基于GAS5/miR-21介导的细胞增殖探讨心康冲剂抗心力衰竭大鼠心肌纤维化的作用机制

Exploration on the Mechanism of Xinkang Granule against Myocardial Fibrosis in Rats with Heart Failure Based on Cell Proliferation Mediated by GAS5/miR-21

目的基于GAS5/miR-21探讨心康冲剂抗心力衰竭大鼠心肌纤维化的作用机制。方法46只SD大鼠选取6只作为正常组,剩余40只通过腹腔注射阿霉素(2 mg/mL)诱导慢性心力衰竭并分为模型组、缬沙坦组及心康冲剂组。心康冲剂组予心康冲剂0.5g/kg灌胃,缬沙坦组予缬沙坦0.03g/kg灌胃,正常组和模型组予等体积蒸馏水灌胃,每日1次,连续4周。心脏彩超检测大鼠心功能,ELISA检测血清Ⅰ型胶原(ColⅠ)和基质金属蛋白酶9(MMP9)含量,HE和Masson染色观察左心室病理变化,免疫荧光染色检测左心室α-平滑肌肌动蛋白(α-SMA)表达,qRT-PCR检测磷酸酶及张力蛋白同源物(PTEN)、生长阻滞特异性转录本5(GAS5)、miR-21基因表达,Westernblot检测PTEN、Akt、周期蛋白D1(CyclinD1)和周期蛋白依赖性激酶4(CDK4)表达。结果与正常组比较,模型组大鼠左室射血分数(LVEF)、左室短轴缩短率(LVFS)降低(P<0.05),血清MMP9、ColⅠ含量增加(P<0.05),心肌细胞肿胀变性、胞浆溶解,细胞排列紊乱,可见大量蓝色胶原纤维,胶原容积分数增加(P<0.05),左心室α-SMA表达升高(P<0.05),PTEN、GAS5基因表达降低(P<0.05),miR-21表达升高(P<0.05),Akt、CDK4、CyclinD1蛋白表达升高(P<0.05),PTEN蛋白表达降低(P<0.05);与模型组比较,心康冲剂组和缬沙坦组大鼠LVEF、LVFS升高(P<0.05),血清MMP9、ColⅠ含量减少(P<0.05),心肌细胞肿胀、胞浆溶解程度相对较轻,蓝色胶原纤维较少,胶原容积分数减少(P<0.05),左心室α-SMA表达降低(P<0.05),PTEN、GAS5基因表达升高(P<0.05),miR-21表达降低(P<0.05),Akt、CDK4、CyclinD1蛋白表达降低(P<0.05),PTEN蛋白表达升高(P<0.05)。结论心康冲剂可能通过调节GAS5/miR-21、PTEN/Akt及细胞周期相关蛋白表达抑制细胞增殖,降低心力衰竭大鼠心肌纤维化程度。

Objective To explore the mechanism of Xinkang Granule(XKG)against myocardial fibrosis in rats with heart failure based on GAS5/miR-21.Methods 6 of 46 SD rats were selected and set as the normal group,and then 40 rats were replicated the model of chronic heart failure with adriamycin(2 mg/mL)intraperitoned injection.After the successful modeling,they were divided into model group,valsartan group and XKG group.The XKG group were given XKG at the dosage of 0.5 g/kg by gavage,and the valsartan group were given valsartan at the dosage of 0.03 g/kg,while the normal group and model group were given equal volume of distilled water,once a day for 4 weeks.The cardiac function was detected by Doppler echocardiography,serum typeⅠcollagen(ColⅠ)and matrix metalloproteinase 9(MMP9)were detected by ELISA,HE and Masson staining were used to observe the pathological changes of left ventricle,the expression ofα-smooth muscle actin(α-SMA)in left ventricle was detected by immunofluorescence staining,the expression of phosphatase and tensin homologues(PTEN),growth retardation specific transcript 5(GAS5),and miR-21 gene were detected by qRT-PCR,Western blot was used to detect the expressions of PTEN,Akt,CyclinD1 and cyclin dependent kinase 4(CDK4)protein.Results Compared with the normal group,the left ventricular ejection fraction(LVEF)and left ventricular fraction shortening(LVFS)in the model group decreased(P<0.05);the contents of MMP9 and ColⅠin serum increased(P<0.05);the myocardial cells were swollen and degenerated,the cytoplasm was dissolved,the cells were arranged disorderly,a large number of blue collagen fibers were visible,and collagen volume fraction(CVF)increased(P<0.05);the expression ofα-SMA in left ventricle increased(P<0.05),the expressions of GAS5 and PTEN gene decreased(P<0.05),the expression of miR-21 increased(P<0.05),the expressions of Akt,CDK4 and CyclinD1 protein increased(P<0.05),and PTEN protein expression decreased(P<0.05).Compared with the model group,the LVEF and LVFS in XKG group and valsartan group increased(P<0.05);the contents of MMP9 and ColⅠin serum decreased(P<0.05);the degree of myocardial cell swelling and cytoplasmic dissolution was relatively light,there were fewer blue collagen fibers,and CVF decreased(P<0.05);the expression ofα-SMA in left ventricle decreased(P<0.05),the expressions of GAS5 and PTEN gene increased(P<0.05),and the expression of miR-21 decreased(P<0.05),the expressions of Akt,CDK4 and CyclinD1 protein decreased(P<0.05),and PTEN protein expression increased(P<0.05).Conclusion XKG may inhibit cell proliferation by regulating the expression of GAS5/miR-21,PTEN/Akt and cell cycle related proteins,and reduce myocardial fibrosis in rats with heart failure.