黄芪甲苷对心肌细胞缺氧/复氧损伤的保护作用及其自噬机制研究  

AstragalosideⅣ’s Therapeutic Effect on Myocardial Infarction via Affecting Autophagy and the Mechanism Study

作　　者：张斌[1] 马倩 马富珍[1] 王静[1] 刘旭东[1] 何儒华[3] ZHANG Bin;MA Qian;MA Fu-zhen;WANG Jing;LIU Xu-dong;HE Ru-hua(Department of Cardiovascular Surgery,General Hospital of Ningxia Medical University,Yinchuan 750004,China;Department of Anesthesiology,General Hospital of Ningxia Medical University,Yinchuan 750004,China;Department of Internal Medicine-Cardiovascular,General Hospital of Ningxia Medical Universityy Yinchuan 750004,China)

机构地区：[1]宁夏医科大学总医院心脏大血管外科,银川750004 [2]宁夏医科大学总医院肿瘤医院麻醉科,银川750004 [3]宁夏医科大学总医院心血管内科,银川750004

出　　处：《四川大学学报（医学版）》2021年第2期222-228,共7页Journal of Sichuan University(Medical Sciences)

基　　金：宁夏自然科学基金项目(No.NZ15151)资助。

摘　　要：目的研究黄芪甲苷(AS-Ⅳ)对新生大鼠心肌细胞缺氧/复氧(H/R)损伤的保护作用,并探讨其潜在的作用机制。方法提取并培养新生大鼠心肌细胞,随机分为6组:对照组,H/R组,H/R+低、中、高浓度AS-Ⅳ(AS-Ⅳ终浓度0.1μmol/L、1μmol/L、10μmol/L)组,和H/R+高浓度AS-Ⅳ(10μmol/L)+AKT抑制剂(5μmol/L)组。H/R前分组预处理心肌细胞(对照组和H/R组不进行预处理)48 h后,除对照组外,其余各组心肌细胞建立细胞H/R损伤模型。MTT法检测各组心肌细胞活力,实时荧光定量PCR(RT-qPCR)检测自噬基因LC3-Ⅱ、p62的表达,Western blot检测LC3-Ⅱ、P62蛋白和磷脂酰肌醇3激酶/丝氨酸苏氨酸蛋白激酶(PI3K/AKT)通路蛋白[包括AKT、磷酸化(p-)AKT(p-AKT)、p-雷帕霉素(p-mTOR)]和自噬启动因子未配位的51样激酶1(ULK1)的表达,免疫荧光染色检测心肌细胞自噬体P62的表达。结果AS-Ⅳ在本研究低、中、高浓度范围内以浓度依赖的方式改善H/R损伤下心肌细胞活力,并抑制H/R损伤后的心肌细胞自噬基因LC3-Ⅱ和p62 mRNA和蛋白的表达,抑制ULK1蛋白的表达(P<0.05),添加AKT抑制剂后,AS-Ⅳ的以上作用被部分抑制(P<0.05)。AS-Ⅳ对AKT和mTOR蛋白的表达没有影响(P>0.05),但可以促进AKT和mTOR的磷酸化(P<0.05)。免疫荧光染色检测结果显示,高浓度AS-Ⅳ可以逆转H/R损伤诱导的自噬体P62的表达。结论AS-Ⅳ对H/R损伤心肌细胞具有保护作用,其作用机制可能是通过激活PI3K/AKT途径中的m TOR信号降低自噬水平,从而预防H/R损伤。Objective The purpose of this study was to investigate the protective effect of astragalosideⅣ(AS-Ⅳ)on neonatal rats’hypoxic/reoxygenated(H/R)injured myocardial cells and to explore its underlying mechanism.Methods Cardiac cells were extracted from newborn rats and divided into control,H/R,H/R-low AS-Ⅳ(0.1μmol/L AS-Ⅳ),H/R-medium AS-Ⅳ(1μmol/L AS-Ⅳ),H/R-high AS-Ⅳ(10μmol/L AS-Ⅳ)and H/R-high AS-Ⅳ-AKT(10μmol/L AS-Ⅳ+5μmol/L AKT)groups.After 48 h of treatment,the contents of LC3-Ⅱ,p62,AKT,pAKT,rapamycin(mTOR)mammalian targets and uncoordinated 51-like kinase 1(ULK1)in cardiac myocytes were compared.Immunofluorescence staining was used to detect the expression of P62 in myocardium autophagosome.Restults AS-Ⅳimproved the proliferative activity of cardio AS-Ⅳimproved the proliferative activity of cardiomyocytes in H/R injury in a dosedependent manner and inhibited the level of cell autophagy.However,when AKT inhibitors were added,the effect of AS-Ⅳwas partially inhibited(P<0.05).Gene and protein expression showed that AS-Ⅳhad no significant effect on the expression of AKT and mTOR genes(P>0.05),but could significantly promote the phosphorylation of AKT and mTOR(P<0.05).Immunofluorescence staining results showed that high concentrations of the AS-Ⅳcan reverse H/R injury induced the expression of autophagy body P62.Conclusion AS-Ⅳshowed protection effect on H/R injured myocardial cells.The possible mechanism is by reducing the autophagy level via activating the mTOR signal in the PI3 K/AKT pathway,thereby preventing H/R damage in neonatal rat cardiomyocytes.

关 键 词：黄芪甲苷 缺氧/复氧损伤 PI3K/AKT通路 自噬 

分 类 号：R285[医药卫生—中药学]