南方医科大学学报

南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (1): 27-34.doi: 10.12122/j.issn.1673-4254.2025.01.04

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益气养阴化浊通络方通过调控miR-21a-5p/FoxO1/PINK1介导的线粒体自噬减轻糖尿病肾病小鼠的足细胞损伤

郭克磊1,2(), 李颖利1,2, 宣晨光1, 侯紫君1,2, 叶松山1,2, 李林运3, 陈丽平1,2, 韩立1,2, 卞华1,2()   

  1. 1.南阳理工学院，张仲景国医国药学院，河南 南阳 473004
    2.南阳理工学院，河南省张仲景方药与免疫调节重点实验室，河南 南阳 473004
    3.南阳市中医院肾病风湿免疫科，河南 南阳 473004
  • 收稿日期:2024-09-30 出版日期:2025-01-20 发布日期:2025-01-20
  • 通讯作者: 卞华 E-mail:keleiguo318@126.com;biancrown@163.com
  • 作者简介:郭克磊，博士，副教授，E-mail: keleiguo318@126.com
  • 基金资助:
    河南省中医药科学研究专项课题(20-21ZY1069);河南省科技攻关项目(23210231119);南阳市科技攻关项目(KJGG027)

Yiqi Yangyin Huazhuo Tongluo Formula alleviates diabetic podocyte injury by regulating miR-21a-5p/FoxO1/PINK1-mediated mitochondrial autophagy

Kelei GUO1,2(), Yingli LI1,2, Chenguang XUAN1, Zijun HOU1,2, Songshan YE1,2, Linyun LI3, Liping CHEN1,2, Li HAN1,2, Hua BIAN1,2()   

  1. 1.ZHANG Zhongjing School of Chinese Medicine, Rheumatology and Immunology, Nanyang Traditional Chinese Medicine Hospital, Nanyang 473004, China
    2.Henan Provincial Key Laboratory of ZHANG Zhong-jing Formulae and Herbs for Immunoregulation, Nanyang Institute of Technology, Nanyang 473004, China, Rheumatology and Immunology, Nanyang Traditional Chinese Medicine Hospital, Nanyang 473004, China
    3.Department of Nephrology, Rheumatology and Immunology, Nanyang Traditional Chinese Medicine Hospital, Nanyang 473004, China
  • Received:2024-09-30 Online:2025-01-20 Published:2025-01-20
  • Contact: Hua BIAN E-mail:keleiguo318@126.com;biancrown@163.com

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摘要：

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。

关键词: 糖尿病肾病, 益气养阴化浊通络方, 线粒体自噬, 足细胞损伤, miR-21a-5p/FoxO1/PINK1

Abstract:

Objective 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. Conclusion YYHT-medicated sera alleviate high glucose-induced injury in MPC5 cells by regulating miR-21a-5p/FoxO1/PINK1-mediated mitochondrial autophagy.

Key words: diabetic nephropathy, Yiqi Yangyin Huazhuo Tongluo Formula, mitochondrial autophagy, podocyte injury, miR-21/FoxO1/PINK1