Osteoarthritis (OA) is one of the most common refractory degenerative joint diseases worldwide. Synovitis is believed to drive joint cartilage destruction during OA pathogenesis. Cuproptosis is a novel form of copper-induced cell death. However, few studies have examined the correlations between cuproptosis-related genes (CRGs), immune infiltration, and synovitis. Therefore, we analyzed CRGs in synovitis during OA. Microarray datasets (GSE55235, GSE55457, GSE12021, GSE82107 and GSE176308) were downloaded from the Gene Expression Omnibus database. Next, we conducted differential and subtype analyses of CRGs across synovitis. Immune infiltration and correlation analyses were performed to explore the association between CRGs and immune cell abundance in synovitis. Finally, single-cell RNA-seq profiling was performed using the GSE176308 dataset to investigate the expression of CRGs in the various cell clusters. We found that the expression of five CRGs (FDX1, LIPT1, PDHA1, PDHB, and CDKN2A) was significantly increased in the OA synovium. Moreover, abundant and various types of immune cells infiltrated the synovium during OA, which was correlated with the expression of CRGs. Additionally, single-cell RNA-seq profiling revealed that the cellular composition of the synovium was complex and that their proportions varied greatly as OA progressed. The expression of CRGs differed across various cell types in the OA synovium. The current study predicted that cuproptosis may be involved in the pathogenesis of synovitis. The five screened CRGs (FDX1, LIPT1, PDHA1, PDHB, and CDKN2A) could be explored as candidate biomarkers or therapeutic targets for OA synovitis.

Our previous study found that lipoxin A₄ (LXA₄) exerts therapeutic effects on osteoarthritis (OA). In this study, we evaluated the effects of LXA₄ via synovial macrophage M1/M2 subtype polarization on chondrocyte pyroptosis and corresponding mechanisms during mechanical stimulation. Synovial macrophages were subjected to various LXA₄ concentrations and cyclic tensile strain (CTS) conditions to determine optimal co-culture conditions. The effects of LXA₄ on chondrocyte pyroptosis, as represented by macrophage M1/M2 subtype polarization, were detected by western blot, immunofluorescence, and flow cytometry analyses. Forty male Sprague-Dawley rats were randomly divided into four groups (n = 10): control (CG), OA (OAG), OA with moderate-intensity treadmill exercise (OAE), and OAE + BOC-2 (an LXA₄ antagonist). All rats were evaluated using histology, enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, and western blot analyses. We found that with increasing Kellgren-Lawrence grade, LXA₄ expression was downregulated in articular fluid and that CD86 and Arg1 expression was upregulated in the synovium of patients. In vitro, CTS and LXA₄ both promoted M2 subtype polarization of synovial macrophages, which inhibited the nuclear translocation of NF-κB p65 and formation of NLRP3 in chondrocytes. In vivo, the OAE treatment exerted protective effects on articular cartilage and facilitated M2 polarization of synovial macrophages. These effects were suppressed by BOC-2 treatment. We concluded that moderate CTS enhances therapeutic effects of LXA₄ by inhibiting the nuclear translocation of NF-κB p65 and NLRP3. Furthermore, the therapeutic effects of LXA₄ during treadmill exercise in monoiodoacetate-induced OA were driven by promotion of synovial macrophage M2 subtype polarization.

Osteoarthritis (OA) is a degenerative disease that is difficult to cure owing to its complicated pathogenesis. Exercise therapy has been endorsed as a primary treatment option. However, it remains controversial how exercise intensity regulates OA progression. Here, a declining propensity for TGF-β1 was predicted via bioinformatics analysis of microarray GSE57218 and validated in cartilage samples obtained from arthroplasty. Based on this, cyclic tensile strain or TGF-β1 intervention was performed on human OA chondrocytes, and we found that moderate-intensity mechanical loads protected chondrocytes against pyroptosis. During this process, the elevation of TGF-β1 is mechanically stimuli-dependent and exerts an inhibitory effect on chondrocyte pyroptosis. Moreover, we elucidated that TGF-β1 activated Smad2/3 and inhibited the NF-κB signaling pathway to suppress chondrocyte pyroptosis. Furthermore, we established a rat knee OA model by intra-articular injection of monosodium iodoacetate and performed treadmill exercises of different intensities. Similar to the in vitro results, we demonstrated that moderate-intensity treadmill exercise had an outstanding chondroprotective effect. An inappropriate intensity of mechanical stimulation may aggravate OA both in vivo and in vitro. Overall, our findings demonstrated that activation of the TGF-β1/Smad2/Smad3 axis and inhibition of NF-κB coordinately inhibit chondrocyte pyroptosis under mechanical loads. This study sheds light on the future development of safe and effective exercise therapies for OA.

By analyzing the single-cell RNA-Seq libraries, the roles of cartilage intermediate layer protein (CILP) and the cartilage intermediate zone in early osteoarthritis (OA) exercise therapy were explored. An early OA rat model was established via a 4-week anterior cruciate ligament transection. The effect of moderate exercise was confirmed using histology, the open-field test, and gait analysis. The response of the cartilage intermediate zone to mechanical stimulation was explored using multiplex fluorescent immunohistochemical staining. Radiomics was used to evaluate the relatively damaged and undamaged areas in the cartilage of patients with OA. CILP was OE and KD in early OA chondrocytes, and quantitative proteomics, yeast one-hybrid, co-immunoprecipitation, Nrf2 and ubiquitination assays were used to investigate its mechanism. We found that moderate exercise upregulates CILP in the cartilage intermediate zone. CILP recovers the type II/I collagen, Sox9, and α-SMA expression ratios, and reduces Keap1-Nrf2 dimer stability, inhibiting Nrf2 ubiquitination and promoting Nrf2 nuclear translocation. Nrf2 nuclear translocation activates SLC7A11, HO-1, GPX4, and SOD-1 expression, decreases MDA content, and increases GSH content, inhibiting chondrocyte ferroptosis and promoting fibrocartilage hyalinization. In conclusion, the exercise-induced cartilage intermediate zone and CILP-Keap1-Nrf2 axis inhibit hyaline cartilage fibrosis and chondrocyte ferroptosis to alleviate early OA.

Background By analyzing the single-cell RNA-Seq libraries we established of OA joints during exercise therapy, we found cartilage intermediate zone might participate in early OA exercise therapy. Methods Early OA rat model was established by 4-week anterior cruciate ligament transection (ACLT). The radiomics was used to evaluate the relative damaged and undamaged area in OA patients’ cartilage. We overexpressed and knocked down CILP in early OA chondrocyte to explore its potential mechanism. The quantitative proteomics was used to examine the protein profiles of the CILP-treated chondorcyte. The Yeast One-Hybrid Assay, Co-Immunoprecipitation (Co-IP), Nrf2 cytosol-nuclei fractionation and ubiquitination assay were used to investigate the potential mechanism in CILP intervention. Western blot, ROS, JC-1, Ferrous ion, MDA and GSH detection, transmission electron microscopy (TEM) were used to explored the therapeutic effect of CILP on OA. Results Moderate exercise up-regulates CILP in the articular cartilage intermediate zone. CILP recovers the ratio of type II / I collagen, Sox9, α-SMA expression and competitively bind to Keap1 protein and reduce the stability of Keap1-Nrf2 dimer, thereby reducing the degree of Nrf2 ubiquitination and promoting Nrf2 nuclear translocation. Nrf2 nuclear translocation activated SLC7A11, HO-1, GPX4 and SOD-1 expression, then decreased the MDA contents, but increased GSH content, which ultimately inhibited chondrocytes ferroptosis and promoted hyalinization of fibrocartilage. Conclusion Exercise induced cartilage intermediate zone and CILP-Keap1-Nrf2 axis inhibits hyaline cartilage fibrosis and chondrocyte ferroptosis to alleviate early osteoarthritis.