气血并治方有效组分干预H/R损伤心肌细胞AMPK相关糖脂代谢通路的分析

AMPK Related Glucose Metabolism and Fatty Acid Oxidation Pathways in Hypoxia/Reoxygenation Myocardial Cells: Intervened by Qixue Bingzhi Recipe

目的：观察气血并治方有效组分对缺氧/复氧（H/R）损伤心肌细胞一磷酸腺苷酸活化蛋白激酶（AMPK）相关糖脂代谢通路的作用机制。方法：分离、提取、培养出生1~2 d SD乳鼠原代心肌细胞,于常规倒置相差显微镜下观察原代心肌细胞形态及生长状态,经α-横纹肌辅肌动蛋白（α-actinin）免疫荧光染色鉴定为心肌细胞后,进行缺氧3 h复氧2 h处理制作H/R损伤模型,随机分为正常组（正常氧）,模型组（缺氧/复氧）,曲美他嗪组（缺氧/复氧＋100μmol·L-1盐酸曲美他嗪,TMZ）,气血并治方有效组分组（缺氧/复氧＋1 mmol·L-1气血并治方有效组分,CWQB）。采用实时荧光定量聚合酶链反应（Real-time PCR）和蛋白免疫印迹法（Western blot）测定AMPK代表性亚基心肌一磷酸腺苷酸活化蛋白激酶α（AMPKα）,及其糖代谢通路中葡萄糖转运体4（GLUT4）,磷酸果糖激酶2（PFK2）,脂肪酸代谢通路中乙酰辅酶A羧化酶（ACC2）,脂肪酸移位酶（FAT/CD36）的基因及蛋白表达情况。结果：与正常组比较,模型组,TMZ组,CWQB组的AMPKα,GLUT4,PFK2基因和蛋白表达上调,ACC2,FAT/CD36基因和蛋白表达下调（P〈0.05）;与模型组比较,TMZ组,CWQB组AMPKα,GLUT4,PFK2,ACC2,FAT/CD36基因和蛋白表达均上调（P〈0.05）,其中TMZ组上调AMPKα,FAT/CD36基因和蛋白,上调GLUT4,PFK2基因表达的效果更为显著（P〈0.05）。结论：气血并治方有效组分可以激活H/R损伤心肌细胞的AMPK信号通路,增强GLUT4介导的葡萄糖转送,PFK2参与的糖酵解,同时促进FAT/CD36调控的脂肪酸转运,上调ACC2抑制脂肪酸氧化过程,进而提高缺氧/复氧条件下心肌细胞对葡萄糖、脂肪酸等产能底物的利用能力,改善H/R损伤心肌细胞的能量代谢。

Objective： To observe the action mechanism of components of water extract from Qixue Bingzhi recipe（ CWQB） for adenosine monophosphate activated protein kinase（ AMPK） related glucose metabolism and fatty acid oxidation pathways in hypoxia/reoxygenation（ H/R） myocardial cells. Method： The myocardial cells from 1-2 d newborn SD rats were separated and extracted. Morphology of primary cardiacmyocyte under different duration of culture was observed by an optical microscope. After α-actinin immunofluorescence identified cardiomyocytes,the H/R models were made by depriving oxygen for 3 hours and then regaining oxygen for 2 hours. Then the cardiomyocytes were randomly divided into four groups： control group（ with normal oxygen）,H/R group（ the H/R model）,trimetazidine（ TMZ） group,and CWQB group. The H/R cardiomyocytes in TMZ group were treated by TMZ 100 μmol·L-1; H/R cardiomyocytesin CWQB group were treated by CWQB1 μmol·L-1. Finally,the mRNA and proteinexpressions of adenosine monophosphate activated protein kinase-α（ AMPKα） together with its related glucose metabolism pathway including glucose transporter4（ GLUT4） and phosphate fructose kinase 2（ PFK2）,fatty acid metabolism pathway including fatty acid translocase/CD36（ FAT/CD36） and acetyl-Co A carboxylase 2（ ACC2） were figured out by Real-time polymerase chain reaction（ Real-time PCR） and Western blot. Result： As compared with the control group,both mRNA and proteinexpressions of AMPKα,GLUT4,PFK2 were increased,while ACC2,FAT/CD36 mRNA and proteinexpressions were decreased in H/R group,TMZ group,and CWQB group（ P〈0. 05）. As compared with H/R group,the mRNA and proteinexpressions of AMPKα,GLUT4,PFK2,ACC2 and FAT/CD36 were increased significantly in TMZ group and CWQB group（ P〈0. 05）. By the way,the up-regulation of mRNA and protein expressionsof AMPKα and FAT/CD36,as well as mRNA expressions of GLUT4 and PFK2 in TMZ group were more obvious than those in CWQB group（ P〈0. 05）. Conclusion： CWQB can activate the AMPK pathway in H/R cardiomyocytes,enhance glucose transport mediated by GLUT4 and glycolysis mediated by PFK2,promote the absorption of fatty acid adjusted by FAT/CD36,and inhibit its oxidation by up-regulating ACC2. Therefore,CWQB recipe can improve the utilization of energy materials in myocardial cells under H/R situation,and then optimize the energy metabolism to prevent damages caused by energy deficiency.