Zhang Le, Dai Qian, Hu Lanlan, Yu Hua, Qiu Jing, Zhou Jiyin, Long Min, Zhou Shiwen, Zhang Kebin
National Drug Clinical Trial Institution, Xinqiao Hospital, Army Medical University, Chongqing 400037, China.
Center of Medical Experiment Technology, Xinqiao Hospital, Army Medical University, Chongqing 400037, China.
Int J Endocrinol. 2020 Jul 21;2020:1361924. doi: 10.1155/2020/1361924. eCollection 2020.
Hyperoside, a flavonoid isolated from conventional medicinal herbs, has been demonstrated to exert a significant protective effect in diabetic nephropathy. This study aimed to determine the underlying mechanisms, by which hyperoside inhibits high glucose-(HG-) induced proliferation in mouse renal mesangial cells.
Mouse glomerular mesangial cells line (SV40-MES13) was used to study the inhibitory effect of hyperoside on cell proliferation induced by 30 mM glucose, which was used to simulate a diabetic condition. Viable cell count was assessed using the Cell Counting Kit-8 and by the 5-ethynyl-20-deoxyuridine incorporation assay. The underlying mechanism involving miRNA-34a was further investigated by quantitative RT-PCR and transfection with miRNA-34a agomir. The phosphorylation levels of extracellular signal-regulated kinases (ERKs) and cAMP-response element-binding protein (CREB) were measured by Western blotting. The binding region and the critical binding sites of CREB in the miRNA-34a promoter were investigated by the chromatin immunoprecipitation assay and luciferase reporter assay, respectively.
We found that hyperoside could significantly decrease HG-induced proliferation of SV40-MES13 cells in a dose-dependent manner, without causing obvious cell death. In addition, hyperoside inhibited the activation of ERK pathway and phosphorylation of its downstream transcriptional factor CREB, as well as the miRNA-34a expression. We further confirmed that CREB-mediated regulation of miRNA-34a is dependent on the direct binding to specific sites in the promoter region of miRNA-34a.
Our cumulative results suggested that hyperoside inhibits the proliferation of SV40-MES13 cells through the suppression of the ERK/CREB/miRNA-34a signaling pathway, which provides new insight to the current investigation on therapeutic strategies for diabetic nephropathy.
金丝桃苷是从传统草药中分离出的一种黄酮类化合物，已被证明在糖尿病肾病中具有显著的保护作用。本研究旨在确定金丝桃苷抑制高糖（HG）诱导的小鼠肾系膜细胞增殖的潜在机制。
采用小鼠肾小球系膜细胞系（SV40-MES13）研究金丝桃苷对30 mM葡萄糖诱导的细胞增殖的抑制作用，30 mM葡萄糖用于模拟糖尿病状态。使用细胞计数试剂盒-8和5-乙炔基-2'-脱氧尿苷掺入试验评估活细胞数量。通过定量RT-PCR和用miRNA-34a激动剂转染进一步研究涉及miRNA-34a的潜在机制。通过蛋白质免疫印迹法测量细胞外信号调节激酶（ERK）和cAMP反应元件结合蛋白（CREB）的磷酸化水平。分别通过染色质免疫沉淀试验和荧光素酶报告基因试验研究CREB在miRNA-34a启动子中的结合区域和关键结合位点。
我们发现金丝桃苷可以显著降低HG诱导的SV40-MES13细胞增殖，呈剂量依赖性，且不会引起明显的细胞死亡。此外，金丝桃苷抑制ERK途径的激活及其下游转录因子CREB的磷酸化，以及miRNA-34a的表达。我们进一步证实，CREB介导的miRNA-34a调节依赖于与miRNA-34a启动子区域中特定位点的直接结合。
我们的累积结果表明，金丝桃苷通过抑制ERK/CREB/miRNA-34a信号通路抑制SV40-MES13细胞的增殖，这为目前糖尿病肾病治疗策略的研究提供了新的见解。
Zotero 插件
