微小RNA-16对急性心肌梗死大鼠心肌细胞凋亡和炎症反应的影响及其机制研究  

Effects of MicroRNA-16 on Cardiomyocyte Apoptosis and Inflammatory Response in Rats With Acute Myocardial Infarction and Related Mechanisms

作　　者：赖震宇 赵展庆 余秉昌 蔡秋燕 林奇栋 LAI Zhenyu;ZHAO Zhanqing;YU Bingchang;CAI Qiuyan;LIN Qidong(Intensive Care Unit,Hainan Western Central Hospital,Danzhou(571700),Hainan,China)

机构地区：[1]海南西部中心医院重症医学科,海南省儋州市571700

出　　处：《中国循环杂志》2022年第10期1048-1057,共10页Chinese Circulation Journal

基　　金：海南省自然科学基金面上项目(820MS154)。

摘　　要：目的:探讨微小RNA(miR)-16对急性心肌梗死(AMI)大鼠心肌细胞凋亡和炎症反应的影响,并分析其作用机制。方法:选取SPF级雄性SD大鼠80只,采用左前降支(LAD)永久结扎法建立AMI模型。实验分为假手术组(sham组)、急性心肌梗死组(AMI组)、重组腺相关病毒血清型9(rAAV9)-miR-16抑制剂阴性对照组(rAAV9-anti-NC组)、rAAV9-miR-16抑制剂组(rAAV9-anti-miR-16组)、rAAV9-miR-16抑制剂+维甲酸受体相关孤核受体A(RORA)短发夹RNA的阴性对照组(rAAV9-anti-miR-16+sh-NC组)、rAAV9-miR-16抑制剂+RORA沉默的短发夹RNA组(rAAV9-antimiR-16+sh-RORA组),每组12只。尾静脉注射含miR-16抑制剂、RORA短发夹RNA(sh-RORA)及其阴性对照的腺病毒进行干预,sham组和AMI组经尾静脉注射等体积的生理盐水,注射2周后,超声心动图检测大鼠左心室舒张末期内径(LVEDD)、左心室收缩末期内径(LVESD)、左心室射血分数(LVEF)和左心室短轴缩短分数(LVFS),评估心功能;酶联免疫吸附(ELISA)法检测血清N末端B型利钠肽原(NT-proBNP)水平、肌酸激酶同工酶(CK-MB)和乳酸脱氢酶(LDH)活性;2,3,5-三苯基氯化四氮唑(TTC)染色检测心肌梗死面积;苏木素-伊红(HE)染色检测心肌组织病理学变化;TUNEL染色检测心肌组织细胞凋亡率;逆转录定量PCR(RT-qPCR)检测心肌组织miR-16、RORA信使RNA(mRNA)和肿瘤坏死因子α(TNF-α)mRNA、白细胞介素(IL)-6 mRNA表达水平;免疫蛋白印迹(Western blot)法检测心肌组织半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)、裂解的Caspase-3(cleaved-Caspase-3)、B细胞淋巴瘤因子2(Bcl-2)、Bcl-2相关X蛋白(Bax)和RORA、TNF-α、核因子(NF)-κB p65蛋白表达。双荧光素酶报告基因实验验证miR-16和RORA之间的靶向关系。结果:敲低miR-16可显著上调AMI大鼠RORA的mRNA和蛋白水平,降低LVEDD、LVESD、血清NT-proBNP水平、CK-MB和LDH活性,升高LVEF、LVFS,改善大鼠心脏功能,并显著减少心肌梗死面积,降低细胞凋亡率、TNF-αmRNA、Objectives:To investigate the impact of MicroRNA-16(miR-16)on myocardial cell apoptosis and inflammatory response in rats with acute myocardial infarction(AMI),and to analyze related mechanisms.Methods:The AMI model was established by permanent ligation of the left anterior descending coronary artery(LAD).Rats were divided into sham operation group(sham group),AMI group,negative control group of recombinant adeno-associated virus serotype 9(rAAV9)-miR-16 inhibitor(rAAV9-anti-NC group),rAAV9-miR-16 inhibitor group(rAAV9-anti-miR-16 group),negative control group of rAAV9-mir-16 inhibitor+retinoic acid receptor related solitary nuclear receptor A(RORA)short hairpin RNA group(rAAV9-anti-miR-16+sh-NC group),and rAAV9-anti-miR-16+sh-RORA group,with 12 rats in each group.Adenoviruses containing miR-16 antagomir,sh-RORA and their negative controls were injected into the tail vein for intervention,whereas rats in the sham group and the AMI group were injected with equal volume of normal saline through the tail vein.After 2 weeks of injection,the left ventricular end-diastolic diameter(LVEDD),left ventricular end-systolic diameter(LVESD),left ventricular ejection fraction(LVEF)and shortaxis left ventricular fractional shortening(LVFS)were measured by echocardiography;the serum levels of N terminal pro B type natriuretic peptide(NT-proBNP),creatine kinase isoenzyme(CK-MB)and lactate dehydrogenase(LDH)activities were detected by ELISA;myocardial infarct size was measured by TTC staining;histopathological changes of myocardial tissue were detected by hematoxylin-eosin(HE)staining;the apoptosis rate of myocardial tissue was detected by TUNEL staining;the expression levels of miR-16,RORA mRNA,tumor necrosis factorα(TNF-α)mRNA and interleukin(IL)-6 mRNA in myocardial tissue were detected by reverse transcription quantitative PCR(RT-qPCR);the protein expression of myocardial tissue Caspase-3,cleaved-Caspase-3,B-cell lymphoma/factor 2(Bcl-2),Bcl-2 related Protein X(Bax)and RORA,TNF-α,nuclear factor-κB p65(NF-κB p65)was measur

关 键 词：微小RNA-16 急性心肌梗死 炎症反应 维甲酸受体相关孤核受体A 肿瘤坏死因子α 核因子-ΚB 

分 类 号：R54[医药卫生—心血管疾病]