祛风养心方对AngⅡ诱导H9c2心肌细胞肥大及自噬的影响

Effect of Qufeng Yangxin Prescription(祛风养心方)on Hypertrophy and Autophagy of H9c2 Myocardial Cells Induced by AngⅡ

目的探讨祛风养心方对血管紧张素Ⅱ(AngⅡ)诱导H9c2心肌细胞肥大及自噬的影响。方法用AngⅡ诱导心肌细胞肥大模型,以H9c2心肌细胞为实验对象,实验分为4组:空白血清组(blank):[高糖培养基(DMEM)+10%空白血清];模型组(AngⅡ)(DMEM+10%空白血清+AngⅡ);含药血清组[AngⅡ+祛风养心方(QFYX)](DMEM+AngⅡ+10%含药血清);自噬抑制剂组[AngⅡ+磷脂酰肌醇3-激酶抑制剂(3-Ma)](DMEM+AngⅡ+10%含药血清+3-Ma)。用鬼笔环肽染色评价细胞面积,使用Western Blot方法检测心肌肥厚与自噬相关蛋白心钠肽(ANP)、脑钠肽(BNP)、人骨骼肌肌动蛋白α1(ACTα1)、微管相关蛋白轻链(LC3-Ⅱ)、BECN1基因(Beclin1)、SQSTM1蛋白(P62)的表达含量。结果荧光显微镜下可见AngⅡ组细胞明显增大,心肌纤维增粗,AngⅡ+QFYX和AngⅡ+3-Ma组与AngⅡ组比较细胞面积较小;与空白组比较,AngⅡ造模组心肌细胞面积明显增大(P<0.01),心肌肥厚基因ANP、BNP、Actα1蛋白表达显著升高(P<0.01),自噬代表基因LC3-Ⅱ、Beclin 1和P62蛋白表达显著增强(P<0.01),与AngⅡ组比较,AngⅡ+QFYX组心肌肥厚蛋白的表达下降(P<0.01),自噬代表基因LC3-Ⅱ、Beclin 1、P62蛋白的表达减弱(P<0.01),AngⅡ+QFYX与AngⅡ+3-Ma结果趋势相同。结论祛风养心方可以抑制AngⅡ诱导H9c2细胞肥大,其可能机制为改善了细胞自噬实现的。

Objective To observe the mechanism of drug-containing serum of Qufeng Yangxin Prescription(祛风养心方,QFYX)on hypertrophy and autophagy of angiotensin Ⅱ(AngⅡ)-induced H9c2 myocardial cells.Methods H9c2 myocardial cells were used to establish myocardial hypertrophy models.Four groups were divided in the experiment containing blank serum group(DMEM+10%blank serum),model group(AngⅡ)[DMEM+10%blank serum+AngⅡ],drug-containing serum group(AngⅡ+QFYX)[DMEM+AngⅡ+10%drug-containing serum]and autophagy inhibitor group[AngⅡ+(3-Methyladenine)3-Ma][DMEM+AngⅡ+10%blank serum+3-Ma].The cell area was evaluated by GCP staining,and the proteins expression levels of Recombinant Atrial Natriuretic Peptide(ANP),Brain Natriuretic Peptide(BNP),Actin alpha 1(ACTα1),Microtubule associated pritein-1B(LC3Ⅱ),Myosin-like Bcl-2 interacting protein(Beclin-1)and Sequestosome1(P62)were detected by Western Blot.Results Under fluorescence microscope,cells in AngⅡ group increased significantly and myocardial fibers became thicker,while cell area in AngⅡ+QFYX and AngⅡ+3-Ma groups decreased.Compared with that of the blank group,the area of myocardial cells in AngⅡ group was significantly increased(P<0.01).Meanwhile,cardiac hypertrophy protein expressions of ANP,BNP and ACTα1genes were also significantly increased,accompanied with the significant raising of autophagy representative genes LC3-Ⅱ,Beclin 1 and P62 protein expressions(P<0.01).Compared with that of the AngⅡgroup,the expression of myocardial hypertrophy protein in AngⅡ+QFYX group decreased(P<0.01),similarly,the expression of autophagy representative genes LC3-Ⅱ,Beclin 1 and p62 protein decreased(P<0.01).The results of AngⅡ+QFYX and AngⅡ+3-Ma showed the same trend.Conclusion Qufeng Yangxin Prescription can inhibit the hypertrophy of H9c2 cells induced by AngⅡ,and the possible mechanism is related to the improvement of autophagy.