甘草酸抑制O-GlcNAc糖基化修饰对高糖诱导的小鼠肾系膜细胞和足细胞线粒体自噬的影响

Effect of glycyrrhizic acid on high glucose-induced mitophagy in mouse mesangial cells and podocytes via inhibiting O-GlcNAcylation

摘要

目的 探讨高糖环境下甘草酸(glycyrrhizic acid,GA)抑制氧连接β-N-乙酰葡萄糖胺(O-linked β-N-acetylglucosamine, O-GlcNAc)糖基化修饰对肾脏细胞线粒体自噬的影响。方法 小鼠肾系膜细胞和足细胞分为正常(normal glucose,NG)组、高糖(high glucose,HG)组、HG+GA组、糖基化抑制剂(HG+OSMI-1)组、糖基化诱导剂(HG+GA+TMG)组。CCK-8法筛选GA合适的干预浓度；Western blotting、RT-qRCR、免疫荧光等实验检测细胞糖基化、自噬等相关因子的表达；DHE探针检测活性氧(reactive oxygen species,ROS)；JC-1检测线粒体膜电位。结果 与NG组相比,高糖环境会导致细胞的O-GlcNAc糖基化修饰、O-GlcNAc转移酶(O-GlcNAc transferase, OGT)及P62、TIM23、TOM20等因子水平升高(P＜0.05),ROS表达升高(P＜0.05),O-GlcNAc水解酶(O-GlcNAcase, OGA)及LC3B水平降低(P＜0.05),线粒体膜电位下降；与HG组相比,加入GA或糖基化抑制剂可以下调O-GlcNAc糖基化修饰、OGT、P62、TIM23、TOM20及ROS等水平(P＜0.05),上调OGA及LC3B水平(P＜0.05),线粒体膜电位上升；糖基化诱导剂可以逆转GA对上述因子的调节作用(P＜0.05)。结论 GA可能通过抑制O-GlcNAc糖基化修饰改善高糖环境下小鼠肾系膜细胞和足细胞的线粒体自噬情况,从而对细胞产生保护作用。

Abstract

Objective To investigate the effect of glycyrrhizic acid (GA) on mitophagy in kidney cells in a high-glucose environment via inhibiting O-GlcNAcylation. Methods Mouse mesangial cells and podocytes were cultured into normal glucose (NG) group, high glucose (HG) group, GA (HG+GA) group, O-GlcNAcylation inhibitor (HG + OSMI-1) group, and O-GlcNAcylation inducer (HG + GA + TMG) group. An appropriate intervention concentration of GA was screened with CCK-8 method. Western blotting, RT-qRCR, and immunofluorescence were used to detect the expressions of factors related to O-GlcNAcylation and mitophagy in each group of cells. The DHE probe was used to test reactive oxygen species (ROS). JC-1 kit was used to test mitochondrial membrane potential. Results Compared with normal group, the high-glucose environment could increase the levels of O-GlcNAcylation, O-GlcNAc transferase (OGT), P62, TIM23 and TOM20 (all P＜0.05), as well as the expression of ROS (P＜0.05), but decrease the levels of O-GlcNAcase (OGA) and LC3B (P＜0.05) and the mitochondrial membrane potential. Compared with the high glucose group, adding GA or O-GlcNAcylation inhibitor could lower the levels of O-GlcNAcylation, OGT, P62, TIM23, TOM20 and ROS (P ＜0.05), but increase the levels of OGA and LC3B (P＜0.05) as well as the mitochondrial membrane potential. O-GlcNAcylation inducer could reverse the effect of GA on the above factors (all P＜0.05). Conclusion GA may improve the mitophagy of renal mesangial cells and podocytes in the high-glucose environment by inhibiting O-GlcNAcylation, thus exerting a protective effect on the cells.

关键词

糖尿病肾病(DKD) / 甘草酸(GA) / 系膜细胞 / 足细胞 / O-GlcNAc糖基化 / 线粒体自噬

Key words

diabetic kidney disease (DKD) / glycyrrhizic acid (GA) / mesangial cell / podocyte / O-GlcNAcylation / mitophagy

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基金资助

宁夏自然科学基金资助项目(2024AAC03654)