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

于永慧; 张佩; 刘剑刚; 李澎; 张大武; 王承龙

doi:10.13422/j.cnki.syfjx.20180695

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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

药理 | 更新时间：2024-01-04

- 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
- 中国实验方剂学杂志 2018年24卷第6期页码：89-95
- 作者机构：
- 作者简介：
- 基金信息：
  
  国家自然科学基金面上项目（81273934）
- DOI：10.13422/j.cnki.syfjx.20180695
  中图分类号： R285
- 纸质出版日期：2018
- 稿件说明：
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于永慧, 张佩, 刘剑刚, 等. 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[J]. 中国实验方剂学杂志, 2018,24(6):89-95.

YU Yong-hui, ZHANG Pei, LIU Jian-gang, et al. AMPK Related Glucose Metabolism and Fatty Acid Oxidation Pathways in Hypoxia/Reoxygenation Myocardial Cells: Intervened by Qixue Bingzhi Recipe[J]. Chinese journal of experimental traditional medical formulae, 2018, 24(6): 89-95.
于永慧, 张佩, 刘剑刚, 等. 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[J]. 中国实验方剂学杂志, 2018,24(6):89-95. DOI： 10.13422/j.cnki.syfjx.20180695.

YU Yong-hui, ZHANG Pei, LIU Jian-gang, et al. AMPK Related Glucose Metabolism and Fatty Acid Oxidation Pathways in Hypoxia/Reoxygenation Myocardial Cells: Intervened by Qixue Bingzhi Recipe[J]. Chinese journal of experimental traditional medical formulae, 2018, 24(6): 89-95. DOI： 10.13422/j.cnki.syfjx.20180695.

摘要

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+100 μmol · 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。

Abstract

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 CWQB 1 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-CoA 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.

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