miR-30b通过抑制NLRP3炎症小体激活降低心肌缺血再灌注损伤研究

miR-30b Protects against Myocardial Ischemia-Reperfusion Injury by Targeting NLRP3 Inflammasome

目的:探究miR-30b通过靶向抑制NLRP3炎症小体的激活来降低心肌缺血再灌注大鼠心肌损伤的作用机制。方法:选取50只SD大鼠纳入研究,随机将大鼠分为Sham组(假手术组)、MIRI组(模型组)、miR-30b mimics组(miR-30b过表达组)、CY-09组(NLRP3信号通路抑制剂组)、miR-30b+CY-09组(miR-30b过表达+NLRP3信号通路抑制剂组),每组10只大鼠;RT-PCR检测各组大鼠心肌组织内miR-30b相对表达;超声心动图检测大鼠心功能参数(LVEDP、LVSP、+dp/dtmax、-dp/dtmax);ELISA法检测大鼠心肌组织损伤标志物(CK-MB、LDH、cTnI)水平;HE染色分析大鼠心肌组织病理形态;TUNEL法检测大鼠心肌细胞凋亡;Western-blot检测心肌组织炎症小体NLRP3信号通路相关蛋白NLRP3、ASC、Caspase-1蛋白表达。结果:与Sham组相比,MIRI模型可以下调大鼠心肌组织内miR-30b的mRNA表达(P<0.05);miR-30b的过表达可以上调大鼠心肌组织内miR-30b的mRNA表达(P<0.05);与Sham组相比,MIRI模型的建立可以上调心功能参数LVEDP、心肌损伤因子(CK-MB、LDH、cTnI)水平及心肌细胞凋亡能力,下调心功能参数(LVSP、+dp/dtmax、-dp/dtmax)(P<0.05);miR-30b过表达质粒转染及NLRP3信号通路被阻断均可以下调心功能参数LVEDP、心肌损伤因子(CK-MB、LDH、cTnI)水平及心肌细胞凋亡能力,上调心功能参数(LVSP、+dp/dtmax、-dp/dtmax)(P<0.05);二者联合可以进一步下调心功能参数LVEDP、心肌损伤因子(CK-MB、LDH、cTnI)水平及心肌细胞凋亡能力,上调心功能参数(LVSP、+dp/dtmax、-dp/dtmax)(P<0.05)。Sham组大鼠心肌形态较为正常;MIRI模型的建立可以导致心肌组织排列紊乱,出现心肌纤维断裂、心肌细胞空泡化及炎性细胞浸润的现象;miR-30b过表达质粒转染及NLRP3信号通路被阻断均可以改善MIRI大鼠心肌组织病理形态,二者联合组大鼠心肌形态改善力度最大;与Sham组相比,MIRI模型的建立可以上调大鼠心肌组织内NLPR3炎症小体信号通路关键蛋白NLRP3、ASC、Caspase-1的表达(P<0.05);miR-30b过表达质粒转染及NLRP3信号通路被阻断均可以下调大鼠心肌组织内NLRP3、ASC、Caspase-1的表达(P<0.05);二者联合可以进一步降低大鼠心肌组织内NLRP3、ASC、Caspase-1的表达(P<0.05)。结论:miR-30b通过靶向抑制MLPR3炎症小体的激活改善缺血再灌注损伤所引起的心脏功能低下、心肌细胞损伤加剧的表现,对缺血再灌注损伤的心肌组织起到保护作用,值得临床进一步研究。

Objective:To investigate the mechanism of miR-30b in alleviating myocardial injury in rats with myocardial ischemia-reperfusion(MI/R)by targeting and inhibiting the activation of NLRP3 inflammasome.Methods:Fifty SD rats were selected for inclusion in the study and randomly divided into Sham group(sham surgery group),MIRI group(model group),miR-30b mimics group(miR-30b overexpression group),CY-09 group(NLRP3 signaling pathway inhibitor group),miR-30b+CY-09 group(miR-30b overexpression+NLRP3 signaling pathway inhibitor group),with 10 rats in each group;RT-PCR was used to detect the relative expression of miR-30b in the myocardial tissue of rats in each group;Echocardiography was used to detect rat cardiac function parameters(LVEDP,LVSP,+dp/dtmax,-dp/dtmax);ELISA was used to detect the levels of myocardial tissue damage markers(CK-MB,LDH,cTnI)in rats;HE staining was used to analyze the pathological morphology of rat myocardial tissue;TUNEL method was used to detect myocardial cell apoptosis in rats;Western-blot was used to detect the expression of NLRP3 signaling pathway related proteins NLRP3,ASC,and Caspase-1 in myocardial inflammasome.Results:Compared with the Sham group,the MIRI model downregulated the mRNA expression of miR-30b in rat myocardial tissue(P<0.05).Overexpression of miR-30b upregulated the mRNA expression of miR-30b in rat myocardial tissue(P<0.05).Compared with the Sham group,the establishment of the MIRI model upregulated the levels of cardiac function parameters LVEDP,myocardial injury factors(CK-MB,LDH,cTnI),and myocardial cell apoptosis ability,and downregulated the cardiac function parameters(LVSP,+dp/dtmax,-dp/dtmax)(P<0.05).Both miR-30b overexpression plasmid transfection and NLRP3 signaling pathway blockade could downregulate the levels of cardiac function parameters LVEDP,myocardial injury factors(CK-MB,LDH,cTnI),and myocardial cell apoptosis ability,and upregulate the cardiac function parameters(LVSP,+dp/dtmax,-dp/dtmax)(P<0.05).The combination of the two could further downregulate the levels of cardiac function parameters LVEDP,myocardial injury factors(CK-MB,LDH,cTnI),and myocardial cell apoptosis ability,and upregulate cardiac function parameters(LVSP,+dp/dtmax,-dp/dtmax)(P<0.05).The myocardial morphology of Sham group rats was relatively normal.The establishment of the MIRI model could lead to disordered arrangement of myocardial tissue,resulting in myocardial fiber breakage,myocardial cell vacuolization,and inflammatory cell infiltration.Transfection of miR-30b overexpression plasmid and blockade of NLRP3 signaling pathway could improve the pathological morphology of myocardial tissue in MIRI rats,and the combination of the two groups had the greatest improvement in myocardial morphology in rats.Compared with the Sham group,the establishment of the MIRI model upregulated the expression of key proteins NLRP3,ASC,and Caspase-1 in the NLPR3 inflammasome signaling pathway in rat myocardial tissue(P<0.05).Transfection of miR-30b overexpression plasmid and blockade of NLRP3 signaling pathway downregulated the expression of NLRP3,ASC,and Caspase-1 in rat myocardial tissue(P<0.05).The combination of the two could further reduce the expression of NLRP3,ASC,and Caspase-1 in rat myocardial tissue(P<0.05).Conclusion:miR-30b improves the manifestations of cardiac dysfunction and myocardial cell damage caused by ischemia-reperfusion injury by targeting the activation of NLPR3 inflammasomes,and has a protective effect on myocardial tissue after ischemia-reperfusion injury.It is worth further clinical researching.