflammation, impair the healing process by interfering with angiogenesis and stimulating chondrocyte apoptosis. FOXO1 in diabetic fractures plays a negative role by reducing new blood vessel formation and increasing chondrocyte cell death which is distinct from its role in normal fracture healing.

Collagen-Derived Dipeptide Pro-Hyp Enhanced ATDC5 Chondrocyte Differentiation under Hypoxic Conditions.

Chondrocytes are surrounded by a lower oxygen environment than other well-vascularized tissues with higher oxygenation levels. Prolyl-hydroxyproline (Pro-Hyp), one of the final collagen-derived peptides, has been previously reported to be involved in the early stages of chondrocyte differentiation. However, whether Pro-Hyp can alter chondrocyte differentiation under physiological hypoxic conditions is still unclear. This study aimed to investigate whether Pro-Hyp affects the differentiation of ATDC5 chondrogenic cells under hypoxic conditions. The addition of Pro-Hyp resulted in an approximately 18-fold increase in the glycosaminoglycan staining area compared to the control group under hypoxic conditions. Moreover, Pro-Hyp treatment significantly upregulated the expression of SOX9, Col2a1, Aggrecan, and MMP13 in chondrocytes cultured under hypoxic conditions. These results demonstrate that Pro-Hyp strongly promotes the early differentiation of chondrocytes under physiological hypoxic conditions. Therefore, Pro-Hyp, a bioactive peptide produced during collagen metabolism, may function as a remodeling factor or extracellular matrix remodeling signal that regulates chondrocyte differentiation in hypoxic cartilage.

Teriparatide ameliorates articular cartilage degradation and aberrant subchondral bone remodeling in DMM mice.

Knee osteoarthritis (KOA) is a highly prevalent musculoskeletal disorder characterized by degeneration of cartilage and abnormal remodeling of subchondral bone (SCB). Teriparatide (PTH (1-34)) is an effective anabolic drug for osteoporosis (OP) and regulates osteoprotegerin (OPG)/receptor activator of nuclear factor ligand (RANKL)/RANK signaling, which also has a therapeutic effect on KOA by ameliorating cartilage degradation and inhibiting aberrant remodeling of SCB. However, the mechanisms of PTH (1-34) in treating KOA are still uncertain and remain to be explored. Therefore, we compared the effect of PTH (1-34) on the post-traumatic KOA mouse model to explore the potential therapeutic effect and mechanisms. In vivo study, eight-week-old male mice including wild-type (WT) (n​=​54) and OPG-/- (n​=​54) were investigated and compared. Post-traumatic KOA model was created by destabilization of medial meniscus (DMM). WT mice were randomly assigned into three groups: the sham group (WT-sham; n​=​18), the DMM group (WT-DMM; n​=​18), and the PTH (1-34)-treated group (WT-DMM​+​PTH (1-34); n​=​18). Similarly, the OPG-/- mice were randomly allocated into three groups as well. The designed mice were executed at the 4th, 8th, and 12th weeks to evaluate KOA progression. To further explore the chondro-protective of PTH (1-34), the ATDC5 chondrocytes were stimulated with different concentrations of PTH (1-34) in vitro. Compared with the WT-sham mice, significant wear of cartilage in terms of reduced cartilage thickness and glycosaminoglycan (GAG) loss was detected in the WT-DMM mice. PTH (1-34) exhibited cartilage-protective by alleviating wear, retaining the thickness and GAG contents. Moreover, the deterioration of the SCB was alleviated and the expression of PTH1R/OPG/RANKL/RANK were found to increase after PTH (1-34) treatment. Among the OPG-/- mice, the cartilage of the DMM mice displayed typical KOA change with higher OARSI score and thinner cartilage. The damage of the cartilage was alleviated but the abnormal remodeling of SCB didn't show any response to the PTH (1-34) treatment. Compared with the WT-DMM mice, the OPG-/--DMM mice caught more aggressive KOA with thinner cartilage, sever cartilage damage, and more abnormal remodeling of SCB. Moreover, both the damaged cartilage from the WT-DMM mice and the OPG-/--DMM mice were alleviated but only the deterioration of SCB in WT-DMM mice was alleviated after the administration of PTH (1-34). In vitro study, PTH (1-34) could promote the viability of chondrocytes, enhance the synthesis of extracellular matrix (ECM) (AGC, COLII, and SOX9) at the mRNA and protein level, but inhibit the secretion of inflammatory cytokines (TNF-α and IL-6). Both wear of the cartilage was alleviated and aberrant remodeling of the SCB was inhibited in the WT mice, but only the cartilage-protective effect was observed in the OPG-/- mice. PTH (1-34) exhibited chondro-protective effect by decelerating cartilage degeneration in vivo as well as by promoting the proliferation and enhancing ECM synthesis of chondrocytes in vitro. The current investigation implied that the rescue of the disturbed SCB is dependent on the regulation of OPG while the chondro-protective effect is independent of modulation of OPG, which provides proof for the treatment of KOA. Systemic administration of PTH (1-34) could exert a therapeutic effect on both cartilage and SCB in different mechanisms to alleviate KOA progression, which might be a novel therapy for KOA.

AMPK activator decelerates osteoarthritis development by inhibition of β-catenin signaling in chondrocytes.

Osteoarthritis (OA) is a common degenerative joint disease with significant negative impact on the quality of life. It has been reported that abnormal upregulation of β-catenin signaling could lead to OA development; however, the upstream regulatory mechanisms of β-catenin signaling have not been determined. Primary rat chondrocytes and ATDC5 chondrocyte cell line were stimulated with AKT2 and treated with or without metformin, an adenosine 5'-monophosphate-activated protein kinase (AMPK) activator. Westerrn blot analysis, luciferase reporter assay and immunofluorescent (IF) staining were performed to examine changes in β-cateninS552 phosphorylation and β-catenin nuclear translocation in ATDC5 cells and in primary chondrocytes. We found that metformin inhibited β-cateninS552 phosphorylation in ATDC5 cells and in primary chondrocytes in a time-dependent manner. Metformin inhibited β-catenin nuclear translocation and β-catenin reporter activity. In addition, metformin also attenuated the expression of β-catenin downstream target genes. We also demonstrated that metformin inhibited β-cateninS552 phosphorylation in articular cartilage in mice. These findings suggest that metformin may exert its chondro-protective effect at least in part through the inhibition of β-catenin signaling in chondrocytes. This study demonstrated the interaction between AMPK and β-catenin signaling in chondrocytes and defined novel molecular targets for the treatment of OA disease.

Daphnoretin relieves IL-1β-mediated chondrocytes apoptosis via repressing endoplasmic reticulum stress and NLRP3 inflammasome

BackgroundOsteoarthritis (OA), mainly caused by severe joint degeneration, is often accompanied by joint pain and dysfunction syndrome. Inflammatory mediators and apoptosis play key roles in the evolution of OA. It is reported that daphnoretin has significant antiviral and anti-tumor values. The present study aims at investigating the role of daphnoretin in OA.MethodsThe OA mouse model was constructed by performing the destabilization of the medial meniscus through surgery, and the OA cell model was induced in ATDC5 chondrocytes with IL-1β (10 ng/mL) in vitro. Chondrocyte viability and apoptosis were measured by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT), Caspase-3 activity, and flow cytometry. The levels of COX-2, iNOS, TNF-α, IL-6, Bax, Bcl2, cleaved-Caspase3, endoplasmic reticulum stress (ERS) proteins (GRP78, CHOP, ATF6, and Caspase-12), and NLRP3-ASC-Caspase1 inflammasome were determined by quantitative real-time PCR or western blot. The concentrations of TNF-α, IL-6, and PGE2 were tested by enzyme-linked immunosorbent assay. The content of nitrates was detected by the Griess method. In vivo, morphologic differences in knee joint sections and the thickness of the subchondral bone density plate in mice were observed by hematoxylin–eosin (H&E) staining and safranin O-fast green staining.ResultsDaphnoretin effectively choked IL-1β-induced chondrocyte apoptosis and facilitated cell viability. Daphnoretin dose-dependently abated ERS, inflammatory mediators, and the activation of NLRP3 inflammasomes in IL-1β-induced chondrocytes. What’s more, in vivo experiments confirmed that daphnoretin alleviated OA progression in a murine OA model by mitigating inflammation and ERS.ConclusionDaphnoretin alleviated IL-1β-induced chondrocyte apoptosis by hindering ERS and NLRP3 inflammasome.Graphical abstract

Neratinib exerts dual effects on cartilage degradation and osteoclast production in Osteoarthritis by inhibiting the activation of the MAPK/NF-κB signaling pathways

Osteoarthritis (OA) is a degenerative disease caused by the progressive destruction of cartilage and subchondral bone [1]. Studies have shown that by inhibiting the degradation of cartilage cells and the loss of subchondral bone, OA can be prevented and treated. Neratinib, as a small molecule compound with anti-inflammatory and anti-tumor properties, is a very effective inhibitor of IL-1β-induced chondrocyte inflammation and anabolic metabolism. By investigating the effect of neratinib in ATDC5 chondrocytes, the study finds that neratinib reduces inflammation by inhibiting the MAPK and NF-κB signaling pathways, and at the same time reduces pyrolysis (indicated by the results of reverse transcription quantitative PCR and western blotting). For anabolic metabolism, after high-density cell culture, IL-1β-induced catalytic changes and degradation of the extracellular matrix were evaluated by toluidine blue staining. Since osteoclasts are key participants in the process of subchondral bone remodeling in OA, we also studied the effect of neratinib on the maturation of osteoclasts. The results showed that neratinib also acts as an anti-osteoclast agent in vitro. By inhibiting the NF-κB and MAPK pathways, it reduces the expression of osteoclast-related genes, thereby inhibiting RANKL-induced osteoclastogenesis. The results of in vivo animal experiments supported the conclusions from the experiments in vitro. Neratinib inhibited both the destruction of medial meniscus induced cartilage degradation and osteoclast formation, which proves that neratinib has a dual effect, protecting cartilage and inhibiting osteoclast formation. These results indicate that neratinib can be a brand-new latent strategy for the treatment of OA.

Monocyte Chemotactic Proteins Mediate the Effects of Hyperglycemia in Chondrocytes: In Vitro Studies.

Chemokines are secreted by a large variety of cells. They are involved in controlling cell trafficking, maturation, and differentiation. However, the specific responses and effects of chemokines on specific skeletal cell types under high glucose conditions have not been investigated. Chondrocytes play an important role in osteoarthritis and fracture healing. Delayed fracture healing is one of the major health complications caused by diabetes, so the goal of this study was to evaluate the response of several chemokines to high glucose conditions in chondrocyte cells and analyze their role in the catabolic effect of hyperglycemia. ATDC5 chondrocytes were cultured in normal and high glucose media, and mRNA expression levels of several chemokines and chondrocyte differentiation markers were quantified. Bindarit, a specific inhibitor of monocyte chemotactic proteins (MCPs), was used to determine the role of MCPs in mediating the effects of high glucose conditions in chondrocyte cells. High glucose treatment upregulated the expression of three Mcps, as well as the expression of matrix metalloproteinase 13 (Mmp13) and Osteocalcin (Oc). Furthermore, bindarit treatment downregulated Mmp13 and Oc but upregulated Collagen 2 (Col2) mRNA levels in chondrocytes treated with high glucose. Moreover, treatment of chondrocytes with ascorbic acid reduced the effect of high glucose conditions on the expression of chemokines and Mmps. These data together suggest that MCPs mediate the catabolic effect of high glucose in chondrocytes.

Chsy1 deficiency reduces extracellular matrix productions and aggravates cartilage injury in osteoarthritis.

Osteoarthritis (OA) is a kind of degenerative joint disease marked by the destruction of articular cartilage due to the degeneration of chondrocytes. CHSY1, one of the glycosyltransferases, is involved in the synthesis of chondroitin sulfate. Herein, we found that the expression of Chsy1 was decreased in the knee cartilage of OA rats. In order to investigate the role of CHSY1 in chondrogenesis and OA, we established a Chsy1 stable knockdown cell line in mouse ATDC5 chondrocytes by lentivirus. It was found that Chsy1 deficiency resulted in a reduction of extracellular matrix production in chondrocytes and a promotion of endochondral osteogenesis, which was indicated by the decreased expression of early chondrocytes genes (Col2a1, Sox9), and the increased expression of cartilage hypertrophy genes (Col10a1, Runx2, Mmp13, Mmp3). The expression trend of these genes is considered to be the characteristic of osteoarthritis. In addition, knockdown of Chsy1 could upregulate BMP signaling in differentiated chondrocytes, whereas Chsy1 overexpression had opposite effects. The reduction of extracellular matrix production and the promotion of endochondral osteogenesis by Chsy1 knockdown could be rescued by BMP signaling inhibitor LDN193189. Furthermore, the abnormally enhanced BMP signaling and the high expression of OA biomarker Mmp3 in primary cells of OA rats could be rescued by either LDN193189 or Chsy1 overexpression. These results implicate a role for Chsy1 in regulating extracellular matrix production and endochondral osteogenesis through BMP signaling; and a lack of Chsy1 could aggravate the cartilage damage of osteoarthritis.

Galactose Enhances Chondrogenic Differentiation of ATDC5 and Cartilage Matrix Formation by Chondrocytes.

Galactose, an important carbohydrate nutrient, is involved in several types of cellular metabolism, participating in physiological activities such as glycosaminoglycan (GAG) synthesis, glycosylation, and intercellular recognition. The regulatory effects of galactose on osteoarthritis have attracted increased attention. In this study, in vitro cell models of ATDC5 and chondrocytes were prepared and cultured with different concentrations of galactose to evaluate its capacity on chondrogenesis and cartilage matrix formation. The cell proliferation assay demonstrated that galactose was nontoxic to both ATDC5 cells and chondrocytes. RT-PCR and immunofluorescence staining indicated that the gene expressions of cartilage matrix type II collagen and aggrecan were significantly upregulated with increasing galactose concentration and the expression and accumulation of the extracellular matrix (ECM) protein. Overall, these results indicated that a galactose concentration below 8 mM exhibited the best effect on promoting chondrogenesis, which entitles galactose as having considerable potential for cartilage repair and regeneration.

Bilobalide Exerts Anti-Inflammatory Effects on Chondrocytes Through the AMPK/SIRT1/mTOR Pathway to Attenuate ACLT-Induced Post-Traumatic Osteoarthritis in Rats.

Although osteoarthritis (OA) significantly affects the quality of life of the elderly, there is still no effective treatment strategy. The standardized Ginkgo biloba L. extract preparation has been shown to have a wide range of therapeutic effects. Bilobalide, a unique ingredient of Ginkgo biloba, has anti-inflammatory and antioxidant pharmacological properties, but its mechanism of action on OA remains unknown. In this study, we investigated the effects of bilobalide on the development of OA through in vivo and in vitro experiments, as well as its potential anti-inflammatory mechanisms. The in vitro experiments demonstrated that bilobalide significantly inhibited the production of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and matrix metalloproteinase 13 (MMP13) in ATDC5 chondrocytes induced by Interleukin-1β (IL-1β). At the molecular level, bilobalide induced chondrocyte autophagy by activating the AMPK/SIRT1/mTOR signaling pathway, which increased the expression of autophagy-related Atg genes, up-regulated the expression of LC3 protein, and reduced the expression of the p62 protein. In vivo, bilobalide exerted significant anti-inflammatory and anti-extracellular matrix (ECM) degradation effects in a rat model of post-traumatic OA (PTOA) induced by anterior cruciate ligament transection (ACLT). Bilobalide could relieve joint pain in PTOA rats, inhibit the expression of iNOS and COX-2 protein in cartilage via the AMPK/SIRT1/mTOR pathway, and reduce the level of ECM degradation biomarkers in serum. In conclusion, bilobalide exhibits vigorous anti-inflammatory activity, presenting it as an interesting potential therapeutic agent for OA.

Fxyd5 activates the NF‑κB pathway and is involved in chondrocytes inflammation and extracellular matrix degradation.

It is known that increased inflammation and extracellular matrix (ECM) degradation in chondrocytes can promote the development of osteoarthritis (OA). The FXYD domain containing ion transport regulator 5 (Fxyd5) has been found to promote chronic inflammatory responses. The present study aimed to investigate the role of Fxyd5 in OA. Murine ATDC5 chondrocytes were transfected with short hairpin RNAs specifically targeting Fxyd5 to silence its expression. Subsequently, cells were induced with lipopolysaccharide (LPS). The protein expression levels of Fxyd5, MMPs and proteins related to ECM, apoptosis and NF-κB signaling were detected using western blot analysis. In addition, cell viability was assessed using a Cell Counting Kit-8 assay, while the secretion of the proinflammatory factors and those of the oxidative stress-related markers were measured using the corresponding kits. Finally, cells were treated with the NF-κB activator, betulinic acid (BA) and the above experiments were repeated. The results demonstrated that Fxyd5 was significantly upregulated in ATDC5 cells treated with LPS. Additionally, Fxyd5 knockdown increased cell viability, enhanced the protein expression of Bcl-2, Aggrecan and collagen II, while reduced the expression of Bax, cleaved caspase-3/caspase-3, MMP3 and MMP13 in LPS-induced ATDC5 cells. The production of IL-1β, IL-6 and IL-18 as well as reactive oxygen species and malondialdehyde, and the reduction of superoxide dismutase caused by LPS in ATDC5 cells, were also reversed by Fxyd5 silencing. Fxyd5 silencing inhibited the phosphorylation of p65 and IκBα induced by LPS. Finally, BA reversed the protective effect of Fxyd5 silencing on LPS induced chondrocytes injury. In conclusion, Fxyd5 could enhance chondrocyte inflammation and ECM degradation via activating the NF-κB signaling.

Differences in pathways contributing to thyroid hormone effects on postnatal cartilage calcification versus secondary ossification center development.

The proximal and distal femur epiphyses of mice are both weight-bearing structures derived from chondrocytes but differ in development. Mineralization at the distal epiphysis occurs in an osteoblast-rich secondary ossification center (SOC), while the chondrocytes of the proximal femur head (FH), in particular, are directly mineralized. Thyroid hormone (TH) plays important roles in distal knee SOC formation, but whether TH also affects proximal FH development remains unexplored. Here, we found that TH controls chondrocyte maturation and mineralization at the FH in vivo through studies in thyroid stimulating hormone receptor (Tshr-/-) hypothyroid mice by X-ray, histology, transcriptional profiling, and immunofluorescence staining. Both in vivo and in vitro studies conducted in ATDC5 chondrocyte progenitors concur that TH regulates expression of genes that modulate mineralization (Ibsp, Bglap2, Dmp1, Spp1, and Alpl). Our work also delineates differences in prominent transcription factor regulation of genes involved in the different mechanisms leading to proximal FH cartilage calcification and endochondral ossification at the distal femur. The information on the molecular pathways contributing to postnatal cartilage calcification can provide insights on therapeutic strategies to treat pathological calcification that occurs in soft tissues such as aorta, kidney, and articular cartilage.

Semaphorin 3A mitigates lipopolysaccharide-induced chondrocyte inflammation, apoptosis and extracellular matrix degradation by binding to Neuropilin-1

ABSTRACT Semaphorin 3A (SEMA3A) and its receptor neuropilin-1 (NRP-1) are expressed low in chondrocytes under stress, and overexpressing SEMA3A reduces pro-inflammatory cytokine release. This study was aimed at exploring whether SEMA3A participates in lipopolysaccharide (LPS)-induced chondrocyte inflammation, apoptosis and extracellular matrix (ECM) degradation. SEMA3A and NRP-1 expression in LPS-induced ATDC5 cells was determined with RT-qPCR and western blotting. Following stimulation with LPS in the absence or presence of SEMA3A overexpression, the viability of ATDC5 cells was observed through CCK-8 assay. RT-qPCR and western blot were performed to detect the expression of pro-inflammatory cytokines. ATDC5 cell apoptosis was observed through TUNEL, and apoptosis-related proteins were assayed. Expression of ECM-related proteins was measured by RT-qPCR and western blotting. Additionally, the binding of SEMA3A to NRP-1 was verified by co-immunoprecipitation. After interference with NRP-1, cell viability, inflammation and ECM degradation were examined in LPS-induced ATDC5 cells with SEMA3A overexpression. Results revealed that SEMA3A expression in ATDC5 cells decreased following stimulation with LPS. Overexpressing SEMA3A improved cell viability and reduced the inflammatory injury of LPS-stimulated ATDC5 cells. Moreover, SEMA3A overexpression alleviated LPS-induced apoptosis and ECM degradation of ATDC5 chondrocytes. SEMA3A and NRP-1 bound to each other in ATDC5 cells. NRP-1 interference crippled the ameliorative effect of SEMA3A overexpression on LPS-induced chondrocyte inflammation, apoptosis and ECM degradation. To conclude, SEMA3A binds to NRP-1, mitigating LPS-induced chondrocyte inflammation, apoptosis and ECM degradation. This study elucidated the role of SEMA3A in osteoarthritis and illustrated its action mechanism involving NRP-1.