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

Q3 Medicine 南方医科大学学报杂志 Pub Date : 2025-01-20 DOI:10.12122/j.issn.1673-4254.2025.01.04

Kelei Guo, Yingli Li, Chenguang Xuan, Zijun Hou, Songshan Ye, Linyun Li, Liping Chen, Li Han, Hua Bian

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Abstract

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.

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益气养阴化浊通络方通过调节miR-21a-5p/FoxO1/ pink1介导的线粒体自噬减轻糖尿病足细胞损伤。

目的：探讨益气养阴化浊通络方对高糖所致小鼠肾足细胞（MPC5）损伤的保护作用及其可能机制。方法：在转染miR-21a-5p抑制剂或miR-21a-5p模拟物之前，成年Wistar大鼠分别以19、38和76 g/kg YYHT或生理盐水灌胃7天，制备YYHT药物或空白血清，用于高糖（30 mmol/L）培养的MPC5细胞。qRT-PCR检测处理细胞中miR-21a-5p表达及FoxO1、PINK1、Parkin mRNA水平的变化，Western blotting检测nephrin、podocin、FoxO1、PINK1、Parkin蛋白水平。采用MDC染色法检测细胞自噬活性。采用荧光素酶报告基因法和放射免疫沉淀法检测miR-21a-5p模拟物对fox01转录的影响以及miR-21a-5p与fox01的结合。结果：高糖环境下MPC5细胞miR-21a-5p表达显著升高，FoxO1、PINK1、Parkin1 mrna表达降低，FoxO1、PINK1、parkin、nephrin、podocin蛋白水平降低，自噬活性降低。yyht给药血清和miR-21a-5p抑制剂处理暴露细胞，均能显著提高nephrin和podocin的蛋白表达，抑制miR-21a-5p的表达，增加FoxO1、PINK1和Parkin mRNA和蛋白表达，上调细胞自噬活性。转染miR-21a-5p模拟物可有效抑制MPC5细胞中FoxO1的转录，促进miR-21a-5p与FoxO1的结合，而这些作用在经yyt治疗的血清中明显减弱。结论：yyht给药血清通过调节miR-21a-5p/FoxO1/ pink1介导的线粒体自噬，减轻高糖诱导的MPC5细胞损伤。

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