摘要
目的探讨黄芪甲苷(AS-Ⅳ)对缺氧/复氧(H/R)诱导大鼠心肌细胞(H9c2细胞)能量代谢重编程的影响及机制。方法常规培养H9c2细胞,细胞贴壁后随机分组。空白对照组正常培养,缺氧/复氧组(H/R组)放至95%N2+5%CO_(2)培养箱缺氧3 h、95%O_(2)+5%CO_(2)培养箱复氧3 h。H/R+黄芪甲苷低剂量组(H/R+AS-ⅣL组)、H/R+黄芪甲苷高剂量组(H/R+AS-ⅣH组)分别加入800、1600μmol/L干预6 h后再H/R处理;H/R+黄芪甲苷高剂量+腺苷酸活化蛋白激酶(AMPK)抑制剂组(H/R+AS-ⅣH+AMPKI组)加入AS-Ⅳ1600μmol/L干预6 h+10μmol/L Compound C干预2 h后再行H/R处理。用紫外分光度法检测各组二磷酸腺苷(ADP)、三磷酸腺苷(ATP)、乙酰辅酶A(Acetyl-CoA),Seahorse XF能量代谢分析法检测细胞外酸化率(ECAR)、氧气消耗速率(OCR),用qRT-PCR检测细胞内AMPK mRNA表达,用Western blotting法检测细胞内AMPKα、磷酸化AMPKα(p-AMPKα)蛋白表达。结果与空白对照组比较,H/R组ADP、ATP低,Acetyl-CoA高,ECAR高,AMPK mRNA及AMPKα、p-AMPKα蛋白相对表达量低(P均<0.05);与H/R组比较,H/R+AS-ⅣL组、H/R+AS-ⅣH组ADP、ATP高,Acetyl-CoA低,OCR高,ECAR低,AMPK mRNA及AMPKα、p-AMPKα蛋白相对表达量高(P均<0.05);与H/R+AS-IVH组比较,H/R+AS-ⅣH+AMPKI组ADP、ATP低,Acetyl-CoA高,OCR低,ECAR高,AMPK mRNA及AMPKα、p-AMPKα蛋白相对表达量低(P均<0.05)。结论黄芪甲苷可改善H/R诱导的大鼠心肌细胞能量代谢重编程,其作用机制可能与激活AMPK信号有关。
Objective To investigate the effect and mechanism of astragalosideⅣ(AS-Ⅳ)on energy metabolic re‑programming of rat cardiomyocytes(H9c2 cells)induced by hypoxia/reoxygenation(H/R).Methods H9c2 cells were cultured routinely and randomly divided into different groups after cell adhesion.The cells in the blank control group were cultured normally,and the cells in the H/R group were placed in 95%N_(2)+5%CO_(2) incubator for hypoxia for 3 h and 95%O_(2)+5%CO_(2) incubator for reoxygenation for 3 h.The cells in the H/R+AS-Ⅳlow-dose group(H/R+AS-ⅣL group)and H/R+AS-Ⅳhigh-dose group(H/R+AS-ⅣH group)were treated with 800 and 1600μmol/L AS-Ⅳ,respectively,for 6 hours.The cells in the H/R+high-dose AS-Ⅳ+adenylate activated protein kinase(AMPK)inhibitor group(H/R+AS-ⅣH+AMPKI group)were intervened with 1600μmol/L AS-Ⅳfor 6 h+10μmol/L compound C for 2 h,and then were treated with H/R.The levels of adenosine diphosphate(ADP),adenosine triphosphate(ATP)and acetyl CoA were detected by UV spectrophotometry,extracellular acidification rate(ECAR)and oxygen consumption rate(OCR)were detect‑ed by Seahorse XF energy metabolism analysis,intracellular AMPK mRNA expression was detected by qRT-PCR,and the protein expression levels of AMPKα and phosphorylated AMPKα(p-AMPKα)were detected by Western blotting.Re⁃sults Compared with the blank control group,the H/R group had lower ADP and ATP,higher acetyl CoA,higher ECAR,and lower relative expression levels of AMPK mRNA and AMPKαand p-AMPKαprotein(all P<0.05).Compared with the H/R group,H/R+AS-ⅣL group and H/R+AS-ⅣH group had higher ADP and ATP,lower acetyl CoA,higher OCR,lower ECAR,and higher relative expression levels of AMPK mRNA and AMPKαand p-AMPKαprotein(all P<0.05).Compared with the H/R+AS-ⅣH group,H/R+AS-ⅣH+AMPKI group had lower ADP and ATP,higher acetyl CoA,lower OCR,higher ECAR,and lower relative expression levels of AMPK mRNA and AMPKα and p-AMPKαprotein(all P<0.05).Conclusion AstragalosideⅣcan improve H/R-induced energy metabolic reprogramming of cardiomyocytes,and its mechanism may be related to the activation of AMPK signal.
作者
张蒙
王新东
ZHANG Meng;WANG Xindong(The Third Clinical Medical College,Nanjing University of Chinese Medicine,Nanjing 210028,China;不详)
出处
《山东医药》
CAS
2021年第31期13-16,共4页
Shandong Medical Journal
基金
国家自然科学基金资助项目(81973766)
江苏省研究生科研与实践创新计划项目(SJCX21-0801)
江苏省名老中医专家董其美传承工作室建设项目(苏中医科教〔2019〕10号)。
关键词
黄芪甲苷
心肌细胞
细胞能量代谢重编程
缺氧/复氧
大鼠
astragalosideⅣ
cardiomyocytes
energy metabolism reprogramming
hypoxia/reoxygenation
rats
