GA结合蛋白转录因子亚基β1的反义RNA对过氧化氢诱导的大鼠脑微血管内皮细胞损伤的影响  

Effect of GA binding protein transcription factor subunitβ1 antisense RNA1 on hydrogen peroxide-induced brain microvascular endothelial cells injury in rats

作　　者：马迎辉 潘冬梅 叶继业 胡骁 Ma Yinghui;Pan Dongmei;Ye Jiye;Hu Xiao(Neurosurgery Department,2Geriatric Department,Central Hospital of Edong Healthcare,Huangshi City,Hubei Province,Huangshi 435000,China)

机构地区：[1]湖北省黄石市鄂东医疗集团市中心医院神经外科,435000 [2]湖北省黄石市鄂东医疗集团市中心医院老年病科,435000

出　　处：《心脑血管病防治》2023年第4期4-10,共7页CARDIO-CEREBROVASCULAR DISEASE PREVENTION AND TREATMENT

摘　　要：目的探讨GA结合蛋白转录因子亚基β1的反义RNA(GABPB1-AS1)对过氧化氢(H2O2)诱导的大鼠脑微血管内皮细胞损伤的影响及可能机制。方法体外培养大鼠脑微血管内皮细胞,分为对照(Con)组、模型(Model)组、A组(Model加用si-NC)、B组(Model加用si-GABPB1-AS1)、C组(Model加用si-GABPB1-AS1及anti-miR-NC)和D组(Model加用si-GABPB1-AS1及anti-miR-101-3p)。RT-qPCR检测细胞中GABPB1-AS1和miR-101-3p表达水平,流式细胞术检测细胞凋亡,Western Blot检测细胞中B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关蛋白(Bax)、活化的半胱天冬酶3(Cleaved-caspase 3)和活化的半胱天冬酶9(Cleaved-caspase 9)蛋白表达,试剂盒检测上清液中白细胞介素1β(IL-1β)和肿瘤坏死因子α(TNF-α)水平及细胞中丙二醛(MDA)和超氧化物歧化酶(SOD)水平。采用双荧光素酶报告基因实验验证GABPB1-AS1和miR-101-3p靶向调控关系。结果Con组、Model组、A组及B组GABPB1-AS1、miR-101-3p、细胞凋亡率、Bax、Bcl-2、Cleaved-caspase 3、Cleaved-caspase 9、MDA、SOD、IL-1β和TNF-α水平差异均有统计学意义(F=724.541、450.866、368.481、154.595、107.850、103.824、62.514、371.871、210.219、1726.871、488.217,P<0.05)。与Con组比较,Model组细胞中GABPB1-AS1表达升高,miR-101-3p表达降低,细胞凋亡率、Bax、Cleaved-caspase 3和Cleaved-caspase 9蛋白水平、MDA含量及IL-1β和TNF-α水平升高(P<0.05),Bcl-2蛋白水平和SOD活性降低(P<0.05)。与A组和Model组比较,B组GABPB1-AS1表达、细胞凋亡率、Bax、Cleaved-caspase 3和Cleaved-caspase 9蛋白水平、MDA含量及IL-1β和TNF-α水平降低,miR-101-3p表达、Bcl-2蛋白水平和SOD活性升高(P<0.05)。GABPB1-AS1靶向负调控miR-101-3p表达。与C组比较,D组细胞凋亡率、Bax、Cleaved-caspase 3和Cleaved-caspase 9蛋白水平、MDA含量及IL-1β和TNF-α水平升高(t=13.360、9.779、5.644、5.156、13.545、37.810、25.132,P<0.05),Bcl-2蛋白水平和SOD活性降低(t=9.311、12.752,P<0.05)。结论干扰GABObjective To investigate the effect of GA binding protein transcription factor subunitβ1 antisense RNA1(GABPB1-AS1)on the injury of rat brain microvascular endothelial cells induced by hydrogen peroxide(H2O2)and its possible mechanism.Methods Rat brain microvascular endothelial cells were cultured in vitro and divided into control group(Con group),model group(Model group),group A(Model add si-NC),group B(Model add si-GABPB1-AS1),group C(Model add si-GABPB1-AS and anti-miR-NC)and group D(Model add si-GABPB1-AS and anti-miR-101-3p).RT-qPCR was used to detect the expression level of GABPB1-AS1 and miR-101-3p in cells,flow cytometry was used to detect cell apoptosis.Western Blot was used to detect the protein expression levels of B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),Cleaved-caspase 3 and Cleaved-caspase 9 in cells,and the kits were used to detect the levels of interleukin 1β(IL-1β)and tumor necrosis factorα(TNF-α)in the supernatant and the levels of malondialdehyde(MDA)and superoxide dismutase(SOD)in the cells.The dual luciferase reporter gene experiment was used to verify the targeted regulation relationship between GABPB1-AS1 and miR-101-3p.Results The differences of expressions of GABPB1-AS1 and miR-101-3p,the apoptosis rate,the levels of Bax,Bcl-2,Cleaved-caspase 3,Cleaved-caspase 9,MDA,SOD,IL-1βand TNF-αamong the Con group,Model group,group A and group B were statistically significant(F=724.541,450.866,368.481,154.595,107.850,103.824,62.514,371.871,210.219,1726.871,488.217;P<0.05).Compared with the Con group,the expression of GABPB1-AS1 in the Model group increased,while the expression of miR-101-3p decreased.Compared with the Con group,the apoptosis rate,the protein levels of Bax,Cleaved-caspase 3 and Cleaved-caspase 9,the MDA content and levels of IL-1βand TNF-αin the Model group increased(P<0.05),while the level of Bcl-2 protein and the activity of SOD decreased(P<0.05).Compared with the Model group and group A,the expression of GABPB1-AS1,the apoptosis rate,the protein levels of

关 键 词：脑微血管内皮细胞 GA结合蛋白转录因子亚基β1的反义RNA miR-101-3p 细胞凋亡 氧化应激 炎症 

分 类 号：R743.3[医药卫生—神经病学与精神病学]