整合素β3在异丙肾上腺素诱导的心肌细胞损伤与凋亡中的作用

Role of integrinβ3 in isoproterenol induced injury and apoptosis in cardiomyocytes

目的:探讨整合素β3(integrinβ3,ITGB3)在β-肾上腺素受体(β-adrenergic receptor,β-AR)激动剂异丙肾上腺素(isoprenaline,ISO)诱导的心肌细胞损伤与凋亡中的作用及其机制。方法:心肌细胞随机分为对照组(Con组)、siRNA-ITGB3处理组(Con+si-ITGB3组)、ISO组、ISO+siRNA-ITGB3处理组(ISO+si-ITGB3组)。采用CCK-8检测细胞活性,TUNEL染色检测细胞凋亡,GFP-LC3腺病毒检测细胞自噬流强度,RT-PCR检测ITGB3 mRNA的表达;蛋白质印迹法检测细胞蛋白质水平。结果:在10μmol/L的ISO刺激后,心肌细胞活力下降、且随刺激时间的延长,细胞活力下降程度增加。siRNA-ITGB3干扰能够下调ISO诱导的ITGB3表达,抑制ISO所致的细胞活力下降。siRNAITGB3干扰能够抑制ISO诱导细胞凋亡,抑制促凋亡蛋白cleaved-caspase-3和Bax蛋白表达,上调抗凋亡蛋白Bcl-2表达;siRNA-ITGB3干扰能够增强GFP-LC3表达、促进自噬蛋白Beclin1,LC3-I/LC3-II积累,降低p62水平,抑制ISO所致的细胞自噬水平的下降。结论:ITGB3表达下调能激活细胞自噬,抑制ISO诱导的心肌细胞损伤与凋亡。

Objective:To investigate the role and mechanism of integrin beta 3(ITGB3)inβ‐agonist,adrenoceptor agonist ISO-induced cardiomyocyte injury and apoptosis.Methods:Cardiac myocytes were randomly divided into a control group(Con),a Con+si-ITGB3 group,an isoprinosine(ISO)group,and an ISO+si-ITGB3 group.CCK-8 assay was used to detect cell viability.TUNEL staining was used to detect cell apoptosis.GFP-LC3 adenovirus were used to detect the level of cell autophagy.RT-qPCR was used to detect the mRNA expression of ITGB3.Western blot was used to detect the protein level.Results:After stimulation with 10μmol/L ISO,the cell viability of cardiomyocytes was reduced gradually with the prolongation of time.After pretreatment with siRNA-ITGB3,siRNA-ITGB3 treatment inhibited the decline of cell viability induced by ISO.Additional,siRNA-ITGB3 treatment inhibited the cell apoptosis induced by ISO,as evidenced by decreased the expression of cleaved caspase 3 and Bax,and increased the expression of Bcl-2.Consistently,siRNA-ITGB3 treatment decreased p62,increased the number of GFP-LC3 positive dots as well as LC3-II/LC3-I ratio and Beclin1 expression.Conclusion:Downregulation of ITGB3 partially attenuates isoproterenol induced injury and apoptosis via activation of autophagy in cardiomyocytes.