保肾通络方含药血清调节线粒体自噬对高糖诱导下足细胞氧化损伤的影响

目的 观察保肾通络方含药血清对高糖培养下足细胞氧化损伤的影响,并对保肾通络方的作用机制进行探讨。方法 以体外培养的肾小球足细胞为研究对象,给予高浓度葡萄糖刺激,采用CCK-8法分析足细胞活力以确定最佳模型及给药浓度,分为正常对照组、高糖组、保肾通络方组,采用鬼笔环肽进行细胞骨架染色,Western blotting法检测足细胞裂孔膜蛋白nephrin,自噬相关蛋白5(autophagy-related protein 5,ATG5)、ATG7、微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)的表达,流式细胞术检测足细胞活性氧(reactive oxygen species,ROS)及线粒体膜电位,免疫荧光共标LC3B及线粒体,观察各组足细胞内线粒体自噬泡的生成情况。结果 30 mmol/L高糖干预48 h可显著降低足细胞活力(P＜0.05),保肾通络方含药血清可使高糖诱导的足细胞活力明显提升(P＜0.01);与正常对照组相比,高糖组足细胞骨架褶皱呈多边形,应力纤维束被破坏,ROS生成显著增多(P＜0.01),足细胞nephrin、ATG5、ATG7表达显著降低,LC3Ⅱ/LC3Ⅰ比值降低(P＜0.05),LC3B与线粒体共标减少,线粒体膜电位下降(P＜0.01);与高糖组相比,保肾通络方组足细胞骨架改善,ROS生成减少(P＜0.01),nephrin、ATG5、ATG7表达显著增加(P＜0.05),LC3Ⅱ/LC3Ⅰ比值上升(P＜0.05),LC3B与线粒体荧光共标增多,线粒体膜电位升高(P＜0.01)。结论 保肾通络方含药血清能够减轻高糖诱导的足细胞氧化损伤,其作用可能与改善足细胞线粒体自噬有关。

Research of Baoshentongluo formula-medicated serum on regulating mitophagy and reducing oxidative damage of podocytes induced by high glucose

Objective To observe the effect of Baoshentongluo formula-medicated serum on oxidative damage of podocytes cultured with high glucose, and explore its underlying mechanism. Methods The podocytes in vitro were used as the research object, and stimulated with high concentration of glucose. CCK-8 method was used to determine optimal model and drug concentration, and then the podocytes were divided into the normal control group, high glucose group and Baoshentongluo formula group. Cytoskeleton of podocytes was stained with phalloidin.The expression levels of podocyte-specific protein nephrin and autophagy-related proteins autophagy-related protein 5(ATG5), ATG7 and microtubule-associated protein 1 light chain 3(LC3) was detected with Western blotting.Flow cytometry was used to detect the level of reactive oxygen species (ROS) and mitochondrial membrane potential of podocytes. Colocalization of LC3B and mitochondria were examined with immunofluorescence to observe the generation of mitochondrial autophagy vesicles in podocytes of each group. Results The podocyte viability was significantly decreased with 30 mmol/L high glucose intervention for 48 h (P<0.05), and Baoshentongluo formula-medicated serum could significantly increase the viability of high glucose-induced podocyte (P<0.01).Compared with normal control group, the podocytes of high glucose group were demonstrated with folded skeleton into a polygon and destroyed stress fiber bundles with increased ROS production (P<0.01), reduced expression of nephrin, ATG5, ATG7 and LC3Ⅱ/LC3Ⅰ ratio (P<0.05), decreased co-labeling of LC3B with mitochondria, lowerer mitochondrial membrane potential (P<0.01). Compared with high glucose group, the podocytes of Baoshentongluo formula group showed improved podocyte structure,decreased production of ROS (P＜0.01), elevated expression of nephrin, ATG5, ATG7 and LC3Ⅱ/LC3Ⅰ ratio (P＜0.05) ,increased co-labeling of LC3B with mitochondria, higher mitochondrial membrane potential (P＜0.01). Conclusion Baoshentongluo formula-medicated serum can alleviate oxidative damage to podocytes induced by high glucose and the mechanism may be related to improvement of mitophagy.