苍术素调节PINK1/Parkin信号通路对膝骨关节炎大鼠线粒体自噬的影响

Effect of Atractylodin on Mitochondrial Autophagy in Rats with Knee Osteoarthritis by Regulating PINK1/Parkin Signal Pathway

目的:探讨苍术素对膝骨关节炎(KOA)大鼠线粒体自噬的影响以及PINK1/Parkin信号通路在此过程中的作用。方法:采用关节腔内注射碘乙酸钠法构建KOA模型。将SD大鼠按随机数表法分为对照组、模型组、(低、高)剂量苍术素组(KOA模型建模成功后腹腔分别注射3mg/kg、6mg/kg苍术素)和高剂量苍术素+自噬抑制剂(3-MA)组(KOA模型建模成功后腹腔注射6mg/kg苍术素+15mg/kg 3-MA),每组16只。ELISA法检测大鼠血清中TNF-α、IL-1β、软骨寡聚基质蛋白(COMP)水平;番红O-固绿染色检测大鼠膝关节软骨组织病理学改变,并进行Mankin’s评分;TUNEL染色检测大鼠膝关节软骨细胞凋亡率;DHE荧光探针检测大鼠膝关节软骨组织中ROS水平;免疫组化检测大鼠膝关节软骨组织中cleaved Caspase-3、LC3阳性表达;Western Blot检测大鼠膝关节软骨组织中自噬以及PINK1/Parkin通路相关蛋白表达。结果:与对照组比较,模型组大鼠血清中TNF-α、IL-1β、COMP水平、膝关节软骨组织中Mankin’s评分、细胞凋亡率、ROS水平、cleaved Caspase-3和LC3阳性细胞比例以及Beclin1、LC3Ⅱ/LC3Ⅰ、PINK1、Parkin蛋白表达水平均升高(P<0.05),且存在明显的膝关节软骨病理损伤;与模型组比较,(低、高)剂量苍术素组大鼠血清中TNF-α、IL-1β、COMP水平以及膝关节软骨组织中Mankin’s评分、细胞凋亡率、ROS水平、cleaved Caspase-3阳性细胞比例降低(P<0.05),而LC3阳性细胞比例以及Beclin1、LC3Ⅱ/LC3Ⅰ、PINK1、Parkin蛋白表达水平进一步升高(P<0.05),且膝关节软骨病理损伤有所减轻;与高剂量苍术素组比较,高剂量苍术素+3-MA组大鼠血清中TNF-α、IL-1β、COMP水平以及膝关节软骨组织中Mankin’s评分、细胞凋亡率、ROS水平、cleaved Caspase-3阳性细胞比例升高(P<0.05),而LC3阳性细胞比例以及Beclin1、LC3Ⅱ/LC3Ⅰ、PINK1、Parkin蛋白表达水平降低(P<0.05),且膝关节软骨病理损伤加重。结论:苍术素可能通过激活PINK1/Parkin信号通路增强KOA大鼠膝关节软骨线粒体自噬,并减少软骨细胞凋亡,从而改善膝关节软骨退变。

Objective:To investigate the effect of Atractylodin on mitochondrial autophagy in knee osteoarthritis(KOA)rats and the role of PINK1/Parkin signaling pathway in this process.Method:KOA model was established by intra-articular injection of sodium iodoacetate.SD rats were divided into control group(normal saline),model group(normal saline),(low and high)dose Atractylodin groups(3mg/kg,6mg/kg Atractylodin),and high dose Atractylodin+autophagy inhibitor(3-MA)group(6mg/kg Atractylodin+15mg/kg 3-MA),with 16 rats in each group.The levels of serum TNF-α,IL-1β and cartilage oligomeric matrix protein(COMP)were detected by ELISA.The histopathological changes of rat knee joint cartilage were detected by safranine O-solid green staining,and Mankin's score was performed,TUNEL staining was used to detect the apoptosis rate of chondrocytes in rat knee joint,DHE fluorescence probe was used to detect the level of ROS in rat knee cartilage,The expression of cleaved Caspase-3 and LC3 was detected by immunohistochemistry in the cartilage of rat knee joint,and Western blot was used to detect the expression of autophagy and PINK1/Parkin pathway related proteins in rat knee cartilage.Results:Compared with the control group,the levels of TNF-α,IL-1β and COMP in serum,Mankin's score,apoptosis rate,the level of ROS,the proportions of cleaved Caspase-3 and LC3 positive cells,and the expression levels of Beclin1,LC3Ⅱ/LC3Ⅰ,PINK1,Parkin proteins in knee cartilage tissue in the model group were increased(P<0.05).There was obvious pathological damage of knee cartilage.Compared with the model group,the levels of TNF-α,IL-1β and COMP in serum,Mankin's score,apoptosis rate,the level of ROS,the proportions of cleaved Caspase-3 and LC3 positive cells of rats in the(low and high)dose Atractylodin groups were decreased(P<0.05),while the proportion of LC3 positive cells and the expression levels of Beclin1,LC3Ⅱ/LC3Ⅰ,PINK1,Parkin proteins were increased(P<0.05),the pathological damage of knee cartilage was alleviated.Compared with the high-dose Atractylodin group,the levels of TNF-α,IL-1β and COMP in serum,Mankin's score,apoptosis rate,the level of ROS,the proportions of cleaved Caspase-3 and LC3 positive cells of rats in high dose Atractylodin+3-MA group were increased(P<0.05),the proportion of LC3 positive cells and the expression levels of Beclin1,LC3Ⅱ/LC3Ⅰ,PINK1,Parkin proteins were decreased(P<0.05),the pathological damage of knee cartilage was aggravated.Conclusion:Atractylodin can enhance mitochondrial autophagy of knee joint cartilage in KOA rats by activating PINK1/Parkin signal pathway,and reduce chondrocyte apoptosis,thereby improving the degeneration of knee joint cartilage.