冠心丹参制剂通过miR-330-3p/ALDH2保护大鼠心脏免受缺血/再灌注损伤

Guanxin Salvia Miltiorrhiza Preparation protects rat cardiomyocytes against ischemia/reperfusion injury through miR-330-3p/ALDH2

目的探讨冠心丹参(Guanxin Salvia miltiorrhiza, GXDS)的心脏保护作用与肥大细胞脱粒抑制是否有关及其机制。方法使用miR-330-3p mimics、ALDH2 Vector转染或者肥大细胞促分泌素化合物48/80(C48/80)处理心肌细胞,采用PCR和Western blot检测miR-330-3p和ALDH2的表达,MTT和流式细胞仪检测细胞增殖和凋亡,ELISA检测肥大细胞脱粒和类胰蛋白酶释放,双荧光素酶报告基因检测两者的靶向结合。通过结扎大鼠左前降支冠状动脉30 min并再灌注120 min建立缺血再灌注(I/R)损伤模型,使用肥大细胞促分泌素化合物48/80(C48/80)、GXDS治疗,通过免疫组化检测不同处理组小鼠心肌组织损伤。结果 C48/80抑制心肌细胞H9C2(2-1)和肥大细胞RBL-2H3的活力并促进其凋亡(P<0.05),增加cTnI和类胰蛋白酶的释放(P<0.05),而GXDS处理可通过miR-330-3p/ALDH2促进心肌细胞的活力并抑制其凋亡,同时抑制肥大细胞脱粒(P<0.05);进一步的体内实验结果显示:与假手术组相比,I/R组血流动力学异常,心律不齐,心脏水肿,梗死面积、组织病理学损伤以及cTnI和miR-330-3p的水平均显著升高(P<0.05),ALDH2表达水平下调(P<0.05),而GXDS预处理可以通过抑制肥大细胞脱粒和类胰蛋白酶释放发挥心肌保护作用。结论 GXDS通过miR-330-3p/ALDH2抑制大鼠心脏肥大细胞脱粒,从而改善缺血/再灌注引起的损伤。

Objective To explore the cardioprotective effectiveness of Guanxin Salvia miltiorrhiza(GXDS)Preparation(a prescribed Chinese herbal formula)and its relationship with inhibition of mast cell degranulation,and investigate the underlying mechanism.Methods Cardiomyocytes H9C2(2-1)cocultured with mast RBL-2H3 cells cells were transfected with miR-330-3p mimics or ALDH2 Vector,or treated with GXDS or Compound 48/80,a mast cell degranulator.The expression of miR-330-3p and ALDH2 was detected by PCR and Western blotting,and cell proliferation and apoptosis were detected by MTT assay and flow cytometry.Mast cell degranulation and trypsin release were detected by ELISA,and the targeted binding of mast cell degranulation and trypsin release was detected by double luciferase reporter gene.Then,a rat model of myocardial ischemia/reperfusion injury was established by ligating the left anterior descending coronary artery for 30 min followed by reperfusion for 120 min.Then Compound 48/80 or GXDS was used to treat the rats with myocardial injury or sham operation.The myocardial injury was detected by immunohistochemistry in different treatment groups.Results Compound 48/80 inhibited the viability and promoted the apoptosis of H9C2(2-1)and RBL-2H3 cells,and increased the releases of cTnI and trypsin(P<0.05).GXDS treatment could promote the viability and inhibit the apoptosis of cardiomyocytes through miR-330-3p/ALDH2(P<0.05),and inhibit mast cell degranulation(P<0.05).Compared with the sham operation rats,the hemodynamic abnormalities,arrhythmia,cardiac edema,infarct size,histopathological damages and levels of cTnI and miR-330-3p were significantly increased and the expression of ALDH2 was down-regulated in the rats of the model group(P<0.05).GXDS pretreatment protected myocardium by inhibiting mast cell degranulation and trypsin release.Conclusion GXDS inhibits the degranulation of rat cardiac mast cells through miR-330-3p/ALDH2,and thus improves the myocardial ischemia/reperfusion injury.