虫草素调节环磷酸腺苷/蛋白激酶A信号通路对异丙肾上腺素诱导的心肌细胞凋亡的影响

Impacts of cordycepin on isoproterenol-induced cardiomyocyte apoptosis by regulating cAMP/PKA signaling pathway

目的探讨虫草素对异丙肾上腺素(isoproterenol,ISO)诱导的心肌细胞凋亡及环磷酸腺苷(cyclic adenosine monophosphate,cAMP)/蛋白激酶A(protein kinase A,PKA)信号通路的影响。方法采用10μmol/L的ISO处理H9c2细胞,再用1、5、10、15μg/ml的虫草素处理H9c2细胞,检测细胞增殖情况。将H9c2细胞分为对照组、ISO组、低虫草素组、高虫草素组、联合组。流式细胞术检测细胞凋亡率;鬼笔环肽染色检测心肌细胞表面积;定量聚合酶链反应检测肥大相关基因心房钠尿肽(atrial natriuretic peptide,ANP)、B型钠尿肽(brain natriuretic peptide,BNP)、β-肌球蛋白重链(β-myosin heavy chain,β-MHC)表达。酶联免疫吸附法检测肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素(interleukin,IL)-6、IL-1β以及cAMP表达;Western blot法检测B淋巴细胞瘤(B cell lymphoma/leukemia-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)、半胱氨酸天冬氨酸蛋白酶3(cysteinyl aspartate specific proteinase-3,Caspase-3)、PKA表达。结果与对照组比较,ISO组细胞凋亡率[(33.89±3.68)%vs(2.57±0.26)%]、细胞表面积、TNF-α、IL-6、IL-1β、Bax、Caspase-3、ANP、BNP、β-MHC表达明显升高,Bcl-2、cAMP、PKA表达明显降低(P<0.05)。与ISO组比较,低虫草素组和高虫草素组细胞凋亡率[(20.58±1.93)%、(7.58±0.76)%vs(33.89±3.68)%]、细胞表面积、TNF-α、IL-6、IL-1β、Bax、Caspase-3、ANP、BNP、β-MHC表达明显降低,Bcl-2、cAMP、PKA表达明显升高(P<0.05)。与高虫草素组比较,联合细胞凋亡率[(28.69±2.82)%vs(7.58±0.76)%]、细胞表面积、TNF-α、IL-6、IL-1β、Bax、Caspase-3、ANP、BNP、β-MHC表达明显升高,Bcl-2、cAMP、PKA表达明显降低(P<0.05)。结论虫草素通过激活cAMP/PKA信号通路抑制ISO诱导的心肌细胞凋亡。

Objective To investigate the impacts of cordycepin(Cor)on isoproterenol(ISO)-induced cardiomyocyte apoptosis and cyclic AMP/protein kinase A(cAMP/PKA)signaling pathway.Methods After H9c2 cardiomyocytes were treated with ISO(10μmol/L),the cells were further treated with Cor(1,5,10,15μg/ml),respectively to detect cell proliferation.Then the H9c2 cells were grouped into Control group,ISO group,5μg/ml Cor group(Cor-L group),10μg/ml Cor group(Cor-H group),and 10μg/ml Cor+20μmol/L H-89 group(combined group).Flow cytometry was applied to detect cell apoptotic rate in above groups.Phalloidin staining was used to detect the surface area of the cells.RT-PCR was used to detect the expression of hypertrophy related genes,such as ANP,BNP,andβ-myosin heavy chain(β-MHC)in the cardiomyocytes.ELISA was conducted on test the levels of inflammatory factors,TNF-α,IL-6 and IL-1βand cAMP.Western blot assay was employed to measure the expression levels of B cell lymphoma/leukemia-2(Bcl-2),Bcl-2 associated X protein(Bax),Caspase-3,and PKA.Results Compared with the Control group,the apoptotic rate[(33.89±3.68)%vs(2.57±0.26)%],cell surface area,and the expression levels of TNF-α,IL-6,IL-1β,Bax,Caspase-3,ANP,BNP,andβ-MHC were increased,and the expression levels of Bcl-2,cAMP,and PKA were decreased in the ISO group(P<0.05).The Cor-L and Cor-H groups had significantly lower apoptotic rate[(20.58±1.93)%and(7.58±0.76)%vs(33.89±3.68)%],smaller cell surface area,and decreased expression levels of TNF-α,IL-6,IL-1β,Bax,Caspase-3,ANP,BNP,andβ-MHC,and increased levels of Bcl-2,cAMP,and PKA when compared with the ISO group(P<0.05).The apoptotic rate[(28.69±2.82)%vs(7.58±0.76)%],cell surface area,and expression levels of TNF-α,IL-6,IL-1β,Bax,Caspase-3,ANP,BNP,andβ-MHC were elevated,and the expression levels of Bcl-2,cAMP and PKA protein were decreased in the combined group than the Cor-H group(P<0.05).Conclusion Cor inhibits ISO-induced cardiomyocyte apoptosis by activating the cAMP/PKA signaling pathway.