miR-145过表达对缺氧诱导心肌细胞焦亡的抑制作用及其机制  

Inhibitory effect of miR-145 overexpression on hypoxia-induced pyroptosis of cardiomyocytes and its mechanism

作　　者：浦湧 崔胜宇 吴浩亮 陶波[2] 孟祥平 夏豪[2] 徐林[2] PU Yong;CUI Shengyu;WU Haoliang;TAO Bo;MENG Xiangping;XIA Hao;XU Lin(不详;Department of Cardiology,Wuhan Economic and Technological Development Zone(Hannan District)People's Hospital,Wuhan 430090,China)

机构地区：[1]武汉经济技术开发区(汉南区)人民医院武汉大学人民医院汉南医院心内科,武汉430090 [2]武汉大学人民医院武汉大学心血管病研究所心血管病湖北省重点实验室心内科 [3]武汉市第四医院老年医学科

出　　处：《山东医药》2022年第29期1-5,共5页Shandong Medical Journal

基　　金：国家自然科学基金项目(81100130,81370283);武汉市卫生和计划生育委员会科研项目(WX18C30,WX21Z67)。

摘　　要：目的观察miR-145过表达对缺氧诱导心肌细胞焦亡的抑制作用并探讨其机制。方法将大鼠心肌细胞H9C2分为正常组、缺氧组、缺氧+miR-145组、缺氧+阴性对照组,缺氧+miR-145组、缺氧+阴性对照组分别使用腺病毒包装的miR-145及腺病毒包装的阴性对照序列进行腺病毒感染,正常组、缺氧组不做腺病毒感染处理;24 h后将缺氧组、缺氧+miR-145组、缺氧+阴性对照组细胞转移至缺氧培养箱中培养24 h,正常组不做缺氧诱导。采用实时荧光定量PCR法检测细胞miR-145水平,超氧化物阴离子探针检测细胞内活性氧水平,TUNEL染色及流式细胞术观察细胞焦亡情况,免疫组织化学及实时荧光定量PCR法检测细胞焦亡相关因子NOD样受体热蛋白结构域相关蛋白3(NLRP3)、(IL-1β)、白细胞介素1β(IL-18)水平,Western blotting法检测钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)/核转录因子κB(NF-κB)信号通路相关蛋白磷酸化CaMKⅡ、磷酸化NF-κB、NLRP3、Caspase-1、IL-1β、IL-18表达。结果心肌细胞miR-145 mRNA相对表达量缺氧+miR-145组>正常组>缺氧组、缺氧+阴性对照组,心肌细胞ROS水平缺氧组、缺氧+阴性对照组>缺氧+miR-145组>正常组,心肌细胞TUNEL阳性率及焦亡率缺氧组、缺氧+阴性对照组>缺氧+miR-145组>正常组,心肌细胞NLRP3蛋白及NLRP3、IL-1β、IL-18 mRNA表达缺氧组、缺氧+阴性对照组>缺氧+miR-145组>正常组,心肌细胞CaMKⅡ、磷酸化CaMKⅡ、NF-κB、磷酸化NF-κB、NLRP3、Caspase-1、IL-1β、IL-18蛋白表达缺氧组、缺氧+阴性对照组>缺氧+miR-145组>正常组(P均<0.05)。结论miR-145过表达可减轻缺氧诱导的心肌细胞焦亡,其机制可能与抑制CaMKⅡ/NF-κB信号通路有关。Objective To investigate the inhibitory effect of miR-145 overexpression on hypoxia-induced pyroptosis of cardiomyocytes and to analyze its mechanism.Methods Rat cardiomyocytes H9C2 were divided into the normal group,hypoxia group,hypoxia+miR-145 group,and hypoxia+negative control group.Cells in the hypoxia+miR-145 group and hypoxia+negative control group were infected with adenovirus by miR-145 packaged with adenovirus and adeno-virus-packaged negative control sequence,respectively.After 24 h,the cells in the hypoxia group,hypoxia+miR-145 group and hypoxia+negative control group were transferred to the hypoxia incubator for 24 h,and the normal group was not induced.The levels of miR-145 and reactive oxygen species in cells were detected by quantitative real-time PCR and superoxide anion probe.Pyroptosis was observed by TUNEL staining and flow cytometry.The levels of NOD-like receptor family pyrin domain containing 3(NLRP3),interleukin(IL)-1βand IL-18 were detected by immunohistochemistry and quantitative real-time PCR.Western blotting was used to detect the expression levels of calmodulin-dependent protein ki-naseⅡ(CaMKⅡ)/nuclear factorκB(NF-κB)-related CaMKⅡ,phosphorylated CaMKⅡ,NF-κB,phosphorylated NL-RP3,Caspase-1,IL-1β,and IL-18.Results The relative expression of miR-145 mRNA in cardiomyocytes was as fol-lows:hypoxia+miR-145 group>normal group>hypoxia group and hypoxia+negative control group;the ROS level was as follows:hypoxia group,hypoxia+negative control group>hypoxia+miR-145 group>normal group.TUNEL positive rate and pyroptosis rate of cardiomyocytes were in the following order:the hypoxia group,hypoxia+negative control group>hypoxia+miR-145 group>normal group;NLRP3 protein and NLRP3,IL-1βand IL-18 mRNA expression in car-diomyocytes were as follows:the hypoxia group,hypoxia+negative control group>hypoxia+miR-145 group>normal group;the cardiomyocyte CaMKⅡ,phosphorylated CaMKⅡ,NF-κB,phosphorylated NF-κB,NLRP3,Caspase-1,IL-1β,and IL-18 protein expression levels were as follows:the hypo

关 键 词：微小RNA MIR-145 心肌细胞 细胞焦亡 CaMKⅡ/NF-κB信号通路 

分 类 号：R542.2[医药卫生—心血管疾病]