Department of Endocrinology, Suizhou Hospital Affiliated to Hubei Medical College, China.
Department of Endocrinology, Suizhou Hospital Affiliated to Hubei Medical College, China.
Life Sci. 2020 Apr 1;246:117382. doi: 10.1016/j.lfs.2020.117382. Epub 2020 Jan 28.
Our preliminary research revealed that metformin, a classic anti-diabetic drug, could rescue Parkin protein expression and mitophagy in high glucose-challenged human renal epithelial cells in vitro, but the molecular mechanism remains to be explored. In the study, Human Renal Cortical Epithelial Cells (HRCEpiC) and Human Renal Proximal Tubular Epithelial Cells (HRPTEpic) were challenged with high glucose with or without metformin pre-treatment to monitor Parkin mRNA and protein expression level change. PRKN gene knockdown was performed by lentiviral-based shRNA delivery. Cell viability, apoptosis and mitophagy were monitored after treatment. Mitochondrial damage was evaluated by analyzing mitochondrial permeability transition pore opening, membrane potential change, mitochondrial superoxide accumulation and cytochrome C release. Protein levels of activating transcription factor 4 (ATF4), p53 phospho-Ser15, IκBα phosphor-Ser32, IKKα phosphor-Ser176/180 in whole cell lysate and nuclear entry of p50/p65 were assessed by western blot. Okadaic acid was used to inhibit protein phosphatase 2A (PP2A). The data suggested high glucose challenge significantly reduced PRKN gene expression, mitophagy, mitochondria integrity and cell viability in vitro, which was rescued by metformin co-treatment. The effects of metformin were crippled by PRKN gene knockdown. Metformin increased PRKN gene transcription while reducednuclear factor kappa B (NF-κB) activation but not that of p53 or ATF4. Inhibiting PP2A weakened NF-κB inhibition and PRKN induction by metformin in high glucose-challenged cells, reducing its mitochondrial protective and cytoprotective effect. So, we concluded thatmetformin protects human renal epithelial cells from high glucose-induced apoptosis by restoring Parkin protein expression and mitophagy via PP2A activation and NF-κB inhibition.
我们的初步研究表明，经典的抗糖尿病药物二甲双胍可以在体外挽救高糖挑战的人肾上皮细胞中的 Parkin 蛋白表达和线粒体自噬，但分子机制仍有待探索。在这项研究中，用人肾皮质上皮细胞（HRCEpiC）和人近端肾小管上皮细胞（HRPTEpic）进行高糖挑战，并用或不用二甲双胍预处理来监测 Parkin mRNA 和蛋白表达水平的变化。通过基于慢病毒的 shRNA 递送进行 PRKN 基因敲低。处理后监测细胞活力、凋亡和线粒体自噬。通过分析线粒体通透性转换孔开放、膜电位变化、线粒体超氧化物积累和细胞色素 C 释放来评估线粒体损伤。通过 Western blot 评估全细胞裂解物中激活转录因子 4 (ATF4)、p53 磷酸化 Ser15、IκBα 磷酸化 Ser32、IKKα 磷酸化 Ser176/180 的蛋白水平，以及 p50/p65 的核内进入。使用冈田酸抑制蛋白磷酸酶 2A（PP2A）。数据表明，高糖挑战显著降低了 PRKN 基因表达、线粒体自噬、线粒体完整性和细胞活力，二甲双胍共处理可挽救这一结果。PRKN 基因敲低削弱了二甲双胍的作用。二甲双胍增加了 PRKN 基因转录，同时降低了核因子 kappa B (NF-κB) 的激活，但不降低 p53 或 ATF4 的激活。在高糖挑战的细胞中，抑制 PP2A 减弱了二甲双胍对 NF-κB 的抑制和对 PRKN 的诱导，降低了其对线粒体的保护和细胞保护作用。因此，我们得出结论，二甲双胍通过激活 PP2A 和抑制 NF-κB 来恢复 Parkin 蛋白表达和线粒体自噬，从而保护人肾上皮细胞免受高糖诱导的细胞凋亡。
