ALKBH5介导NLRP3的m^(6)A修饰促进心肌梗死小鼠心肌细胞焦亡  

ALKBH5 mediated m^(6)A modification of NLRP3 promotes cardiomyocytespyroptosis in mice with myocardial infarction

作　　者：翟苗苗 尹俭俭 王智墨 周月姣 于庆文 王沛[1] 张莉蓉[1] 韩圣娜 ZHAI Miao-miao;YIN Jian-jian;WANG Zhi-mo;ZHOU Yue-jiao;YU Qing-wen;WANG Pei;ZHANG Li-rong;HAN Sheng-na(Dept of Pharmacology,School of Basic Medical Sciences,Zhengzhou University,Zhengzhou 450001,China)

机构地区：[1]郑州大学基础医学院药理学系,河南郑州450001

出　　处：《中国药理学通报》2025年第3期434-444,共11页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金面上项目(No 82270287)。

摘　　要：目的N 6-甲基腺苷修饰(N 6-methyladenosine,m^(6)A)去甲基化酶ALKBH5对心肌梗死(myocardial infarction,MI)小鼠心肌细胞焦亡的影响。方法分别利用腺相关病毒敲低ALKBH5建立左冠状动脉前降支结扎手术的MI模型和采用小干扰RNA敲低目的基因的方法建立小鼠心肌细胞(HL-1)缺氧模型,观察ALKBH5对MI小鼠和缺氧HL-1细胞焦亡的影响。随后在细胞水平进行机制研究,通过RNA pull-down和RNA免疫共沉淀(RIP)实验检测ALKBH5和阅读蛋白IGF2BP2与NLRP3 mRNA的相互结合,应用MeRIP-qPCR方法测定ALKBH5对NLRP3的mRNA的m^(6)A水平的影响。RNA稳定性实验检测ALKBH5和IGF2BP2对NLRP3 mRNA稳定性的影响。结果体内和体外敲低ALKBH5均可抑制NLRP3炎性小体活化缓解MI小鼠和缺氧HL-1细胞的焦亡。机制上,结果显示在HL-1细胞中NLRP3 mRNA能够和ALKBH5蛋白相互结合;敲低ALKBH5可以增加NLRP3的m^(6)A水平和降低NLRP3 mRNA稳定性;随后证实NLRP3 mRNA和IGF2BP2的蛋白相互结合;敲低IGF2BP2增加NLRP3的mRNA稳定性。Rescue实验表明敲低IGF2BP2可以逆转敲低ALKBH5引起的NLRP3 mRNA表达的减少。结论ALKBH5通过m^(6)A修饰的方式介导NLRP3炎性小体活化参与MI小鼠的心肌细胞焦亡。Aim To investigate the effects of m^(6)A demethylase ALKBH5 on cardiomyocytes pyroptosis in mice with myocardial infarction(MI).Methods The MI model of left anterior descending coronary artery ligation surgery was established by knocking down ALKBH5 using adeno-associated virus,and the hypoxia model of mouse cardiomyocytes(HL-1)was established by knocking down small interfering RNA.The effects of ALKBH5 on the pyroptosis of MI mice and hypoxic HL-1 cells were observed.Subsequently,mechanism studies were conducted at the cellular level,and the binding of ALKBH5 and IGF2BP2 to NLRP3 mRNA was detected through RNA pull down and RNA immunoprecipitation(RIP)experiments.The MeRIP-qPCR method was used to determine the effects of ALKBH5 on the mRNA m^(6)A level of NLRP3.Actinomycin D for RNA stability experiments were conducted to detect the effects of ALKBH5 and IGF2BP2 on the stability of NLRP3 mRNA.Results Knocking down ALKBH5 in vivo and in vitro both inhibited NLRP3 inflammasome activation and alleviated pyroptosis in MI mice and hypoxic HL-1 cells.Mechanistically,the results showed that NLRP3 mRNA could bind to ALKBH5 protein in HL-1 cells;knocking down ALKBH5 could increase the m^(6)A level of NLRP3 and reduce the stability of NLRP3 mRNA;subsequently,it was confirmed that NLRP3 mRNA and IGF2BP2 protein bound to each other;knocking down IGF2BP2 increased the mRNA stability of NLRP3.The Rescue experiment showed that knocking down IGF2BP2 reversed the decrease in NLRP3 mRNA expression caused by knocking down ALKBH5.Conclusions ALKBH5 mediated m^(6)A modification of NLRP3 promotes cardiomyocytes pyroptosis in mice with myocardial infarction.

关 键 词：心肌梗死 NLRP3炎性小体 ALKBH5 m^(6)A修饰 IGF2BP2 细胞焦亡 

分 类 号：R-332[医药卫生] R329.24R329.411R342.2R364.5R542.2