基于IncRNA-MIAT/miR-324-3p/Notch通路探究大鼠脑缺血再灌注损伤的作用机制  

Study on the mechanism of cerebral ischemia-reperfusion injury in rats based on Incrna miat/mir-324-3p/notchpathway

作　　者：王玉松[1] 李培[2] 饶国敏 Wang Yusong;Li Pei;Rao(Guomin Department of Neurology,982 Hospital of Joint Service Support Force of Chinese People's Liberation Army,Hebei 063000,China)

机构地区：[1]中国人民解放军联勤保障部队第九八二医院神经内科,河北063000 [2]唐山市工人医院神经内科

出　　处：《脑与神经疾病杂志》2023年第8期488-494,共7页Journal of Brain and Nervous Diseases

基　　金：河北省科技计划项目(16397747D)。

摘　　要：目的 探讨长链非编码RNA心肌梗死相关转录(lncRNA-MIAT)在大鼠脑缺血再灌注损伤(I/R)模型的表达及其对低氧缺糖复供(OGD/R)诱导的PC12细胞凋亡及氧化应激的影响。方法 SD大鼠10只,随机分为I/R模型组及假手术组,采用大脑中动脉闭塞法(MCAO)建立脑损伤I/R模型。建立大鼠肾上腺髓质嗜铬细胞瘤的细胞系(PC12细胞)的氧糖剥夺/复氧(OGD/R)模型,检测相关指标。采用RT-PCR法检测LncRNA MIAT和miR-324-3p在SD大鼠脑组织和PC12细胞中的表达量。采用黄嘌呤氧化酶法(WST-8V)、硫代巴比妥酸法(TBA test)及5,5’—二硫代双(2—硝基苯甲酸)法(DTNB)分别检测PC12细胞中超氧化物歧化酶(SOD)、丙二醛(MDA)和谷胱甘肽(GSH)的含量;酶联免疫吸附法(ELISE)检测PC12细胞的肿瘤坏死因子—α (TNF-α)、白介素—6 (IL-6)及白介素—1β (IL-1β)等炎性因子的含量;流式细胞法检测PC12细胞的凋亡率,蛋白印迹法(Western blot)法检测细胞凋亡相关蛋白Bax(Bcl-2家族促凋亡蛋白)、Bcl-xl(Bcl-2家族抗凋亡蛋白)、Bcl-2(B淋巴细胞瘤—2),Notch信号通路相关蛋白Notch1 (Notch家族1型跨膜糖蛋白)、Hes1 (多毛增强子1)、Hes5表达量。结果 I/R组大鼠、OGD/R组PC12细胞中LncRNA MIAT水平分别显著低于假手术组、对照组(P<0.05),miR-324-3p水平呈相反变化趋势。过表达LncRNA MIAT显著上调OGD/R组LncRNA MIAT、Bcl-xl、Bcl-2表达量,抑制Bax表达量(P<0.05)。过表达LncRNA MIAT能显著降低Vetor组MDA含量,提高SOD和GSH含量(P<0.05)。ELISA结果显示,过表达LncRNA MIAT组TNF-α、IL-6及IL-1 β含量显著低于Vector组,同时显著高于过表达LncRNA MIAT+过表达miR-324-3p组(P<0.05)。Western-blot检测结果显示,过表达LncRNA MIAT组Notch1、Hes1、Hes5表达量显著低于模型组和过表达LncRNA MIAT+过表达miR-324-3p组(P<0.05)。结论 LncRNA-MIAT的过度表达可抑制OGD/R模型中PC12细胞Notch1、Hes1和Hes5(Notch通路相关蛋白)的表达,降低PC12细胞Objective To investigate the expression of long chain noncoding RNA myocardial infarction related transcripts(lncRNA MIAT)in the rat model of cerebral ischemia-reperfusion injury(I/R)and its effect on PC12 cell apoptosis and oxidative stress induced by hypoxia glucose deprivation reperfusion(OGD/R).Methods10 SD rats were randomly divided into I/R model group and sham operation group.I/R model of brain injury was established by middle cerebral artery occlusion(MCAO).The oxygen glucose deprivation/reoxygenation(OGD/R)model of rat adrenal medullary pheochromocytoma cell line(PC 12 cells)was established,and the related indexes were detected.RT-PCR was used to detect the expression of IncRNA MIAT and miR-324-3p in SD rat brain and PC12 cells.The contents of superoxide dismutase(SOD),malondialdehyde(MDA)and glutathione(GSH)in PC12cells were detected by xanthine oxidase method(wst-8v),thiobarbituric acid method(TBA test)and 5,5'-dithio bis(2-nitrobenzoic acid)method(DTNB);Detection of TNF-α(tumor necrosis factor-a),IL-6(interleukin-6),IL-1β(IL-1β)and other inflammatory factors in PC 12 cells by enzyme linked immunosorbent assay(ELISA);The apoptosis rate of PC 12 cells was detected by flow cytometry,and the expression of apoptosis related proteins Bax(Bcl-2 family Pro apoptotic protein),BCL-xl(Bcl-2 family anti apoptotic protein),Bcl-2(B lymphoma-2),Notch signaling pathway related proteins Notch 1(Notch family type 1 transmembrane glycoprotein),Hesl(hairy enhancer1)and Hes5(hairy enhancer 5)were detected by Western blot.Results The level of lncrna MIAT in PC 12 cells in I/R group and OGD/R group was significantly lower than that in sham operation group and control group respectively(P<0.05),and the level of mir-324-3p showed an opposite trend.Overexpression of lncrna MIAT significantly increased the expression of lncrna MIAT,Bcl-xl and Bcl-2 in OGD/R group,and inhibited the expression of Bax(P<0.05).Overexpression of lncrna MIAT could significantly reduce MDA content and increase SOD and GSH content in vetor group(P<0

关 键 词：LncRNA MIAT miR-324-3p NOTCH信号通路 脑缺血再灌注损伤 氧化应激 炎症 

分 类 号：R743.32[医药卫生—神经病学与精神病学]