高糖环境下法舒地尔调控自噬对原代心肌细胞凋亡的作用  

Role of Fasudil in Primary Cardiomyocyte Apoptosis via Autophagy under High Glucose Environment

作　　者：高惠宽[1] 侯斐 GAO Huikuan;HOU Fei(Department of Cardiology,Beijing Friendship Hospital,Capital Medical University,Beijing 100050,China;不详)

机构地区：[1]首都医科大学附属北京友谊医院心内科,100050 [2]首都医科大学附属北京地坛医院,1001022

出　　处：《医学研究杂志》2023年第6期36-41,共6页Journal of Medical Research

基　　金：北京市临床重点专科项目{京卫医[2018]204号};北京友谊医院科研启动基金资助项目(yyqdkt2017-17)。

摘　　要：目的探讨高糖环境下,法舒地尔对原代心肌细胞凋亡的作用及自噬在其中的作用。方法分离培养原代心肌细胞,使用不同葡萄糖浓度培养基培养细胞,并使用法舒地尔及3-MA干预。检测细胞凋亡,测定Bcl-2、Bax、MYPT-1、p-MYPT1、LC3、P62等蛋白的表达,并检测ROCK1和ROCK2的mRNA表达量。结果高糖环境下,原代心肌细胞凋亡比例升高,Bcl-2表达下降、Bax表达增多,ROCK1、ROCK2 mRNA表达增加,p-MYPT1/MYPT1比值降低;法舒地尔可抑制高糖环境下原代心肌细胞凋亡比例,Bcl-2升高、Bax水平降低,ROCK1、ROCK2 mRNA表达减少、p-MYPT1/MYPT1比值降低。高糖环境下不可溶性P62表达增多;法舒地尔干预后,不可溶性P62蛋白表达减少、可溶性P62蛋白表达增多、同时LC3-Ⅱ/LC3-Ⅰ比值增大。在法舒地尔干预基础上应用自噬流抑制剂3-甲基腺嘌呤,可见到细胞凋亡比例升高、Bcl-2下降及Bax水平的升高、不可溶性P62表达增多(P<0.05)。结论高糖环境下,法舒地尔通过活化自噬流抑制原代心肌细胞凋亡。Objective To investigate the effect of fasudil on apoptosis of primary cardiomyocytes under high glucose environment and the role of autophagy.Methods Primary cardiomyocytes was isolated and cultured.Cells were cultured with different glucose concentration medium,and then intervened with fasudil and 3-MA.Cell apoptosis was examined.ROCK1 and ROCK2 mRNA levels were detected.Bcl-2,Bax,myosin phosphatase target subunit-1(MYPT-1),phosphorylated(p)-MYPT1,LC3,soluble and insoluble P62 protein levels were determined by Western blot analysis.Results Under high glucose environment,we observed increased apoptosis ratio of primary cardiomyocytes,expression of Bax,mRNA expressions of ROCK1 and ROCK2,while decreased expression of Bcl-2 and the ratio of p-MYPT1/MYPT1.Fasudil could inhibit the apoptosis rate of primary cardiomyocytes under high glucose environment,increase Bcl-2,decrease Bax level,ROCK1,ROCK2 mRNA expression,and p-MYPT1/MYPT1 ratio.The expression of insoluble P62 increased under high glucose environment;after fasudil intervention,the expression of insoluble P62 protein decreased,the expression of soluble P62 protein increased,and the ratio of LC3-Ⅱ/LC3-Ⅰincreased.On the basis of fasudil intervention,the autophagy flux inhibitor 3-methyladenine was applied.We observed increased proportion of apoptosis,Bax level,and insoluble P62 expression,while decreased Bcl-2(P<0.05).Conclusion Fasudil could inhibit primary cardiomyocyte apoptosis by activating autophagic flux under high glucose environment.

关 键 词：糖尿病心肌病 原代心肌细胞 凋亡 自噬流 法舒地尔 

分 类 号：R34[医药卫生—基础医学]