m6A去甲基化酶在脑梗死病人体内表达及过表达FTO对神经元细胞凋亡和缝隙连接蛋白的影响

Expression of m6A Demethylase in Cerebral Infarction Patients and Overexpression of FTO on Neuronal Cell Apoptosis and Gap Junction Proteins

目的探讨N6-甲基腺苷(m6A)去甲基化酶肥胖相关蛋白(FTO)对脑梗死神经元细胞凋亡、缝隙连接蛋白43(Cx43)和缝隙连接蛋白36(Cx36)表达的影响。方法收集2017年1月—2018年12月海南医学院第二附属医院神经内科病房收治的63例脑梗死病人的外周血血液样本,从体检中心收集68名健康体检者的外周血血液样本,聚合酶链反应(qPCR)检测血液中m6A去甲基酶(ALKBH5)和FTO的mRNA水平;酶联免疫吸附试验(ELISA)检测ALKBH5和FTO的蛋白水平。使用颈内动脉结扎手术法建立脑梗死模型小鼠,以假手术处理为假手术组,苏木精-伊红(HE)染色观察脑组织变化;蛋白免疫印迹法检测小鼠脑组织中FTO、ALKBH5、半胱氨酸天冬氨酸蛋白酶3(caspase-3)、活性caspase-3(cleaved-caspase-3)、Bcl-2相关蛋白x(Bax)、B细胞淋巴瘤/白血病x基因长片段(Bcl-xl)、Cx43、Cx36、磷酸化Cx43(p-Cx43)和磷酸化Cx36(p-Cx36)的表达。体外培养小鼠海马神经元细胞(HT-22),进行缺血缺氧处理并转染FTO的过表达质粒(pcDNA3.1-FTO,pcFTO)及其阴性对照组(pcNull)。HT-22细胞分为对照组、缺血组、缺血+pcFTO组、缺血+pcNull组、pcFTO组、pcNull组。蛋白免疫印迹法检测HT-22细胞中的FTO、ALKBH5、caspase-3、cleaved-caspase-3、Bax、Bcl-xl、Cx43、Cx36、p-Cx43和p-Cx36的表达,流式细胞术检测HT-22细胞凋亡率变化。结果FTO在脑梗死病人体内表达下调(P<0.05),ALKBH5无变化(P>0.05);小鼠脑梗死模型组脑部凋亡特征增加,凋亡标记物cleaved-caspase-3、Bax,缝隙连接蛋白Cx43表达上调,而Bcl-xl表达下调,差异均有统计学意义(P<0.05);模型组中FTO、磷酸化的Cx43表达下调(P<0.05),但磷酸化的Cx36水平差异无统计学意义(P>0.05)。缺血诱导的HT-22细胞过表达FTO后,细胞凋亡率明显降低(P<0.05),cleaved-caspase-3、Bax、Cx43表达下调,Bcl-xl表达上调,差异均有统计学意义(P<0.05),磷酸化的Cx43表达上调(P<0.05)。结论过表达m6A去甲基化酶FTO可改善缺血诱导的脑梗死和神经元细胞的凋亡并抑制Cx43介导的缝隙连接。

Objective To investigate the effect of m6A demethylase FTO on neuronal cell apoptosis and expression of connexin 43(Cx43)and Cx36 in cerebral infarction patients.Methods Blood samples were collected from patients with acute cerebral infarction and healthy patients.Quantitative polymerase chain reaction(qPCR)was used to detect the mRNA levels of m6A demethylase(ALKBH5)and FTO in the blood.Enzyme-linked immuno sorbent assay(ELISA)was used to detect the protein levels of ALKBH5 and FTO.The cerebral infarction model mice were established using the internal carotid artery ligation method,with sham operation as the sham group.The brain tissues were observed by hematoxylin-eosin(HE)staining.Western blotting was used to detect FTO,ALKBH5,caspase-3,active caspase-3(cleaved-caspase-3),Bax,Bcl-xl,Cx43,Cx36,phosphorylated Cx43(p-Cx43)and phosphorylated Cx36(p-Cx36)expression.Mice hippocampal neuronal cells(HT-22)were cultured in vitro,treated with hypoxia and ischemia,and transfected with FTO overexpression plasmid(pcDNA3.1-FTO,pcFTO)and its negative control group(pcNull).HT-22 cells were divided into control group,ischemia group,ischemia+pcFTO group,ischemia+pcNull group,pcFTO group,pcNull group.Western blot was used to detect the expression of FTO,ALKBH5,caspase-3,cleaved-caspase-3,Bax,Bcl-xl,Cx43,Cx36,p-Cx43,and p-Cx36 in HT-22 cells.Flow cytometry was used to detect the expression of HT-22 cells changes in apoptosis rate.Results The expression of FTO was down-regulated in patients with cerebral infarction(P<0.05),and there was no change in ALKBH5(P>0.05).In the mice cerebral infarction model group,the brain apoptosis features increased,and the expressions of apoptosis markers cleaved-caspase-3,Bax,gap junction protein Cx43 up-regulated,and Bcl-xl expression down-regulated;In addition,the expressions of FTO and p-Cx43 down-regulated in the model group(P<0.05),but there was no statistically significant difference in the level of p-CX36(P>0.05).After ischemia-induced HT-22 cells overexpress FTO,the apoptosis rate significantly reduced(P<0.05),the expressions of cleaved-caspase-3,Bax,Cx43 down-regulated,and the expression of Bcl-xl up-regulated(P<0.05),the expression of p-Cx43 up-regulated(P<0.05).Conclusion Overexpression of m6A demethylase FTO can improve ischemia-induced cerebral infarction and neuronal cell apoptosis,and inhibit Cx43-mediated gap junctions.