褪黑素上调SIRT1改善高糖诱导原代心肌细胞损伤的机制研究

Melatonin ameliorates high glucose-induced primary cardiomyocyte injury via up-regulating SIRT1 expression

目的探讨褪黑素在高糖诱导的原代心肌细胞损伤中的作用,并明确SIRT1在其中的作用及其机制。方法将原代心肌细胞分为对照组(Con组)、褪黑素对照组(Con+Mel组)、高糖组(HG组)、高糖+褪黑素组(HG+Mel组)和SIRT1抑制剂理组(HG+Mel+EX527组)。采用Cell Counting Kit-8(CCK-8)试剂盒检测细胞活性,丙二醛(MDA)和超氧化物歧化酶(SOD)试剂盒检测细胞氧化应激水平,TUNEL染色检测细胞凋亡,免疫印迹检测SIRT1、Beclin1、Atg5、cleaved Caspase-3、Bax和Bcl-2蛋白表达水平。结果与Con组相比,HG组原代心肌细胞活力减低,氧化应激水平升高,细胞凋亡水平增加,SIRT1表达下调(P<0.05)。给予褪黑素干预后,与Con组相比,Con+Mel组SIRT1表达量增加(P<0.05),而两组间细胞活力、氧化应激水平和细胞凋亡水平无显著差异(P>0.05)。与HG组相比,HG+Mel组原代心肌细胞SIRT1表达量提高(P<0.05),细胞活力增加(P<0.05),氧化应激水平和细胞凋亡水平下降(P<0.05)。免疫印迹结果显示,与Con组相比,HG组原代心肌细胞自噬相关蛋白Beclin1和Atg5表达减低(P<0.05),Con+Mel组Beclin1和Atg5表达明显上升(P<0.05);与HG组相比,HG+Mel组Beclin1和Atg5表达上升(P<0.05)。此外,给予SIRT1特异性抑制剂EX527干预后,相比于HG+Mel组,HG+Mel+EX527组细胞凋亡比率增加(P<0.05),凋亡蛋白cleaved Caspase-3和Bax上调(P<0.05),抗凋亡蛋白Bcl-2下调(P<0.05),细胞自噬蛋白Beclin1和Atg5表达减低(P<0.05)。结论褪黑素可能通过上调SIRT1信号增强细胞自噬,改善高糖诱导的原代心肌细胞损伤。

Objective To investigate the effect of melatonin on high glucose-induced cardiomyocyte injury,and to clarify the role and mechanism of SIRT1.Methods Primary cardiomyocytes were divided into control group,melatonin control group(Con+Mel group),high glucose(HG)group,high glucose and melatonin group(HG+Mel group)and SIRT1 inhibitor treatment group(HG+Mel+EX527 group).CCK-8 was used to test the cell viability,MDA and SOD kits were used to detect the level of cell oxidative stress,TUNEL staining was used to detect the apoptosis,and Western blot method was used to detect the protein expression of SIRT1,Beclin1,ATG5,cleaved Caspase-3,Bcl-2 and Bax.Results Compared with control group,the cell viability was significantly decreased in HG group(P<0.05),the levels of oxidative stress and apoptosis were up-regulated(P<0.05),and the expression of SIRT1 was significantly declined(P<0.05).Compared with control group,the expression of SIRT1 was increased in Con+Mel group(P<0.05),but there was no significant difference in cell viability,oxidative stress and apoptosis between the two groups(P<0.05).Compared with HG group,the expression of SIRT1 in primary cardiomyocytes was significantly increased in HG+Mel group(P<0.05),the cell viability was significantly improved(P<0.05),and the levels of oxidative stress and apoptosis were significantly lessened(P<0.05).Western blot results also showed that the protein expression levels of Beclin1 and ATG5 were reduced in HG group and increased in Con+Mel group compared to control group(P<0.05).Compared with HG group,the expression of Beclin1 and ATG5 were significantly augmented in HG+Mel group(P<0.05).The apoptosis rate was increased after intervention with SIRT1 specific inhibitor EX527 in HG+Mel+EX527 group compared with HG+Mel group(P<0.05),the expression levels of cleaved Caspase 3 and Bax were improved(P<0.05),and the protein expression levels of Bcl-2,Beclin1 and ATG5 were decreased(P<0.05).Conclusion Melatonin can protect cardiomyocytes against high glucose-induced primary cardiomyocyte injury via promotion of SIRT1 expression and restoring autophagy.