Department of Pharmaceutical Analysis, Dalian Medical University, Western 9 Lvshunnan Road, Dalian 116044, China.
Department of Pharmaceutical Analysis, Dalian Medical University, Western 9 Lvshunnan Road, Dalian 116044, China; Key Laboratory for Basic and Applied Research on Pharmacodynamic Substances of Traditional Chinese Medicine of Liaoning Province, Dalian Medical University, Dalian, China; National-Local Joint Engineering Research Center for Drug Development (R&D) of Neurodegenerative Diseases, Dalian Medical University, Dalian, China.
Pharmacol Res. 2020 May;155:104718. doi: 10.1016/j.phrs.2020.104718. Epub 2020 Feb 19.
Renal ischemia-reperfusion (RI/R) injury with high morbidity and mortality is one common clinical disease. Development of drug targets to treat the disorder is critical important. MiR-27a-3p plays important roles in regulating oxidative stress. However, its effects on RI/R injury have not been reported. In this paper, hypoxia/reoxygenation (H/R) models on NRK-52E and HK-2 cells, and RI/R model in C57BL/6 mice were established. The results showed that H/R in vitro decreased cell viability and increased ROS levels in cells, and RI/R caused renal injury and oxidative damage in mice. The expression levels of miR-27a-3p were up-regulated based on real-time PCR and FISH assays in model groups compared with control groups, which directly targeted Grb2 based on dual luciferase reporter assay and co-transfaction test. In addition, miR-27a- 3p markedly reduced Grb2 expression to down-regulate the expression levels of p-PI3K, p-AKT, Nrf2, HO-1, and up-regulate Keap1 expression in model groups. MiR-27a-3p mimics in vitro enhanced H/R-caused oxidative stress via increasing ROS levels and decreasing Grb2 expression to down-regulate PI3K-AKT signal. In contrary, miR-27a-3p inhibitor in vitro significantly reduced H/R-caused oxidative damage via decreasing ROS levels and increasing Grb2 expression to up-regulate PI3K-AKT signal. In vivo, miR-27a- 3p agomir exacerbated RI/R-caused renal damage by decreasing SOD level and increasing Cr, BUN, MDA levels via suppressing Grb2 expression to down-regulate PI3K- AKT signal. However, miR-27a -3p antagomir alleviated RI/R-caused oxidative damage via increasing Grb2 expression to up-regulate PI3k-AKT signal. Grb2siRNA in mice further enhanced RI/R-caused renal injury by increasing Cr, BUN, MDA levels and decreasing SOD level via inhibiting the expression levels of Grb2, Nrf2, HO-1, and increasing Keap1 expression. Our data showed that miR-27a-3p aggravated RI/R injury by promoting oxidative stress via targeting Grb2, which should be considered as one new drug target to treat RI/R injury.
肾缺血再灌注（RI/R）损伤具有高发病率和死亡率，是一种常见的临床疾病。开发治疗该疾病的药物靶点至关重要。miR-27a-3p 在调节氧化应激方面发挥着重要作用。然而，其在 RI/R 损伤中的作用尚未被报道。在本文中，建立了 NRK-52E 和 HK-2 细胞的缺氧/复氧（H/R）模型以及 C57BL/6 小鼠的 RI/R 模型。结果表明，体外 H/R 降低了细胞活力并增加了细胞内 ROS 水平，而 RI/R 在小鼠中引起了肾损伤和氧化损伤。与对照组相比，实时 PCR 和 FISH 检测显示模型组中 miR-27a-3p 的表达水平上调，双荧光素酶报告基因检测和共转染实验表明 miR-27a-3p 直接靶向 Grb2。此外，在模型组中，miR-27a-3p 明显降低了 Grb2 的表达，从而下调了 p-PI3K、p-AKT、Nrf2、HO-1 的表达水平，并上调了 Keap1 的表达。体外 miR-27a-3p 模拟物通过增加 ROS 水平和降低 Grb2 表达来增强 H/R 引起的氧化应激，从而下调 PI3K-AKT 信号。相反，体外 miR-27a-3p 抑制剂通过降低 ROS 水平和增加 Grb2 表达来显著减少 H/R 引起的氧化损伤，从而上调 PI3K-AKT 信号。在体内，miR-27a-3p agomir 通过抑制 Grb2 表达下调 PI3K-AKT 信号，从而降低 SOD 水平并增加 Cr、BUN、MDA 水平，加剧 RI/R 引起的肾损伤。然而，miR-27a-3p antagomir 通过增加 Grb2 表达上调 PI3k-AKT 信号，从而减轻 RI/R 引起的氧化损伤。在小鼠中使用 Grb2siRNA 通过抑制 Grb2、Nrf2、HO-1 的表达水平并增加 Keap1 的表达，进一步增加 Cr、BUN、MDA 水平并降低 SOD 水平，从而加剧 RI/R 引起的肾损伤。我们的数据表明，miR-27a-3p 通过靶向 Grb2 促进氧化应激从而加重 RI/R 损伤，这可能成为治疗 RI/R 损伤的一个新的药物靶点。
