摘要
目的采用血清剥夺/复灌的大鼠H9c2心肌细胞模拟心肌缺血/再灌注损伤(I/R),从细胞层面探讨天麻素(gastrodin)抗H9c2心肌细胞凋亡的作用及其机制。方法体外培养H9c2心肌细胞随机分为对照组、血清剥夺(0.5~6 h)处理组、血清剥夺/复灌(2/4 h)处理组、天麻素(10、20和40μmol/L)处理组、天麻素(20μmol/L)+渥曼青霉素(wortmannin)(1μmol/L)处理组、wortmannin (1μmol/L)处理组。采用免疫印迹法检测p38丝裂原活化蛋白激酶(p38MAPK)、磷酸化p38MAPK(p-p38MAPK)、蛋白激酶B(Akt)、p-Akt以及凋亡相关蛋白cleaved Caspase-3、Bcl-2和Bax的表达变化;采用流式细胞术检测细胞凋亡;通过实时定量聚合酶链反应(Real-time PCR)检测Caspase-3、Bcl-2和Bax的mRNA表达变化;采用免疫荧光双标记法检测细胞内p-Akt蛋白水平表达的变化。结果血清剥夺(0.5~6 h)和血清剥夺/复灌(2/4 h)处理诱导细胞凋亡水平增加;加入不同浓度天麻素处理,与血清剥夺(0.5~6 h)和血清剥夺/复灌(2/4 h)处理组比较,凋亡相关蛋白cleaved Caspase-3表达降低,Bcl-2/Bax蛋白表达的比值上调(P<0.05),基因转录水平检测的Caspase-3和Bax表达量降低,Bcl-2表达量上调((P<0.05),流式细胞术凋亡检测结果也同样显示,天麻素能抑制血清剥夺/复灌(2/4 h)诱导的细胞凋亡(P<0.05);同时,血清剥夺(0.5~6 h)和血清剥夺/复灌(2/4 h)处理能抑制Akt/p38MAPK信号通路;加入不同浓度的天麻素(10、20和40μmol/L)处理后,p-Akt的表达水平增多,p-p38MAPK蛋白表达减少(P <0.05)。加入Akt的特异性抑制剂wortmannin(1μmol/L)处理后,天麻素对上述因子的调控作用被反转(P<0.05)。结论在H9c2心肌细胞中,天麻素通过激活Akt/p38MAPK信号通路,抑制血清剥夺/复灌诱导的细胞凋亡,从而发挥抗心肌I/R损伤的保护作用。
Objective To investigate the effect and precise molecular mechanisms for explaining how gastrodin suppresses the apoptosis in the serum deprivation/reperfusion-induced H9 c2 cardiomyocytes.Methods Rat H9 c2 cardiomyocytes were cultured and treated differently in vitro and randomly divided into following groups,including the control group,the serum deprivation-stimulated(0.5-6 hours) groups,the serum deprivation/reperfusion(2/4 hours)group,gastrodin(10,20 and 40 μmol/L) treatment groups,wortmannin(1 μmol/L) and gastrodin(20 μmol/L) cotreatment group,wortmannin(1 μmol/L) treatment group.The variation of protein expression levels of p-p38 mitogen actived protein kinase(MAPK),p-Akt and apoptosis related cleaved caspase-3,Bcl-2 and Bax were analyzed by Western blotting.H9 c2 cardiomyocytes were analyzed by flow cytometry.The level of RNA expression levels of apoptosis relatedCaspase-3,Bcl-2 and Bax were analyzed by Real-time PCR assay.The expression level of p-Akt was assayed by double immunofluorescence labeling.Results The results showed that serum deprivation(0.5-6 hours) and serum deprivation/reperfusion(2/4 hours) induced apoptosis increasing;Using Western blotting and Real-time PCR assay,we found that treatment with gastrodin significantly alleviated cleaved Caspase-3 activation and increased the ratio of Bcl-2/Bax in a concentration-dependent manner after serum deprivation and serum deprivation/reperfusion exposure.The same result has been showed by flow cytometer.Serum deprivation and serum deprivation/reperfusion inhibited the Akt/p38 MAPK signaling pathway;After treatment with gastrodin in a concentration-dependent manner induced the expression of p-Akt and decreased the expression of p-p38 MAPK.The regulation effects of gastrodin were reversed by the Akt specific inhibitor wortmannin.Conclusion In conclusion,the present study demonstrated that gastrodin exerted protective effects against serum deprivation/reperfusion-induced apoptosis in H9 c2 cardiomyocytes,at least partly via the Akt/p38 MAPK pathway.
作者
张玲
杨萍
姜永良
普俞维
谢立秋
孙林
陆地
ZHANG Ling;YANG Ping;JIANG Yong-liang;PU Yu-wei;XIE Li-qiu;SUN Lin;LU Di(Biomedical Engineering Research Center,Kunming Medical University,Kunming 650500,China;Department of Human Anatomy and Histology,Kunming Medical University College of Basic Medicine,Kunming 650500,China;Department of Cardiology,the Second Affiliated Hospital of Kunming Medical University,Kunming 650101,China)
出处
《解剖学报》
CAS
CSCD
北大核心
2019年第1期40-48,共9页
Acta Anatomica Sinica
基金
国家自然科学基金(31760292
81760087
81560050)
云南省应用基础研究重点项目[2017FA035
2017FE467(-008)]
昆明医科大学研究生创新基金(2017S009)
