| 王小亚,杜旭辉,何晓刚.微小 RNA-199a-5p调控 Klotho表达对体外脑缺血再灌注大鼠氧 -葡萄糖剥夺 /再灌注诱导的肾上腺嗜铬瘤细胞损伤的保护作用[J].安徽医药,2022,26(11):2285-2291. |
| 微小 RNA-199a-5p调控 Klotho表达对体外脑缺血再灌注大鼠氧 -葡萄糖剥夺 /再灌注诱导的肾上腺嗜铬瘤细胞损伤的保护作用 |
| Protective mechanism of Klotho expression regulated by mir-199a-5p on OGD/R injury of PC12 cells |
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| DOI：10.3969/j.issn.1009-6469.2022.11.037 |
| 中文关键词: PC12细胞 再灌注损伤 嗜铬细胞瘤 微小 RNA-199a-5p 氧 -葡萄糖剥夺 /再灌注损伤 氧化应激 炎症反应 Klotho |
| 英文关键词: PC12 cells Reperfusion injury Pheochromocytoma miR-199a-5p Oxygen-glucose deprivation/reperfusion inju. ry Oxidative stress Inflammatory response Klotho |
| 基金项目:河南省医学科技攻关项目( 20170101210) |
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| 中文摘要: |
| 目的探究抑制微小 RNA-199a-5p(miR-199a-5p)表达对体外脑缺血再灌注( I/R)损伤大鼠模型氧 -葡萄糖剥夺 /再灌注(OGD/R)诱导的肾上腺嗜铬瘤细胞( PC12)损伤的保护作用，并进一步探讨 Klotho在该过程中的作用。方法将 PC12细胞分为 Control组、 OGD/R组、 miR-NC inhibitor组、 miR-199a-5p inhibitor组、 miR-199a-5p inhibitor+shRNA-pLKO.1组和 miR-199a-5p inhibitor+shRNA-Klotho组。 RT-qPCR或蛋白质印迹法( Western blotting)确定各组 PC12细胞转染效率；胆囊收缩素 /缩胆囊素八肽( CCK-8)和流式细胞术确定各组 PC12细胞活力和凋亡率；试剂盒测量各组 PC12细胞中活性氧( ROS)释放量、丙二醛(MDA)含量以及超氧化物歧化酶( SOD)和谷胱甘肽过氧化物酶( GSH-Px)活性；酶联免疫吸附试验( ELISA)法检测各组 PC12细胞中肿瘤坏死因子 -α(TNF-α)、白细胞介素 -1β(IL-1β)、单核细胞趋化蛋白 -1(MCP-1)和白细胞介素 -6(IL-6)水平；双荧光素酶报告基因实验检测 miR-199a-5p与 Klotho靶向关系。结果在 OGD/R诱导的 PC12细胞中， miR-199a-5p表达增高， Klotho表达降低；细胞活力降低，凋亡率增高； ROS释放量和 MDA含量以及 TNF-α、IL-1β、MCP-1和 IL-6水平升高， SOD和 GSH-Px活性降低( P<0.05)。转染 miR-199a-5p抑制物减弱了 OGD/R的诱导 PC12细胞活力下降和细胞凋亡率增高；削弱了暴露于 OGD/R的 PC12细胞中 ROS释放量和 MDA含量以及 TNF-α、IL-1β、MCP-1和 IL-6水平，并提高了 SOD与 GSH-Px活性(P<0.05)而 miR-199a-5p抑制物对 OGD/R诱导 PC12细胞的这些作用可被敲低的 Klotho逆转( P<0.05)。另外， miR-199a-5p负靶向调控，Klotho表达(P<0.05)。结论抑制 miR-199a-5p通过靶向 Klotho减轻氧化应激和炎症反应来改善 OGD/R诱导的 PC12细胞损伤。 |
| 英文摘要: |
| Objective To explore the protective effect of inhibiting the expression of microRNA-199a-5p (miR-199a-5p) on the inju. ry of rat adrenal pheochromoma cells (PC12) induced by oxygen-glucose deprivation/reperfusion (OGD/R) in an in vitro brain ischemia-reperfusion (I/R) model, and further explore the role of Klotho in this process.Methods PC12 cells were separated into Control group, OGD/R group, miR-NC inhibitor group, miR-199a-5p inhibitor group, miR-199a-5p inhibitor+shRNA-pLKO.1 group and miR-199a-5p inhibitor+ shRNA-Klotho group. RT-qPCR or Western blot was performed to determine the transfection efficiency of PC12 cells in eachgroup; cholecystokinin/cholecystokinin octapeptide (CCK-8) and flow cytometry were performed to determine the viability and apoptosisrate of PC12 cells in each group; the kit was performed to measure Reactive oxygen species (ROS) release, malondialdehyde (MDA)content and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities in PC12 cells of each group; enzyme-linked im. munosorbent assay (ELISA) method was performed to determine the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) in PC12 cells of each group; dual luciferase reporter gene experi.ment was performed to determine the targeting relationship between miR-199a-5p and Klotho.Results In PC12 cells induced by OGD/R, the expression of miR-199a-5p increased, and the expression of Klotho decreased; cell viability decreased and apoptosis rate in.creased; the release of ROS, the content of MDA and the levels of TNF-α, IL-1β, MCP-1 and IL-6 increased, and the activities of SOD and GSH-Px decreased (P<0.05). Transfection of miR-199a-5p inhibitor attenuated the decrease in PC12 cell viability and increase inapoptosis rate induced by OGD/R; weakened the release of ROS, the content of MDA and the levels of TNF-α, IL-1β, MCP-1 and IL-6 in PC12 cells exposed to OGD/R, and increased the activities of SOD and GSH-Px (P<0.05), these effects of miR-199a-5p inhibitor on OGD/R-induced PC12 cells was able to be reversed by knockdown Klotho (P<0.05). In addition, miR-199a-5p negatively targeted Klotho expression (P<0.05).Conclusion Inhibition of miR-199a-5p can improve PC12 cell injury induced by OGD/R by targeting Klotho to reduce oxidative stress and inflammation. |
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