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。
Objective To explore the protective effect of inhibiting the expression of micro RNA-199a-5p(miR-199a-5p)on the injury of rat adrenal pheochromoma cells(PC12)induced by oxygen-glucose deprivation/reperfusion(OGD/R)in an in vitro brain ischemiareperfusion(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+sh RNA-p LKO.1 group and mi R-199a-5p inhibitor+sh RNA-Klotho group.RT-qPCR or Western blot was performed to determine the transfection efficiency of PC12 cells in each group;cholecystokinin/cholecystokinin octapeptide(CCK-8)and flow cytometry were performed to determine the viability and apoptosis rate 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 immunosorbent 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 experiment was performed to determine the targeting relationship between mi R-199a-5p and Klotho.Results In PC12 cells induced by OGD/R,the expression of mi R-199a-5p increased,and the expression of Klotho decreased;cell viability decreased and apoptosis rate increased;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 mi R-199a-5p inhibitor attenuated the decrease in PC12 cell viability and increase in apoptosis 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 P
作者
王小亚
杜旭辉
何晓刚
WANG Xiaoya;DU Xuhui;HE Xiaogang(Department of Neurology,The Second People's Hospital of Pingdingshan,Pingdingshan,Henan 467000,China;Department of Neurology,Xuchang Central Hospital,Xuchang,Henan 461000,China)
出处
《安徽医药》
CAS
2022年第11期2285-2291,共7页
Anhui Medical and Pharmaceutical Journal
基金
河南省医学科技攻关项目(20170101210)。
