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Abstract：
With the increasing number of patients with hypertensive nephropathy worldwide, it has posed a major threat to health and studies on its treatment and pathogenesis are imminent. The present study investigated the mechanism through which microRNA (miR)-98-5p in microvesicles (MVs) secreted by endothelial progenitor cells (EPCs) is involved in the repair of angiotensin II (Ang II)-induced injury of rat primary renal kidney cells (PRKs). After isolation of rat renal cortical sections, PRKs were isolated by density gradient centrifugation and identified by immunofluorescence staining. Transmission electron microscopy identifies successful separation of Mvs. An in vitro cell injury model was constructed using Ang II. The Gene Expression Omnibus was used to analyze the differentially expressed genes between diabetic rats and normal rats, and the Kyoto Encyclopedia of Genes and Genomes was used to analyze the signaling pathways involved in these differentially expressed genes. Reverse transcription-quantitative PCR was used to analyze the effect of EPC-MVs on the expression of miRNA induced by Ang II, and the levels of target genes and signaling pathway-related proteins involved were analyzed by western blot. luciferase was used to detect the targeted binding of miR-98-5p to insulin-like growth factor 1 receptor (IGF1R). Enzyme-linked immunosorbent assay was used to analyze the effect of EPC-MVs on Ang II-induced oxidative stress and inflammation levels on PRKs. Cell Counting Kit-8 was used to analyze the effect of EPC-MVs on the cell viability of PRKs induced by Ang II. The results showed that treatment of PRKs with Ang II decreased cell viability, whereas oxidative stress and inflammation were increased. However, EPC-MVs alleviated Ang II-induced damage of the PRKs. During this process, the Ang-II-induced downregulation of miR-98-5p was reversed by EPC-MVs, so miR-98-5p may be a key factor regulating the action of EPC-MVs. Dual-luciferase assay confirmed that miR-98-5p targets IGF1R. It was subsequently demonstrated that EPC-MVs overexpressing miR-98-5p promoted phosphorylation of PI3K/Akt/endothelial nitric oxide synthase (eNOS), and inhibited the oxidative stress and inflammation in PRKs, which were reversed by the overexpression of IGF1R. In conclusion, the results of the present study demonstrated that EPC-MVs with high expression of miR-98-5p can activate the PI3K/Akt/eNOS pathway by regulating IGF1R, as well as protect PRKs from Ang II-induced oxidative stress, inflammation and inhibition of cell viability.
摘要：
随着世界范围内高血压肾病患者数量的不断增加,它对健康构成了重大威胁，对其治疗和发病机制的研究迫在眉睫。本研究研究了内皮祖细胞（EPCs）分泌的微泡（MV）中的microRNA（miR）-98-5p参与血管紧张素II（AngII）诱导的大鼠原发性肾肾损伤的修复机制。细胞（PRKs）。分离大鼠肾皮质切片后，通过密度梯度离心法分离PRK，并通过免疫荧光染色进行鉴定。透射电子显微镜鉴定了Mvs的成功分离。使用AngII构建了体外细胞损伤模型。用基因表达Omnibus分析糖尿病大鼠与正常大鼠的差异表达基因,京都基因和基因组百科全书被用来分析这些差异表达基因的信号通路。逆转录-定量PCR分析EPC-MVs对AngⅡ诱导miRNA表达的影响,蛋白印迹分析靶基因和信号通路相关蛋白的水平。荧光素酶用于检测miR-98-5p与胰岛素样生长因子1受体(IGF1R)的靶向结合。酶联免疫吸附试验用于分析EPC-MVs对AngII诱导的PRK氧化应激和炎症水平的影响。细胞计数试剂盒-8用于分析EPC-MV对AngII诱导的PRK细胞活力的影响。结果表明，用AngII处理PRKs降低了细胞活力，而氧化应激和炎症增加。然而,EPC-MV减轻了AngII引起的PRK损伤。在这个过程中,Ang-II诱导的miR-98-5p下调被EPC-MV逆转，因此miR-98-5p可能是调节EPC-MV作用的关键因素。双荧光素酶测定证实miR-98-5p靶向IGF1R。随后证明，EPC-MV过表达miR-98-5p促进PI3K/Akt/内皮一氧化氮合酶（eNOS）的磷酸化，并抑制PRKs的氧化应激和炎症，通过IGF1R的过表达而逆转。总之,本研究结果表明,miR-98-5p高表达的EPC-MV可以通过调节IGF1R激活PI3K/Akt/eNOS通路,以及保护PRKs免受AngII诱导的氧化应激，炎症和细胞活力的抑制。
