摘要
目的 探讨益母草碱对骨关节炎(OA)大鼠软骨细胞自噬和凋亡的影响以及PTEN诱导假定激酶1(PINK1)/帕金森病蛋白(Parkin)信号通路发挥作用.方法 采用标准前交叉韧带横断术(ACLT)构建OA大鼠模型,将建模成功的大鼠分为OA组、益母草碱-L组、益母草碱-M组、益母草碱-H组,每组12只,另取12只为假手术组;HE染色检测各组大鼠软骨细胞形态变化;透射电镜观察各组大鼠软骨细胞超微结构;TUNEL法检测各组大鼠软骨细胞凋亡;RT-qPCR检测各组大鼠软骨组织PINK1、Parkin、P62、LC3B mRNA表达水平;Western blot检测各组大鼠软骨组织PINK1、Parkin、P62、LC3B蛋白表达水平.结果 与假手术组相比,OA组大鼠软骨形态异常、结构模糊、表面出现裂缝、软骨细胞线粒体数量减少、少量自噬体存在,软骨细胞凋亡率、软骨组织P62 mRNA和蛋白水平显著升高,软骨组织PINK1、Parkin、LC3B mRNA和蛋白水平显著降低(P＜0.05).与OA组比较,益母草碱-L、M、H组大鼠软骨形态逐渐恢复、结构逐渐清晰、表面裂缝减少、软骨细胞数量恢复、线粒体增多、自噬体数量增加,软骨细胞凋亡率、软骨组织P62 mRNA和蛋白水平显著降低,软骨组织PINK1、Parkin、LC3B mRNA和蛋白水平显著升高,呈剂量依赖性(P＜0.05).结论 益母草碱可能通过激活PINK1/Parkin信号通路促进OA大鼠软骨细胞自噬,抑制软骨细胞凋亡.
Abstract
Objective To investigate the effects of leonurine on autophagy and apoptosis of chondrocytes and the role of PTEN-induced putative kinase 1(PINK 1)/Parkinson's disease protein(Park)signaling pathway in osteoarthritis(OA)rats.Methods Standard anterior cruciate ligament transection(ACLT)was applied to construct an OA rat model.The successfully medeled rats were divided into OA group,leonurine L group,leonurine M group,and leonurine H group,with 12 rats in each group.Other 12 rats were collected as the sham operation group.HE staining was applied to detect the morphological changes of chondrocytes of rats in each group.Transmission electron microscopy was applied to observe the ultrastructure of chondrocytes of rats in each group.TUNEL method was applied to detect chondrocyte apoptosis of rats in each group.RT-qPCR was applied to detect the mRNA expression levels of PINK1,Parkin,P62,and LC3B in the cartilage tissue of rats in each group.Western blotting was applied to detect the expression levels of PINK1,Parkin,P62,and LC3B proteins in the cartilage tissue of rats in each group.Results Compared to that in the sham surgery group,the cartilage morphology of rats in the OA group was abnormal,the structure was blurry,the cracks appeared on the surface,the number of mitochondria in chondrocytes reduced,and a small amount of autophagosomes were present.The apoptosis rate of chondrocytes and the levels of P62 mRNA and protein in the cartilage tissue increased significantly.The mRNA and protein levels of PINK1,Parkin,and LC3B in the cartilage tissue reduced significantly(P＜0.05).Compared to those in the OA group,the cartilage morphology of the rats in the leonurine L,M,H groups gradually recovered,the structure gradually became clear,surface cracks decreased,the number of chondrocytes restored,mitochondria increased,and the number of autophagosomes increased.The apoptosis rate of chondrocytes and the mRNA and protein levels of P62 in the cartilage tissue reduced significantly.The mRNA and protein levels of PINK1,Parkin,and LC3B in the cartilage tissue increased with a dose-dependent manner(P＜0.05).Conclusion Leonurine may promote autophagy and inhibit chondrocyte apoptosis in OA rats by activating the PINK1/Parkin signaling pathway.
基金项目
自治区卫生健康科技计划(2022)(202202341)
NETL
NSTL
万方数据