乌头碱调控miR-150-5p表达对心力衰竭模型大鼠心功能及心室重构的影响

Effect of aconitine on cardiac function and ventricular remodeling in heart failure model rats by regulating miR-150-5p expression

目的探究乌头碱对心力衰竭模型大鼠心功能及心室重构的影响及对miR-150-5p的调控作用。方法将SPF级SD雄性大鼠随机分为假手术组、模型组、曲美他嗪组、乌头碱组、乌头碱+antagomir-NC组、乌头碱+miR-150-5p antagomir组,每组15只。除假手术组外,其余组均利用结扎左前降支冠状动脉法建立心力衰竭大鼠模型。测定各组大鼠心功能指标左室舒张末期内径(LVEDD)、左室收缩末期内径(LVESD)、左室射血分数(LVEF);ELISA检测各组大鼠心肌损伤指标心肌肌钙蛋白I(CTnI)、脑钠肽(BNP)和N末端B型脑钠肽前体(NT-pro BNP)的水平;测定各组大鼠心脏和左心室质量指数;Masson染色观察各组大鼠心肌组织形态;TUNEL染色检测各组大鼠心肌细胞凋亡率;RT-qRCR检测各组大鼠心肌组织中miR-150-5p和细胞周期蛋白D2(CCND2)的表达;双荧光素酶报告基因实验验证miR-150-5p与CCND2的靶向关系;Western blotting检测心肌细胞中CCND2蛋白的表达。结果与模型组相比,乌头碱组大鼠心功能指标LVEDD、LVESD、心肌损伤指标CTnI、BNP和NT-pro BNP水平、心脏质量指数、左心室质量指数、心肌纤维化区域、心肌细胞凋亡率和CCND2m RNA水平均降低(P<0.05),LVEF、miR-150-5p水平升高(P<0.05);使用miR-150-5p antagomir进行回补实验,结果显示,乌头碱对心力衰竭大鼠心功能和心室重构的保护作用被逆转,且CCND2 m RNA水平升高(P<0.05);与miR-150-5p mimic-NC组相比,miR-150-5pmimic组心肌细胞CCND2蛋白表达显著降低(P<0.05),且双荧光素酶报告基因实验验证了miR-150-5p与CCND2之间存在靶向关系。结论乌头碱通过上调miR-150-5p表达对心力衰竭大鼠心功能和心室重构发挥保护作用。

Objective To investigate the impacts of aconitine on cardiac function and ventricular remodeling in heart failure model rats,and its regulatory effect on miR-150-5p during this process.Methods SPF grade SD male rats were randomly grouped into sham surgery group,model group,trimetazidine group,aconitine group,aconitine+antagonimir NC group,and aconitine+miR-150-5p antagonimir group,with 15 rats in each group.Except for the sham surgery group,all other groups established heart failure rat models by ligating the left anterior descending coronary artery.The left ventricular end diastolic diameter(LVEDD),left ventricular end systolic diameter(LVESD),and left ventricular ejection fraction(LVEF)were measured for cardiac function indicators of rats in each group.ELISA was applied to detect the levels of myocardial injury indicators cardiac troponin I(CTnI),brain natriuretic peptide(BNP),and N-terminal B-type brain natriuretic peptide precursor(NT-proBNP)of rats in each group.Heart and left ventricular mass index of rats in each group were measured.Masson staining was applied to observe the myocardial tissue morphology of rats in each group,TUNEL staining was applied to detect the apoptosis rate of myocardial cells of rats in each group.RT-qRCR was applied to detect the expression of miR-150-5p and cyclin D2(CCND2)in myocardial tissue of rats in each group.The double luciferase reporter gene experiment verified the targeting relationship between miR-150-5p and CCND2.Western blotting was used to detect the expression of CCND2 protein in myocardial cells.Results Compared with the model group,the cardiac function indicators LVEDD,LVESD,myocardial injury indicators CTnI,BNP,and NT proBNP levels,cardiac mass index,left ventricular mass index,myocardial fibrosis area,myocardial cell apoptosis rate and the level of CCND2 mRNA in the aconitine group decreased(P<0.05).The levels of LVEF and miR-150-5p increased(P<0.05).Supplementation experiments with miR-150-5p antagomir showed that the protective effects of aconitine on cardiac function and ventricular remodeling in rats with heart failure were reversed,and CCND2 mRNA levels were increased(P<0.05).Compared with miR-150-5p mimic-NC group,the expression of CCND2 protein in myocardial cells in miR-150-5p mimic group was significantly decreased(P<0.05),and the double luciferase reporter gene experiment verified that there was a targeted relationship between miR-150-5p and CCND2.Conclusion Aconitine plays a protective role in cardiac function and ventricular remodeling in rats with heart failure by up-regulating the expression of miR-150-5p.