Abstract

Emodin has been shown to exert a renoprotective effect in diabetic nephropathy (DN). In this paper, we investigated whether circular RNAs (circRNAs) might be involved in the renoprotective mechanism of emodin in DN. The levels of malondialdehyde (MDA), reactive oxygen species (ROS), superoxide dismutase (SOD), interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) were measured using the corresponding assay kits. The expression levels of circ_0000064, microRNA (miR)-30c-5p, large multifunctional protease 7 (Lmp7), fibronectin (FN), and collagen type I (Col.1) were gauged by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. Subcellular localization assay was used to assess the cellular localization of circ_0000064. Targeted relationships among circ_0000064, miR-30c-5p and Lmp7 were confirmed by dual-luciferase reporter, RNA pull-down and RNA immunoprecipitation (RIP) assays. Our data showed the alleviative effect of emodin on HG-induced oxidative stress, inflammation and extracellular matrix (ECM) accumulation in SV-MES13 cells. Circ_0000064 was an importantly downstream effector of emodin function in HG-induced SV40-MES13 cells. Moreover, circ_0000064 directly targeted miR-30c-5p, and circ_0000064 modulated Lmp7 expression through miR-30c-5p. Circ_0000064 silencing alleviated HG-induced cell oxidative stress, inflammation and ECM accumulation via up-regulating miR-30c-5p. The enforced expression of miR-30c-5p attenuated HG-induced oxidative stress, inflammation and ECM accumulation in SV40-MES13 cells by targeting Lmp7. Our findings identified that emodin alleviated HG-induced oxidative stress, inflammation and ECM accumulation in SV40-MES13 cells at least partially by the regulation of the circ_0000064/miR-30c-5p/Lmp7 axis.

摘要

大黄素已被证明在糖尿病肾病 (DN) 中发挥肾脏保护作用。在本文中，我们研究了环状 RNA (circRNA) 是否可能参与 DN 中大黄素的肾脏保护机制。使用相应的测定法测量丙二醛（MDA）、活性氧（ROS）、超氧化物歧化酶（SOD）、白细胞介素-1β（IL-1β）、IL-6和肿瘤坏死因子-α（TNF-α）的水平套件。通过定量实时聚合酶链反应（qRT- PCR) 或蛋白质印迹。亚细胞定位分析用于评估 circ_0000064 的细胞定位。circ_0000064、miR-30c-5p 和Lmp7通过双荧光素酶报告基因、RNA 下拉和 RNA 免疫沉淀 (RIP) 测定得到证实。我们的数据显示了大黄素对 SV-MES13 细胞中 HG 诱导的氧化应激、炎症和细胞外基质 (ECM) 积累的缓解作用。Circ_0000064 是 HG 诱导的 SV40-MES13 细胞中大黄素功能的重要下游效应子。此外，circ_0000064 直接靶向 miR-30c-5p，circ_0000064 通过 miR-30c-5p 调节Lmp7的表达。Circ_0000064 沉默通过上调 miR-30c-5p减轻了 HG 诱导的细胞氧化应激、炎症和 ECM 积累。miR-30c-5p 的强制表达通过靶向Lmp7减弱 HG 诱导的 SV40-MES13 细胞中的氧化应激、炎症和 ECM 积累. 我们的研究结果表明，大黄素至少部分地通过调节 circ_0000064/miR-30c-5p/ Lmp7轴来减轻 HG 诱导的 SV40-MES13 细胞中的氧化应激、炎症和 ECM 积累。