Untitled ItemMechanical Stress Protects Against Chondrocyte Pyroptosis through Lipoxin A4 via Synovial Macrophage M2 Subtype Polarization in an Osteoarthritis Model

Our previous study found that lipoxin A4 (LXA4) exerted 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 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 group (CG), OA group (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 (ELISA), quantitative PCR, and western blot analyses. We found that LXA4 was downregulated in articular fluid and that CD 86/Arg 1 was up-regulated in the synovium of patients with increasing Kellgren-Lawrence grade. In vitro, CTS and LXA4 both promoted M2 subtype polarization of synovial macrophages, inhibiting the nuclear translocation of NF-κB p65 and the NLRP3 formation 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 the 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.

本团队前期研究发现，脂氧素A4（lipoxin A4, LXA4）对骨关节炎（osteoarthritis, OA）具有治疗作用。本研究中，我们探究了脂氧素A4通过调控滑膜巨噬细胞M1/M2亚型极化，对机械刺激下软骨细胞焦亡的影响及其潜在作用机制。我们通过设置不同浓度的脂氧素A4与不同周期性张应变（cyclic tensile strain, CTS）条件处理滑膜巨噬细胞，以确定最优的共培养实验条件。本研究以巨噬细胞M1/M2亚型极化为观测指标，通过蛋白质印迹（western blot）、免疫荧光染色与流式细胞术检测，分析脂氧素A4对软骨细胞焦亡的调控效应。将40只雄性斯普拉格-道利（Sprague-Dawley）大鼠随机分为4组，每组10只：空白对照组（control group, CG）、骨关节炎模型组（OA group, OAG）、中等强度跑台运动干预骨关节炎组（OA with moderate-intensity treadmill exercise, OAE）以及OAE联合BOC-2（脂氧素A4拮抗剂）组。所有大鼠均通过组织形态学检测、酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）、定量聚合酶链式反应（quantitative PCR, qPCR）与蛋白质印迹分析完成评估。本研究发现，骨关节炎患者的关节液中脂氧素A4表达下调，且滑膜组织内CD86/Arg1的表达水平随Kellgren-Lawrence分级升高而上调。体外实验结果显示，周期性张应变与脂氧素A4均可促进滑膜巨噬细胞向M2亚型极化，并抑制软骨细胞内NF-κB p65的核转位与NLRP3炎症小体的形成。体内实验结果表明，中等强度跑台运动干预可对关节软骨起到保护作用，并促进滑膜巨噬细胞向M2亚型极化，而BOC-2处理可阻断上述效应。本研究最终证实，适度周期性张应变可通过抑制NF-κB p65的核转位与NLRP3的激活，增强脂氧素A4的治疗效应。此外，在单碘乙酸盐诱导的骨关节炎模型中，跑台运动下脂氧素A4的治疗作用依赖于对滑膜巨噬细胞M2亚型极化的促进作用。