Liang Dan, Li Zhiyang, Feng Zhaowei, Yuan Zhiping, Dai Yunli, Wu Xin, Zhang Fan, Wang Yuanyuan, Zhou Yuxia, Liu Lingling, Shi Mingjun, Xiao Ying, Guo Bing
Guizhou Provincial Key Laboratory of Pathogenesis & Drug Research on Common Chronic Diseases, Guizhou Medical University, Guiyang, China.
Department of Pathophysiology, Guizhou Medical University, Guiyang, China.
Front Pharmacol. 2022 Jun 23;13:926211. doi: 10.3389/fphar.2022.926211. eCollection 2022.
Diabetic kidney disease is a major cause of chronic kidney condition and the most common complication of diabetes. The cellular senescence participates in the process of diabetic kidney disease, but the specific mechanism is not yet clear. Cell cycle-related protein E2F transcription factor 1 (E2F1) is a member of the E2F transcription factor family, it plays a key role in cellular damage under HG conditions. In this study, we explored whether metformin improves a high-glucose-induced senescence and fibrosis of renal tubular epithelial cells through cell cycle-related protein E2F1. In the experiments, the recombinant adeno-associated virus (AAV-shE2F1) knockdown gene was injected into the tail vein of 16-weeks-old mice for 8 weeks. The 16-week-old mice were administered metformin (260 mg/kg/d) continuously for 8 weeks. The normal control group (NC) and diabetic model group (DM) were set up simultaneously. Mice renal tubular epithelial cells (mRTECs) were cultured . The cells were randomly divided into the following groups: normal glucose (NG, containing 5.5 mmol/L glucose), high glucose group (HG, containing 30 mmol/L glucose), NG/HG metformin intervention group (NG/HG + Met), NG/HG negative control siRNA transfection group (NG/HG + Control), NG/HG E2F1 siRNA transfection group (NG/HG + siRNA E2F1), HG metformin intervention and overexpression E2F1 plasmid transfection group (HG + Met + overexpress-E2F1). The expression of related indexes were detected by Western blot, real-time polymerase chain reaction (PCR), immunohistochemistry, and immunofluorescence. The results showed that E2F1 knockdown or metformin reduces the degree of renal fibrosis, DNA damage, and cellular senescence in the DM group; metformin also reduced the expression of E2F1. If E2F1 was overexpressed, the effects of metformin in delaying fibrosis and reducing DNA damage and cellular senescence could be weakened. Thus, metformin alleviates high-glucose-induced senescence and fibrosis of renal tubular epithelial cells by downregulating the expression of E2F1.
糖尿病肾病是慢性肾脏疾病的主要病因，也是糖尿病最常见的并发症。细胞衰老参与了糖尿病肾病的发生过程，但其具体机制尚不清楚。细胞周期相关蛋白E2F转录因子1（E2F1）是E2F转录因子家族的成员，在高糖（HG）条件下的细胞损伤中起关键作用。在本研究中，我们探讨了二甲双胍是否通过细胞周期相关蛋白E2F1改善高糖诱导的肾小管上皮细胞衰老和纤维化。实验中，将重组腺相关病毒（AAV-shE2F1）敲低基因注入16周龄小鼠尾静脉，持续8周。对16周龄小鼠连续8周给予二甲双胍（260 mg/kg/d）。同时设立正常对照组（NC）和糖尿病模型组（DM）。培养小鼠肾小管上皮细胞（mRTECs）。将细胞随机分为以下几组：正常葡萄糖组（NG，含5.5 mmol/L葡萄糖）、高糖组（HG，含30 mmol/L葡萄糖）、NG/HG二甲双胍干预组（NG/HG + Met）、NG/HG阴性对照siRNA转染组（NG/HG + Control）、NG/HG E2F1 siRNA转染组（NG/HG + siRNA E2F1）、HG二甲双胍干预及E2F1质粒过表达组（HG + Met + overexpress-E2F1）。通过蛋白质免疫印迹法、实时聚合酶链反应（PCR）、免疫组织化学和免疫荧光检测相关指标的表达。结果显示，E2F1基因敲低或二甲双胍可降低DM组肾纤维化程度、DNA损伤及细胞衰老程度；二甲双胍还可降低E2F1的表达。若E2F1过表达，二甲双胍延缓纤维化、减少DNA损伤及细胞衰老的作用会减弱。因此，二甲双胍通过下调E2F1的表达减轻高糖诱导的肾小管上皮细胞衰老和纤维化。
