针刀对膝关节骨关节炎兔软骨PINK1/Parkin通路介导的线粒体自噬的影响

Effect of acupotomy on mitophagy mediated by PINK1/Parkin pathway in cartilage of rabbits with knee osteoarthritis

目的:基于PINK1/Parkin通路观察针刀干预对膝关节骨关节炎(KOA)兔软骨线粒体自噬的影响,探讨其抑制软骨损伤的作用机制。方法:新西兰兔随机分为正常组、模型组、针刀组,每组7只。模型组和针刀组参考Videman法建立KOA兔模型。针刀组取膝关节周围结节或条索进行常规针刀治疗,每周1次,连续3周。分别采用HE染色和透射电镜观察兔膝关节软骨组织形态学和细胞超微形态,流式细胞仪检测软骨细胞线粒体膜电位(Δψm)和活性氧(ROS)平均荧光强度并计算低Δψm细胞百分比,免疫荧光染色法检测软骨组织LC3B、PINK1和Parkin免疫荧光表达,Western blot法检测软骨组织p62、LC3Ⅱ/Ⅰ、PINK1和Parkin蛋白的表达水平。结果:与正常组相比,模型组兔膝关节软骨表面出现裂隙、组织纤维化,软骨细胞分布松散,自噬体减少,线粒体形态异常;软骨组织LC3B、PINK1、Parkin荧光强度,LC3Ⅱ/Ⅰ、PINK1、Parkin蛋白表达水平显著降低(P<0.01),软骨细胞低Δψm细胞百分比、ROS平均荧光强度、软骨组织p62蛋白表达水平升高(P<0.01)。与模型组相比,针刀组兔膝关节软骨表面无明显缺损,软骨细胞相对密集,自噬体增多,线粒体形态正常;软骨组织LC3B、PINK1、Parkin荧光强度,LC3Ⅱ/Ⅰ、PINK1、Parkin蛋白表达水平显著升高(P<0.01,P<0.05),软骨细胞低Δψm细胞百分比、ROS平均荧光强度、软骨组织p62蛋白表达水平下降(P<0.01)。结论:针刀可能通过调节PINK1/Parkin通路促进线粒体自噬,改善KOA兔软骨损伤。

Objective To observe the effect of acupotomy on mitophagy mediated by PINK1/Parkin pathway in cartilage of rabbits with knee osteoarthritis(KOA),so as to explore its mechanism in inhibiting cartilage damage.Methods Twenty-one New Zealand rabbits were randomly divided into normal,model,and acupotomy groups,with 7 rabbits in each group.The KOA rabbit model was established by using the Videman method.Rabbits in the acu⁃potomy group received regular acupotomy treatment around the knee joint nodules or tendons once a week for 3 con⁃secutive weeks.HE staining and transmission electron microscopy were used to observe the morphological and ultra⁃structural changes in knee joint cartilage of rabbits.Flow cytometry was used to measure the mitochondrial membrane potential(Δψm)and reactive oxygen species(ROS)average fluorescence intensity in chondrocytes.Immunofluores⁃cence was performed to detect the fluorescence intensity of LC3B,PINK1 and Parkin in cartilage tissue.Western blot was conducted to measure the protein expression levels of p62,LC3Ⅱ/Ⅰ,PINK1,and Parkin in cartilage tissue.Re‐sults Compared to the normal group,the model group showed fissures and tissue fibrosis on the surface of rabbit knee joint cartilages,loose distribution of chondrocytes,decreased autophagosomes,and abnormal mitochondrial morphology.The fluorescence intensity of LC3B,PINK1 and Parkin,the expression levels of LC3Ⅱ/Ⅰ,PINK1 and Parkin proteins in cartilage tissue were significantly decreased(P<0.01),while the percentage of chondrocytes with lowΔψm,the average fluorescence intensity of ROS,and the expression of p62 protein in cartilage tissue were significantly in⁃creased(P<0.01).Compared to the model group,the acupotomy group showed no obvious defects on the surface of rabbit knee joint cartilage,relatively dense distribution of chondrocytes,increased autophagosomes,and relatively nor⁃mal mitochondrial morphology.The fluorescence intensity of LC3B,PINK1 and Parkin,the expression of LC3Ⅱ/Ⅰ,PINK1 and Parkin proteins in cartilage tissue were significantly increased(P<0.01,P<0.05),while the percentage of chondrocytes with lowΔψm,the average fluorescence intensity of ROS,and the expression of p62 protein in cartilage tissue were significantly decreased(P<0.01).Conclusion Acupotomy may promote mitophagy by regulating the PINK1/Parkin pathway,thereby improving cartilage damage in rabbits with KOA.