Background. Glycogen synthase kinase 3β (GSK3B) is reported to be a protective factor for the degradation of chondrocytes by extracellular mechanisms. Nuclear receptor subfamily 4 group A member 3 (NR4A3) is a proinflammatory factor in osteoarthritis. Their regulation mechanism in posttraumatic osteoarthritis (PTOA) is not fully understood. Methods. GSK3B expression in the cartilage tissue of PTOA patients was analyzed by western blotting. IL-1β-induced chondrocytes were transfected with pcDNA-GSK3B, and then, the cell viability, apoptosis, expression of the chondrocyte extracellular matrix degradation-related genes MMP13, aggrecan, and type II collagen, and secretion of inflammatory factors TNF-α and IL-6 were detected. Co-IP was used to analyze the interaction between GSK3B and DNMT1. Ch-IP and methylation-specific PCR assays were used for methylation. Also, cells were transfected with pcDNA-GSK3B or together with pcDNA-NR4A3, as well as transfected with si-NR4A3, and then, cell functions were tested. Then, the mice subjected to destabilization of medial meniscus (DMM) surgery were intra-articular injected with 100 μL of the following adeno-related virus vectors (empty vector, Ad-GSK3B, scrambled shRNA, and sh-NR4A3), respectively, and the virus titer was 2 × 10 8   TU / mL . Cartilage integrity was evaluated by safranin O/fast green staining, HE staining, and Osteoarthritis Research Society International (OARSI) score. Results. The expression of GSK3B protein was downregulated in PTOA patients. GSK3B overexpression alleviated IL-1β-induced chondrocyte apoptosis and extracellular matrix degradation, as well as cartilage mineralization in PTOA model mice. NR4A3 overexpression reversed the effect of GSK3B on IL-1β-induced chondrocyte functions. GSK3B could recruit DNMT1 to the NR4A3 promoter region to promote the methylation of NR4A3 and inhibit the expression of NR4A3 protein. Similarly, NR4A3 interference alleviated cartilage degradation under stimulating conditions by inhibiting the activation of the JAK2/STAT3 signaling pathway. Conclusion. GSK3B recruits DNMT1 to the NR4A3 promoter region and inhibits the activation of the NR4A3-mediated JAK2/STAT3 signaling pathway, thereby alleviating PTOA.

Background Post-traumatic osteoarthritis (PTOA) is closely related to the inflammatory response caused by mechanical injury, leading to joint degeneration. Herein, we aim to evaluate the role and the underlying mechanism of NUMB in PTOA progression. Methods Anterior cruciate ligament transection (ACLT)-induced rats and LPS-treated chondrocytes were used as in vivo and in vitro models of PTOA, respectively. NUMB overexpression plasmid (pcDNA-NUMB) were administered by intra-articular injection in PTOA model rats, and safranin O-fast green staining, the Osteoarthritis Research Society International (OARSI) Scoring System, and HE staining were used to evaluate the severity of cartilage damage. The secretion of inflammatory cytokines (TNF-α, IL-1β, IL-6) and chondrocyte-specific markers (MMP13, COL2A1) was detected by ELISA. Cell viability and apoptosis were evaluated by MTT assay and TUNEL staining. Results The expression of NUMB was lower expressed in ACLT-induced PTOA rats and LPS-treated chondrocytes. NUMB overexpression enhanced cell viability and reduced cell apoptosis, inflammation and cartilage degradation in chondrocytes stimulated by IL-1β. NUMB bound with BTRC to inhibiting p-IκBα expression, resulting in NF-κB pathway inactivation. BTRC overexpression reversed the promoting effect of NUMB overexpression on cell viability and the inhibitory effects of NUMB overexpression on apoptosis, inflammation and cartilage degradation in LPS-induced chondrocytes. Besides, overexpression of NUMB alleviated articular cartilage damage by repression of inflammation and cartilage degradation in ACLT-induced PTOA rats. Conclusion Our data indicated that NUMB negatively regulates BTRC regulated PTOA progression by BTRC/NF-κB pathway, which may be a viable therapeutic target in PTOA.