Objectives: To investigate the protective effect of Yiqi Yangyin Huazhuo Tongluo Formula (YYHT) against high glucose-induced injury in mouse renal podocytes (MPC5 cells) and the possible mechanism.
Methods: Adult Wistar rats were treated with 19, 38, and 76 g/kg YYHT or saline via gavage for 7 days to prepare YYHT-medicated or blank sera for treatment of MPC5 cells cultured in high glucose (30 mmol/L) prior to transfection with a miR-21a-5p inhibitor or a miR-21a-5p mimic. The changes in miR-21a-5p expressions and the mRNA levels of FoxO1, PINK1, and Parkin in the treated cells were detected with qRT-PCR, and the protein levels of nephrin, podocin, FoxO1, PINK1, and Parkin were detected with Western blotting. Autophagic activity in the cells were evaluated with MDC staining. The effect of miR-21a-5p mimic on FoxO1 transcription and the binding of miR-21a-5p to FoxO1 were examined with luciferase reporter gene assay and radioimmunoprecipitation assay.
Results: MPC5 cells exposed to high glucose showed significantly increased miR-21a-5p expression, lowered expressions of FoxO1, PINK1, and Parkin1 mRNAs, and reduced levels of FoxO1, PINK1, parkin, nephrin, and podocin proteins and autophagic activity. Treatment of the exposed cells with YYHT-medicated sera and miR-21a-5p inhibitor both significantly enhanced the protein expressions of nephrin and podocin, inhibited the expression of miR-21a-5p, increased the mRNA and protein expressions of FoxO1, PINK1 and Parkin, and upregulated autophagic activity of the cells. Transfection with miR-21a-5p mimic effectively inhibited the transcription of FoxO1 and promoted the binding of miR-21a-5p to FoxO1 in MPC5 cells, and these effects were obviously attenuated by treatment with YYHT-medicated sera.
Conclusions: YYHT-medicated sera alleviate high glucose-induced injury in MPC5 cells by regulating miR-21a-5p/FoxO1/PINK1-mediated mitochondrial autophagy.
目的: 探讨益气养阴化浊通络方（YYHT）对高糖诱导小鼠肾足细胞（MPC5）损伤的保护作用及潜在机制。方法: 大鼠分别灌胃19、38、76 g/kg YYHT及生理盐水1周制备低、中、高浓度含药血清和空白血清。体外培养MPC5，分为对照组（5.5 mmol /L D-葡萄糖+miR-21a-5p-inhibitor-NC+空白血清）、模型组（30 mmol/L D-葡萄糖+miR-21a-5p-inhibitor-NC+空白血清）、YYHT-L（30 mmol/L D-葡萄糖+miR-21a-5p-inhibitor-NC+低浓度含药血清）、YYHT-M（30 mmol/L D-葡萄糖+miR-21a-5p-inhibitor-NC+中浓度含药血清）、YYHT-H（30 mmol/L D-葡萄糖+NC+高浓度含药血清）、miR-21a-5p抑制剂组（30 mmol/L D-葡萄糖+miR-21a-5pinhibitor+空白血清）。qRT-PCR检测miR-21a-5p表达及FoxO1、PINK1、Parkin 的mRNA表达，Western blotting检测足细胞标志蛋白（Nephrin、Podocin）及FoxO1、PINK1、Parkin蛋白表达，MDC染色检测自噬荧光。将MPC5分为阴性对照组（30 mmol/L D-葡萄糖+miR-21a-5p-NC+空白血清）、miR-21a-5p-mimic组（30 mmol/L D-葡萄糖+miR-21a-5p-mimic+空白血清）、YYHT-L（30 mmol/L D-葡萄糖+miR-21a-5p-mimic+低浓度含药血清）、YYHT-M（30 mmol/L D-葡萄糖+miR-21a-5p-mimic+中浓度含药血清）、YYHT-H（30 mmol/L D-葡萄糖+miR-21a-5p-mimic+高浓度含药血清），荧光素酶报告基因实验检测miR-21a-5p/FoxO1转录调控，RIP检测miR-21a-5p与靶基因FoxO1结合。结果: 与对照组相比，模型组miR-21a-5p表达升高（P<0.05），FoxO1、PINK1、Parkin mRNA表达降低（P<0.05），FoxO1、PINK1、Parkin、Nephrin、Podocin蛋白表达及自噬荧光降低（P<0.05）；与模型组相比，不同剂量YYHT含药血清及miR-21a-5p-inhibitor促进Nephrin及Podocin蛋白表达（P<0.05），抑制miR-21a-5p表达（P<0.05），增强FoxO1、PINK1、Parkin 的mRNA和蛋白表达及自噬荧光（P<0.05）。与miR-21a-5p-NC组相比，miR-21a-5p-mimic组抑制FoxO1转录（P<0.05），促进miR-21a-5p与FoxO1的结合（P<0.05）；与miR-21a-5p组相比，YYHT含药血清组促进FoxO1转录（P<0.05），抑制miR-21a-5p与FoxO1的结合（P<0.05）。结论: 益气养阴化浊通络方可能通过调节miR-21a-5p/FoxO1/PINK1介导的线粒体自噬，减轻高糖诱导的小鼠肾足细胞损伤。.
Keywords: Yiqi Yangyin Huazhuo Tongluo Formula; diabetic nephropathy; miR-21/FoxO1/PINK1; mitochondrial autophagy; podocyte injury.