目的 基于PTEN诱导激酶1（PTEN induced putative kinase 1，PINK1）/帕金蛋白（Parkin）通路探讨鞣酸促进软骨细胞线粒体自噬并减轻小鼠膝骨关节炎（knee osteoarthritis，KOA）的作用机制。方法 采用内侧半月板失稳手术构建小鼠KOA模型，设置假手术组、模型组和鞣酸低、高剂量（25、50 mg/kg）组，给药干预8周。通过影像学、番红O-固绿染色和甲苯胺蓝染色观察关节软骨病理变化；免疫组化检测软骨组织中Ⅱ型胶原蛋白（collagen II，Col-II）、基质金属蛋白酶13（matrix metallopeptidase 13，MMP13）、白细胞介素-1β（interleukin-1β，IL-1β）、PINK1、Parkin、p62和微管相关蛋白轻链3B（microtubule - associated protein light chain 3B，LC3B）蛋白表达。采用IL-1β刺激小鼠ATDC5软骨细胞，给予鞣酸干预后，CCK-8法检测细胞活力；Western blotting和qRT-PCR检测Col-II、MMP13和IL-1β蛋白及mRNA表达；Western blotting检测线粒体自噬相关蛋白PINK1、Parkin、p62和LC3-II/I蛋白表达；免疫荧光检测活性氧（reactive oxygen species，ROS）和线粒体膜电位变化；透射电镜观察线粒体自噬水平；并利用线粒体自噬抑制剂Mdivi-1进行反向机制验证。结果 与假手术组比较，模型组小鼠软骨严重磨损，OARSI评分显著升高（P＜0.01），软骨组织Col-II、PINK1、Parkin和LC3B蛋白表达水平显著降低（P＜0.01）、IL-1β、MMP13和p62蛋白表达水平显著升高（P＜0.01）；与模型组比较，鞣酸组小鼠软骨形态得到改善，OARSI评分显著降低（P＜0.05），软骨组织Col-II、PINK1、Parkin和LC3B蛋白表达水平显著升高（P＜0.01），IL-1β、MMP13和p62蛋白表达水平显著降低（P＜0.01）。细胞实验结果显示，IL-1β诱导的软骨细胞活力显著降低（P＜0.01），IL-1β、MMP13蛋白和mRNA表达水平显著升高（P＜0.01），Col-II蛋白和mRNA表达水平显著降低（P＜0.01），LC3-II/I、PINK1、Parkin蛋白表达水平显著降低（P＜0.05、0.01），p62蛋白表达水平显著升高（P＜0.05），细胞内ROS积累增加（P＜0.01），线粒体膜电位下降（P＜0.01），自噬小体减少；与模型组比较，鞣酸组细胞活力升高（P＜0.05、0.01），ROS积累显著减少（P＜0.01），线粒体膜电位恢复（P＜0.05），IL-1β、MMP13蛋白和mRNA表达水平显著降低（P＜0.05、0.01），Col-II蛋白和mRNA表达水平显著升高（P＜0.05、0.01），p62蛋白表达水平显著降低（P＜0.01），LC3-II/I、PINK1、Parkin蛋白表达水平显著升高（P＜0.01）；联合使用线粒体自噬抑制剂Mdivi-1后，鞣酸对软骨细胞的作用被逆转（P＜0.05、0.01）。结论 鞣酸通过PINK1/Parkin信号通路促进软骨细胞线粒体自噬，缓解关节软骨退变，从而延缓KOA进展。

Objective To investigate the mechanism of tannic acid in promoting mitochondrial autophagy in chondrocytes and improving knee osteoarthritis (KOA) in mice based on PINK1/Parkin signaling pathway. Methods A mouse KOA model was established via destabilization of medial meniscus surgery. Sham group, model group and tannic acid low-, high-dose (25, 50 mg/kg) groups were set up, drugs were given for intervention for eight weeks. Joint cartilage pathology was assessed by imaging, safranin O-fast green staining and toluidine blue staining. Immunohistochemistry was used to detect the expressions of collagen II (Col-II), matrix metalloproteinase 13 (MMP13), interleukin-1β (IL-1β), PINK1, Parkin, p62 and microtubule-associated protein light chain 3B (LC3B) in cartilage tissue. ATDC5 mouse chondrocytes were stimulated with IL-1β and then treated with tannic acid. Cell viability was measured by CCK-8 assay. Western blotting and qRT-PCR were used to detect the protein and mRNA expressions of Col-II, MMP13 and IL-1β. Western blotting was used to detect the expressions of mitophagy-related proteins PINK1, Parkin, p62 and LC3-II/I. Changes in reactive oxygen species (ROS) and mitochondrial membrane potential were detected by immunofluorescence. Mitochondrial autophagy level was observed by transmission electron microscopy. And reverse mechanism validation was performed using the mitochondrial autophagy inhibitor Mdivi-1. Results Compared with sham group, the cartilage of mice in model group was severely worn, OARSI score was significantly increased (P < 0.01), the protein expression levels of Col-II, PINK1, Parkin and LC3B in cartilage tissue were significantly reduced (P < 0.01), while the protein expression levels of IL-1β, MMP13 and p62 were significantly increased (P < 0.01). Compared with model group, the cartilage morphology of mice in tannic acid group was improved, OARSI score was significantly reduced (P < 0.05), the protein expression levels of Col-II, PINK1, Parkin and LC3B in cartilage tissue were significantly increased (P < 0.01), while the protein expression levels of IL-1β, MMP13 and p62 were significantly decreased (P < 0.01). The cell experiment results showed that IL-1β-induced chondrocyte viability was significantly reduced (P < 0.01), IL-1β, MMP13 protein and mRNA expression levels were significantly increased (P < 0.01), Col-II protein and mRNA expression levels were significantly reduced (P < 0.01), LC3-II/I, PINK1, Parkin protein expression levels were significantly reduced (P < 0.05, 0.01), p62 protein expression level was significantly increased (P < 0.05), intracellular ROS accumulation was increased (P < 0.01), mitochondrial membrane potential was decreased (P < 0.01), and autophagosome was decreased. Compared with model group, cell viability in tannic acid group was significantly increased (P < 0.05, 0.01), ROS accumulation significantly was decreased (P < 0.01), mitochondrial membrane potential was recovered (P < 0.05), IL-1β, MMP13 protein and mRNA expression levels were significantly decreased (P < 0.05, 0.01), Col-II protein and mRNA expression levels were significantly increased (P < 0.05, 0.01), p62 protein expression level was significantly decreased (P < 0.01), LC3-II/I, PINK1 and Parkin protein expression levels were significantly increased (P < 0.01). After the combined use of mitochondrial autophagy inhibitor Mdivi-1, the therapeutic effect of tannic acid on chondrocytes was reversed (P < 0.05, 0.01). Conclusion Tannic acid alleviates articular cartilage damage and delays the progression of KOA by promoting chondrocyte mitophagy through PINK1/Parkin signaling pathway.

R285.5

国家自然科学基金资助项目（82104885）；浙江省自然科学基金资助项目（ZCLQN25H2701）；宁波市自然科学基金资助项目（2023J215）；宁波市医学重点学科“运动医学”项目（2026-A01）；宁波市医疗卫生高端团队重大攻坚项目（2022020102）