麝香保心丸调节PINK1/Parkin信号通路对糖尿病视网膜病变大鼠的影响

Effect of Shexiang Baoxin Pill on regulating PINK1/Parkin signaling pathway in diabetic retinopathy rats

关键词:  麝香保心丸  PINK1/Parkin 信号通路  糖尿病视网膜病变  线粒体自噬

Key words:  Shexiang Baoxin Pill  PINK1/Parkin signaling pathway  diabetic retinopathy  mitochondrial autophagy

摘要:

[目的] 探讨麝香保心丸（SBP）对糖尿病视网膜病变（DR）大鼠线粒体自噬及PTEN诱导激酶蛋白1（PINK1）/细胞质E3-泛素连接酶（Parkin）信号通路的影响。[方法] 建立DR大鼠模型，将大鼠随机分为正常组（Control组）、模型组（DR组）、麝香保心丸低剂量组（SBP-L组）、麝香保心丸高剂量组（SBP-H组）、麝香保心丸高剂量+雷帕霉素组（SBP-H+RAP组）。检测各组大鼠空腹血糖（FPG）及总胆固醇（TC）、三酰甘油（TG）、低密度脂蛋白胆固醇（LDL-C）水平；苏木精-伊红（HE）染色观察各组大鼠DR情况；酶联免疫吸附实验（ELISA）检测各组大鼠血管内皮生长因子（VEGF）、高迁移率族蛋白1（HMGB1）水平；透射电镜观察各组大鼠细胞线粒体形态；免疫组化检测线粒体自噬相关蛋白选择性自噬接头蛋白sequestosome-1（SQSTM1/P62）、微管相关蛋白轻链3Ⅱ（LC3-Ⅱ）的表达；蛋白免疫印迹法检测各组大鼠PINK1、Parkin蛋白表达。[结果] 与Control组比较，DR组视网膜结构破坏严重，细胞排列紊乱，水肿明显，细胞间隙变宽，线粒体损伤、肿胀明显，形态异常，FPG、TC、TG、LDL-C、VEGF、HMGB1、LC3-Ⅱ、PINK1、Parkin表达水平显著升高，SQSTM1/P62表达水平显著降低（P＜0.05）；与DR组比较，SBP-L组、SBP-H组视网膜细胞结构改善，细胞排列逐渐整齐，细胞水肿减轻，细胞、线粒体形态均趋于正常，线粒体肿胀减轻，FPG、TC、TG、LDL-C、VEGF、HMGB1、LC3-Ⅱ、PINK1、Parkin表达水平显著降低，SQSTM1/P62表达水平显著升高（P＜0.05）；与SBP-H组相比，SBP-H+RAP组视网膜细胞结构破坏加重，细胞排列紊乱，水肿明显，线粒体肿胀加重，形态异常，FPG、TC、TG、LDL-C、VEGF、HMGB1、LC3-Ⅱ、PINK1、Parkin表达水平显著升高，SQSTM1/P62表达水平显著降低（P＜0.05）。[结论] 麝香保心丸通过抑制PINK1/Parkin信号通路抑制DR大鼠线粒体自噬。

Abstract:

[Objective] To investigate the effect of Shexiang Baoxin Pill (SBP) on mitochondrial autophagy and PTEN-induced putative kinase 1(PINK1)/cytoplasmic E3 ubiquitin-ligase enzyme(Parkin) signaling pathway in diabetic retinopathy(DR) rats. [Methods] DR rats were separated into normal group(control group),model group(DR group),low dose Shexiang Baoxin Pill group(SBP-L group),high dose Shexiang Baoxin Pill group(SBP-H group),and high dose Shexiang Baoxin Pill+rapamycin group(SBP-H+RAP group). The levels of fasting blood sugar (FPG),total cholesterol (TC),triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) of blood lipids were detected in rats of each group;HE staining was applied to observe diabetic retinopathy of rats in each group;transmission electron microscopy was applied to observe the mitochondrial morphology of rat cells in each group;immunohistochemistry was applied to detect the expression of mitochondrial autophagy related proteins SQSTM1/P62,LC3-Ⅱ;Western blot was applied to detect the expression of PINK1 and Parkin proteins of rats in each group. [Results] Compared with the control group,the retinal structure of the DR group was severely damaged,with disordered cell arrangement,obvious edema,widened cell gaps,obvious mitochondrial damage and swelling,and abnormal morphology,the expression levels of FPG,TC,TG,LDL-C,LC3-Ⅱ,PINK1,and Parkin were obviously increased,the expression level of SQSTM1/P62 was obviously reduced(P<0.05);compared with the DR group,the structure of retinal cells in the SBP-L and SBP-H groups improved,the cell arrangement gradually became neat,the cell edema decreased,the morphology of cells and mitochondria tended to be normal,and mitochondrial swelling decreased,the expression levels of FPG,TC,TG,LDL-C,LC3-Ⅱ,PINK1, and Parkin were obviously reduced,the expression level of SQSTM1/P62 was obviously increased (P<0.05);compared with the SBP-H group,the structural damage of retinal cells in SBP-H+RAP group worsened,with disordered cell arrangement,obvious edema,increased mitochondrial swelling,and abnormal morphology,the expression levels of FPG,TC,TG,LDL-C,LC3-Ⅱ,PINK1,and Parkin were obviously increased,the expression level of SQSTM1/P62 was obviously reduced (P<0.05). [Conclusion] Shexiang Baoxin Pill can inhibit mitochondrial autophagy in diabetic retinopathy rats by inhibiting PINK1/Parkin signaling pathway.