参附益心颗粒对AngⅡ诱导的H9c2心肌细胞葡萄糖、脂肪酸代谢相关因子表达的影响

Effects of Shenfu Yixin Granules on the expression of related factors about the glucose and fatty acid metabolism in angiotensin Ⅱ-induced H9c2 cardiomyocytes

目的:明确参附益心颗粒对AngⅡ诱导的H9c2心肌细胞葡萄糖、脂肪酸代谢相关因子表达的影响。方法:血管紧张素Ⅱ(AngⅡ)诱导H9c2心肌细胞构建心肌细胞损伤模型,钙黄绿素标记检测细胞活性,ATP检测试剂盒检测ATP含量,电子显微镜观察线粒体超微结构,RT-PCR检测葡萄糖代谢相关因子AMPK、PDH、GLUT-4基因表达,Western Blot检测AMPK、PDH、GLUT-4蛋白表达,脂肪酸代谢相关因子FAT、MCPT-1、MCAD蛋白表达。结果:与模型组比较,参附高剂量组、氯沙坦组、辅酶Q10组H9c2细胞活性、ATP含量增加(P<0.01,P<0.05);电子显微镜结果显示:参附益心颗粒、氯沙坦、辅酶Q10均可改善线粒体超微结构,与模型组比较,可见细胞膜表面微绒毛样突起减少,细胞膜完整,线粒体嵴丰富,包膜完整,部分细胞内质网、线粒体肿胀。RT-PCR结果显示:AngⅡ诱导下,H9c2心肌细胞AMPK、PDH基因表达增高(P<0.01),GLUT-4基因表达降低(P<0.01);参附高剂量组、氯沙坦组、辅酶Q10组AMPK、PDH基因表达降低(P<0.01,P<0.05),GLUT-4基因显著增高(P<0.01);Western Blot结果显示:AngⅡ诱导下,H9c2心肌细胞AMPK、PDH蛋白表达增高(P<0.01),GLUT-4、FAT、MCPT-1、MCAD蛋白表达降低(P<0.01),参附高剂量组、氯沙坦组、辅酶Q10组AMPK、PDH蛋白表达降低(P<0.01),GLUT-4、FAT(除辅酶Q10组外)、MCPT-1、MCAD蛋白表达显著升高(P<0.01,P<0.05)。结论:参附益心颗粒可增加AngⅡ诱导下H9c2心肌细胞ATP含量,提高H9c2心肌细胞活性及脂肪酸、葡萄糖代谢相关因子蛋白表达。

Objective:To detemine the effects of Shenfu Yixin Granules on the expression of related factors about the glucose and fatty acid metabolism in angiotensinⅡ(AngⅡ)-Induced H9c2 cardiomyocytes.Methods:AngⅡwas used to establish myocardial cell injury model,and Shenfu Yixin granules were used for treatment.The content of Calcein-AM was measured by fluorescence microscope and enzyme labeling instrument.ATP level was measured by ATP Kit.The ultrastructural changes of rat cardiac myocytes were observed by electron microscope.The mRNA expressions of energy metabolism factors,such as AMPK,PDH,GLUT-4 were detected by RT-PCR and the protein expressions of energy metabolism related factors FAT,MCPT-1,MCAD,AMPK,PDH,GLUT-4 were tested by Western Blot.Results:Compared with the model group,high dose Shenfu Yixin Granules,losartan and the coenzyme Q10 can improved the activity of H9c2 cardiacmyocytes,the ATP generation(P<0.01,P<0.05).Electronic microscope results showed that high dose Shenfu Yixin Granules,losartan and the coenzyme Q10 can improve the mitochondrial ultrastructure,And compared with the model group,microvillous projections on the surface of the cell membrane reduced,cytomembrane was intact,mitochondrial cristae was abundant,envelope was intact,part of endoplasmic reticulum and mitochondria were swollen.RT-PCR results showed that the AMPK,PDH gene expression of H9c2 cardiacmyocytes increased,the GLUT-4 gene expression reduced,after the Ang I’s inducing(P<0.01).The AMPK,PDH gene expression of high dose Shenfu Yixin Granules,losartan and the coenzyme Q10 groups reduced(P<0.01,P<0.05),while the GLUT-4 gene expression reduced(P<0.01).Western Blot results showed that the AMPK,PDH protein expressions of H9c2 cardiacmyocytes increased(P<0.01),while the expression of GLUT-4,FAT,MCPT-1,MCAD protein reduced(P<0.01).The AMPK,PDH proteins’expression of high dose Shenfu Yixin Granules,losartan and the coenzyme Q10 groups reduced(P<0.01),while the expression of GLUT-4,FAT(except the coenzyme Q10 group),MCPT-1,MCAD protein increased(P<0.01,P<0.05).Conclusion:Shenfu Yixin Granules can both improved the ATP generation,the activity of cardiac myocytes,and the expression of related factors about the glucose and fatty acid metabolism which induced by Ang I of H9c2 cardiomyocytes.