摘要
目的研究缺氧诱导因子1α(HIF-1α)对高糖作用下缺血再灌注(IR)损伤的心肌细胞凋亡和氧化应激,及其信号通路下游因子的调控作用。方法二甲基乙二酰基甘氨酸激活HIF-1α对高糖作用下IR心肌细胞损伤的干预。实验分四组:对照组(细胞常规培养)、高糖+缺氧复氧组(25 mmol/L葡萄糖的DMEM培养基培养24 h,缺氧2 h,复氧8 h)、HIF-1α处理组(高糖,缺氧加复氧,缺氧前3 h用100μmol/L的二甲基乙二酰基甘氨酸处理)、二甲基亚砜(DMSO)对照组(高糖,缺氧加复氧,缺氧前3 h用100μmol/L的DMSO处理)。流式细胞术测定细胞凋亡率,CCK-8法分析细胞活性;ELISA法检测细胞培养上清液中超氧化物歧化酶活性和丙二醛的表达;ATP含量试剂盒检测各组细胞ATP含量;Western Blot法检测过氧化物酶体增殖物激活受体γ辅助激活因子1α(PGC-1α)、核呼吸因子1,以及能量代谢相关的p-AMPK、AMPK蛋白表达,HIF-1α核蛋白表达。结果高糖合并IR损伤增加心肌细胞的凋亡(P<0.01),降低心肌细胞活力(P<0.01),HIF-1α表达激活抑制心肌细胞的凋亡(P<0.05)和活力降低(P<0.05)。高糖合并IR损伤增加心肌细胞的氧化应激水平,HIF-1α表达激活抑制心肌细胞的氧化应激。和DMSO对照组相比,HIF-1α处理组的超氧化物歧化酶的表达显著增加[(11.56±0.72)U/mL vs(7.79±0.90)U/mL,P<0.05],丙二醛的表达显著降低[(0.69±0.07)nmol/mL vs(0.96±0.11)nmol/mL,P<0.001]。高糖+缺氧复氧组的应激降低了细胞的ATP含量,HIF-1α表达激活减轻了这种损伤。和DMSO对照组相比,HIF-1α处理组的ATP含量显著增加[(5.41±0.23)μmol/g vs(3.10±0.42)μmol/g,P<0.05]。低氧条件下,HIF-1α核蛋白表达增加(P<0.001),低氧激活p-AMPK蛋白表达(P<0.01),线粒体氧化磷酸化的激活因子PGC-1α(P<0.01)、转录因子核呼吸因子1(P<0.01)的表达均显著上调。和DMSO组相比,HIF-1α处理组HIF-1α的表达显著增加(P<0.05)、PGC-1α表达显著增加(P<0.05)。结论
Objective To study the effects of hypoxia inducible factor-1α(HIF-1α)on apoptosis and oxidative stress of myocardial cells induced by ischemia reperfusion injury under high glucose condition,and the regulation of downstream factors of the signaling pathway.Methods The intervention of HIF-1αwas actived by dimethyloxallyl glycine(DMOG)on myocardial cell injury induced by high glucose and ischemia reperfusion.The experiment was divided into four groups:control group(conventional cell culture),high glucose+hypoxia reoxygenation group(DMEM medium culture of 25 mmol/L glucose for 24 h,hypoxia for 2 h,reoxygenation for 8 h),HIF-1αtreatment group(high glucose,hypoxia reoxygenation,DMOG treatment of 100μmol/L for 3 h before hypoxia),Dimethyl sulfoxide(DMSO)control group(high glucose,hypoxia reoxygenation,DMSO treatment of 100μmol/L for 3 h before hypoxia).Flow cytometry was used to detect the apoptosis rate,CCK-8 was used to analyze the cell activity,ELISA was used to detect the activity of SOD and the expression of MDA in the supernatant of cell culture,ATP content kit was used to detect the content of ATP in each group,Western Blot detected the protein expression of peroxisome prdiferator activated receptorγcoactivator 1α(PGC-1α),transcription factor NRF-1,energy metabolism related factor AMPK,p-AMPK and HIF-1αnuclear proteins.Results The high glucose with ischemia reperfusion injury increased the apoptosis of cardiomyocytes(P<0.01),and decreased the viability of cardiomyocytes(P<0.01).Activation of HIF-1αexpression inhibited the apoptosis of cardiomyocytes(P<0.05)and the decrease of the viability of cardiomyocytes(P<0.05).The high glucose with ischemia reperfusion injury increased the oxidative stress of cardiomyocytes,and the activation of HIF-1αinhibited the oxidative stress of cardiomyocytes.Compared with DMSO control group,the activity of SOD in HIF-1αtreatment group increased significantly[(11.56±0.72)U/mL vs(7.79±0.90)U/mL,P<0.05],and the expression of MDA decreased significantly[(0.69±0.07)nmol/m
作者
王雪梅
刘芬
曹莹
邢远
汪洁英
潘涛
WANG Xuemei;LIU Fen;CAO Ying;XING Yuan;WANG Jieying;PAN Tao(Department of Public Health,Xi'an Medical University,Xi'an 710021,Shaanxi,China;Xinjiang Key Laboratory of Medical Animal Model Research,The First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,Xinjiang,China)
出处
《心血管病学进展》
CAS
2020年第10期1085-1090,1095,共7页
Advances in Cardiovascular Diseases
基金
国家自然科学基金地区基金项目(81760083)
国家自然科学基金青年科学基金项目(81900408)
陕西省教育厅重点科学研究计划项目(20JS137)
西安医学院国家基金培育项目(2018GJFY01)。
