乌梅提取物对缺氧复氧诱导的心肌细胞自噬的影响  

Effects of fructus mume extract on autophagy of cardiomyocytes induced by hypoxia/reoxygenation

作　　者：杨萍 刘波[1] 邹勇[1] 刘泰民 郭姣姣 代天 Yang Ping;Liu Bo;Zou Yong;Liu Taimin;Guo Jiaojiao;Dai Tian(Department of Cardiology,Sixth Hospital of Wuhan Affiliated Hospital of Jianghan University,Wuhan 430015,Hubei Province,China)

机构地区：[1]武汉市第六医院(江汉大学附属医院)心血管内科,430015

出　　处：《中华老年心脑血管病杂志》2023年第4期416-420,共5页Chinese Journal of Geriatric Heart,Brain and Vessel Diseases

基　　金：武汉市医学科研项目(WZ20B04)。

摘　　要：目的探讨乌梅提取物(FME)调控磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)/哺乳类动物雷帕霉素靶蛋白(mTOR)通路对缺氧/复氧(H/R)诱导的H9C2细胞自噬反应的影响。方法使用不同浓度FME预处理H/R诱导的H9C2细胞,确定FME给药浓度,将H9C2细胞分为对照组、H/R组、FME组(2.0 mg/ml FME)、FME+PI3K抑制剂组(2.0 mg/ml FME+50.0μmol/L LY294002)、FME+mTOR抑制剂组(2.0 mg/ml FME+50.0μmol/L Rapamycin),流式细胞仪检测H9C2细胞凋亡率,透射电子显微镜观察自噬的形成,激光共聚焦显微镜观察细胞自噬体水平,Western blot检测微管相关蛋白1轻链3-Ⅱ/Ⅰ(LC3Ⅱ/Ⅰ)、自噬降解底物(p62)、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)、B淋巴细胞瘤-2相关X蛋白(Bax)、PI3K/Akt/mTOR通路相关蛋白表达。结果与对照组比较,H/R组细胞凋亡率、自噬体、自噬溶酶体、LC3Ⅱ/Ⅰ、Caspase-3、Bax表达水平显著升高,p62、磷酸化PI3K(p-PI3K)、磷酸化Akt(p-Akt)、磷酸化mTOR(p-mTOR)表达水平明显降低,差异有统计学意义(P<0.05);与H/R组比较,FME组细胞凋亡率、自噬体、自噬溶酶体、LC3Ⅱ/Ⅰ、Caspase-3(0.31±0.05 vs 0.87±0.10)、Bax表达水平明显降低,p62、p-PI3K、p-Akt、p-mTOR表达水平明显升高,差异有统计学意义(P<0.05)。FME+PI3K抑制剂组和FME+mTOR抑制剂组细胞凋亡率、自噬体、自噬溶酶体明显高于FME组[(18.47±3.93)%,(20.61±3.64)%vs(12.26±2.35)%;(17.50±2.15)个/细胞,(16.83±3.64)个/细胞vs(12.50±2.61)个/细胞;(15.20±2.47)个/细胞,(14.67±3.03)个/细胞vs(9.33±1.56)个/细胞,P<0.05]。结论PI3K抑制剂与mTOR抑制剂可逆转FME对H/R诱导的H9C2细胞自噬抑制作用,FME通过激活PI3K/Akt/mTOR信号通路,抑制H/R诱导的H9C2细胞损伤与自噬。Objective To investigate the influence of fructus mume extract(FME)on autophagy response in cardiomyocytes(H9C2cells)induced by hypoxia/reoxygenation(H/R)by regulating phosphatidylinositol 3kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)pathway.Methods H/R-induced H9C2cells were pretreated with different concentrations of FME to determine the optimal dose of FME administration.H9C2cells were grouped into Control group,H/R group,FME group(2.0mg/ml),FME+PI3Kinhibitor group(2.0mg/ml FME+50.0μmol/L LY294002),FME+mTOR inhibitor group(2.0mg/ml FME+50.0μmol/L rapamycin).Cell apoptosis was detected by flow cytometry,autophagy formation was observed by transmission electron microscopy,and autophagosome level was observed by confocal laser microscopy.The expression levels of microtubule-associated protein 1light chain 3-Ⅱ/Ⅰ(LC3Ⅱ/Ⅰ),autophagy degradation substrate(p62),Caspase-3,B-lymphocytoma-2-associated X protein(Bax)and PI3K/Akt/mTOR pathway-related proteins were detected by Western blotting.Results The apoptotic rate,autophagosome,autophagolysome,and expression levels of LC3Ⅱ/Ⅰ,Caspase-3and Bax in H/R group were significantly higher,while the levels of p62,P-PI3K,P-Akt and P-mTOR were significantly lower in the H/R group than the control group(P<0.05).The apoptotic rate,autophagosome,autophagolysosome,and expression levels of LC3Ⅱ/Ⅰ,Caspase-3,and Bax were significantly lower,while the expression levels of p62,p-PI3K,p-Akt and p-mTOR were obviously higher in the FME group than the H/R group(P<0.05).The apoptotic rate,autophagosome,and autophalysome were significantly higher in the FME+PI3Kinhibitor group and FME+mTOR inhibitor group than the FME group(18.47%±3.93%,20.61%±3.64%vs 12.26%±2.35%;17.50±2.15,16.83±3.64 vs 12.50±2.61;15.20±2.47,14.67±3.03 vs 9.33±1.56,P<0.05).Conclusion PI3Kinhibitor and mTOR inhibitor can reverse the inhibition of FME on H/R-induced autophagy in H9C2cells.FME inhibits H/R-induced injury and autophagy in H9C2cells by activating the PI3K/Akt/mTOR

关 键 词：低氧 肌细胞 心脏 自噬 乌梅提取物 

分 类 号：R285[医药卫生—中药学]