Mechanical 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.本课题组既往研究证实，脂氧素A4（lipoxin A4, LXA4）对骨关节炎（osteoarthritis, OA）具有治疗作用。本研究旨在探讨LXA4经滑膜巨噬细胞M1/M2亚型极化对机械刺激下软骨细胞焦亡的影响及相关机制。实验首先设置不同浓度LXA4及不同周期性张应变（cyclic tensile strain, CTS）条件培养滑膜巨噬细胞，以确定最优共培养方案；随后采用蛋白质印迹法（Western Blot）、免疫荧光法及流式细胞术，检测以滑膜巨噬细胞M1/M2亚型极化为表征的LXA4对软骨细胞焦亡的调控效应。动物实验部分：将40只雄性斯普拉格-道莱大鼠随机分为4组（每组n=10）：对照组（CG）、骨关节炎模型组（OAG）、中等强度跑台运动骨关节炎组（OAE）以及OAE+BOC-2（LXA4拮抗剂）组。所有大鼠均通过组织学检测、酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）、定量聚合酶链式反应（quantitative PCR）及蛋白质印迹法进行评估。实验结果显示：患者关节液中LXA4表达下调，且滑膜组织中CD86/Arg1的表达随Kellgren-Lawrence分级升高而上调。体外实验中，周期性张应变与LXA4均可促进滑膜巨噬细胞向M2亚型极化，抑制软骨细胞中核因子κB p65（NF-κB p65）的核转位及NLRP3炎症小体的形成。体内实验表明，中等强度跑台运动对关节软骨具有保护作用，并可促进滑膜巨噬细胞向M2亚型极化，而该效应可被BOC-2处理所阻断。本研究最终证实：中等强度周期性张应变可通过抑制核因子κB p65的核转位及NLRP3炎症小体形成，增强LXA4的治疗效果。此外，在单碘乙酸诱导的骨关节炎模型中，跑台运动下LXA4的治疗效应依赖于对滑膜巨噬细胞M2亚型极化的促进作用。