目的 探讨汉黄芩素通过调控PTEN诱导激酶1（PTEN induced putative kinase 1，PINK1）/帕金蛋白（Parkin）信号通路激活软骨细胞自噬的作用，并阐明其对机械应力影响下的膝骨关节炎（knee osteoarthritis，KOA）软骨损伤的干预作用。方法 通过内侧半月板失稳手术建立小鼠KOA模型，并随机分为假手术组、模型组及汉黄芩素（40、80 mg/kg）组和汉黄芩素（80 mg/kg）＋Piezo1激动剂Yoda1（5 mg/kg）组，采用苏木素-伊红（hematoxylin-eosin，HE）染色和番红O-固绿染色评价软骨组织形态学改变；ELISA检测血清中白细胞介素-1β（interleukin-1β，IL-1β）和肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）水平；Western blotting及qRT-PCR检测软骨组织Ⅱ型胶原蛋白（collagen II）、基质金属蛋白酶13（matrix metallopeptidase 13，MMP13）、Piezo1、微管相关蛋白1轻链3-I/II（microtubule-associated protein 1 light chain 3-I/II，LC3-I/II）、p62、Beclin-1、PINK1、Parkin蛋白及mRNA表达。使用应力加载装置处理小鼠软骨细胞建立应力损伤细胞模型，设置对照组、模型组及汉黄芩素（25、50、100 μmol/L）组和汉黄芩素（100 μmol/L）＋Yoda1（2.5 μmol/L）组，采用CCK-8法检测细胞活力；采用DHE、TUNEL染色检测活性氧（reactive oxygen species，ROS）及细胞凋亡水平；ELISA检测细胞上清液中IL-1β和TNF-α水平；免疫荧光检测Piezo1表达；Western blotting及qRT-PCR检测collagen II、MMP13、Piezo1、LC3-I/II、p62、Beclin-1、PINK1、Parkin蛋白及mRNA表达；透射电镜观察线粒体形态。结果 与假手术组比较，模型组小鼠软骨出现严重损坏，软骨组织collagen II、LC3-II/I、Beclin-1、PINK1、Parkin蛋白和mRNA表达水平显著降低（P＜0.01），MMP13、p62、Piezo1蛋白和mRNA表达水平显著升高（P＜0.01）；与模型组比较，汉黄芩素各剂量组小鼠软骨形态改善明显，软骨组织collagen II、LC3-II/I、Beclin-1、PINK1、Parkin蛋白和mRNA表达水平显著升高（P＜0.01），MMP13、p62、Piezo1蛋白和mRNA表达水平显著降低（P＜0.01）；与汉黄芩素高剂量组比较，汉黄芩素＋Yoda1组软骨组织collagen II、LC3-II/I、Beclin-1、PINK1、Parkin蛋白和mRNA表达水平显著降低（P＜0.05、0.01），MMP13、p62、Piezo1蛋白和mRNA表达水平显著升高（P＜0.01）。细胞实验结果表明，与对照组比较，模型组细胞活力显著降低（P＜0.01），ROS、细胞凋亡、IL-1β、TNF-α水平及MMP13、p62、Piezo1蛋白和mRNA表达水平显著升高（P＜0.01），collagen II、LC3-II/I、Beclin-1、PINK1、Parkin蛋白和mRNA表达水平显著降低（P＜0.01）；与模型组比较，汉黄芩素可呈剂量相关性地增加细胞活力（P＜0.01），促进自噬，并上调collagen II、LC3-II/I、Beclin-1、PINK1、Parkin蛋白和mRNA表达水平（P＜0.01）；与汉黄芩素组比较，汉黄芩素＋Yoda1组软骨损伤及自噬相关指标显著下降（P＜0.01）。结论 汉黄芩素通过调控PINK1/Parkin通路，激活细胞自噬程序并减轻线粒体损伤，对机械应力干预下的KOA软骨损伤起到保护作用。

Objective To explore the therapeutic effect of wogonin on activating chondrocyte autophagy by regulating the PTEN induced putative kinase 1 (PINK1)/Parkin signaling pathway, and elucidate its intervention effect on knee osteoarthritis (KOA) cartilage injury under mechanical stress. Methods A mouse KOA model was established through the destruction of medial meniscus surgery, and randomly divided into sham group, model group, wogonin (40, 80 mg/kg) group, and wogonin (80 mg/kg) + Piezo1 agonist Yoda1 (5 mg/kg) group. Hematoxylin-eosin (HE) staining and safranin O-green staining were used to evaluate the morphological changes of cartilage tissue; ELISA was used to detect the levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in serum; Western blotting and qRT-PCR were used to detect the protein and mRNA expressions of collagen II, matrix metalloproteinase 13 (MMP13), Piezo1, microtubule-associated protein 1 light chain 3-I/II (LC3-I/II), p62, Beclin-1, PINK1 and Parkin in cartilage tissue. A stress injury cell model was established by treating mouse chondrocytes with stress loading. Control group, model group, wogonin (25, 50, 100 μmol/L) group, and wogonin (100 μmol/L) + Yoda1 (2.5 μmol/L) group were set up. CCK-8 method was used to detect cell viability; DHE and TUNEL staining were used to detect levels of reactive oxygen species (ROS) and cell apoptosis; ELISA was used to detect the levels of IL-1β and TNF-α; Immunostaining was used to detect Piezo1 expression; Western blotting and qRT-PCR were used to detect the protein and mRNA expressions of collagen II, MMP13, Piezo1, LC3-I/II, p62, Beclin-1, PINK1 and Parkin; Mitochondrial morphology was observed by transmission electron microscopy. Results Compared with sham group, mice in model group showed severe cartilage damage, with significantly reduced protein and mRNA expressions of collagen II, LC3-II/I, Beclin-1, PINK1 and Parkin in cartilage tissue (P < 0.01), and significantly increased protein and mRNA expressions of MMP13, p62 and Piezo1 (P < 0.01); Compared with model group, the cartilage morphology of wogonin in each dose group was significantly improved, the protein and mRNA expressions of collagen II, LC3-II/I, Beclin-1, PINK1 and Parkin in cartilage tissue were significantly increased (P < 0.01), while the protein and mRNA expressions of MMP13, p62 and Piezo1 were significantly decreased (P < 0.01); Compared with wogonin high-dose group, wogonin + Yoda1 group showed significantly reduced protein and mRNA expressions of collagen II, LC3-II/I, Beclin-1, PINK1 and Parkin in cartilage tissue (P < 0.05, 0.01), while significantly increased protein and mRNA expressions of MMP13, p62, and Piezo1 (P < 0.01). The cell experiment results showed that compared with control group, the cell viability of model group was significantly reduced (P < 0.01), the levels of ROS, apoptosis, IL-1β, TNF-α, MMP13, p62, Piezo1 protein and mRNA expressions were significantly increased (P < 0.01), the protein and mRNA expressions of collagen II, LC3-II/I, Beclin-1, PINK1 and Parkin were significantly reduced (P < 0.01); Compared with model group, wogonin increased cell viability in a dose-dependent manner (P < 0.01), promoted autophagy, and up-regulated the protein and mRNA expression levels of collagen II, LC3-II/I, Beclin-1, PINK1 and Parkin (P < 0.01); Compared with wogonin group, wogonin + Yoda1 group showed a significant decrease in cartilage damage and autophagy related indicators (P < 0.01). Conclusion Wogonin exerts a protective effect on KOA cartilage injury under mechanical stress intervention by regulating PINK1/Parkin pathway, activating cellular autophagy program and reducing mitochondrial damage.

R285.5

国家自然科学基金面上项目(82274545); 江苏省中医院中医膝骨关节炎临床医学创新中心项目(Y2023zx05); 江苏省医学重点学科/实验室建设单位项目(JSDW202252); 南京中医药大学膝骨关节炎临床专病研究院项目(LCZBYJYZZ2024-003)