利拉鲁肽对棕榈酸诱导心肌细胞损伤的保护作用

The protective effect of liraglutide on palmitic acid induced myocardial cell injury

目的:探讨利拉鲁肽(lira)对棕榈酸(PA)所致H9C2大鼠心肌细胞脂毒性损伤的干预效果及其相关机制。方法:在不同浓度的PA(0、50、100、150、200、300μmol/L)和不同时间(6、12、24 h)的条件下分别刺激H9C2大鼠心肌细胞(n=3),建立心肌细胞的脂毒性损伤模型,使用CCK8检测确定使H9C2细胞脂毒性损伤较大、细胞存活数最多的刺激条件。将成功建立模型的H9C2细胞分为对照组(CON)、CON+Lira(1000 nmol/L)组、CON+PA(150μmol/L)组和PA(150μmol/L)+Lira(1000 nmol/L)组进行油红O染色(n=3),检测细胞内脂质含量,以明确PA(150μmol/L)对心肌细胞有无脂毒性。将H9C2细胞根据不同的PA浓度(0、50、100、150、200、300μmol/L)分为6个组(CON、PA1、PA2、PA3、PA4和PA5)(n=3),按照不同的Lira浓度分为6组:CON组、PA组(150μmol/L)、CON+Lira3(1000 nmol/L)组、PA(150μmol/L)+Lira1(100 nmol/L)组、PA(150μmol/L)+Lira2(500 nmol/L)组和PA(150μmol/L)+Lira3(1000 nmol/L)组(n=3),用蛋白印迹测定凋亡相关蛋白的表达量及其与PA、Lira浓度的关系。结果:使用CCK8检测并最终确定PA为150μmol/L、刺激12 h的条件下,H9C2细胞的脂毒性损伤较大、细胞存活数最多。与CON组相比,PA组(150μmol/L)的细胞活性明显下降、脂滴明显增加(t=34.53,P<0.05),经lira预处理后脂滴明显减少(t=19.07,P<0.05);与CON组相比,随着PA浓度的增加,Bax、Caspase-3蛋白表达明显增加(F=12.32、3.307,均P<0.05),Bcl-2蛋白表达明显减少(F=7.618,P<0.05);与PA刺激组(150μmol/L)相比,lira预处理可以使Bcl-2蛋白表达明显增加(F=7.104,P<0.05,),Bax、Caspase-3蛋白表达明显减少(F=12.32、7.104,均P<0.05)。结论:浓度为150μmol/L的PA刺激H9C2心肌细胞12 h后可造成心肌细胞脂毒性损伤模型,lira可以改善甚至逆转PA诱导的H9C2心肌细胞脂毒性损伤,其机制可能与抑制炎症、抑制细胞凋亡有关。

Objective:To explore the protective effect of liraglutide(lira)on palmitic acid(PA)induced H9C2 rat cardiomyocyte lipotoxic injury model and its possible mechanism.Methods:First,H9C2 cells were stimulated with different concentrations of PA(0,50,100,150,200,300μmol/L)and different times(6,12,24 hours)to establish a model of lipotoxic injury in cardiomyocytes(n=3).CCK8 was used to determine the stimulus conditions that resulted in greater lipotoxic damage to H9C2 cells and the highest number of viable cells.The H9C2 cells that had successfully established the model were divided into control group(CON),CON+Lira(1000 nmol/L)group,CON+PA(150μmol/L)group,and PA(150μmol/L)+Lira(1000 nmol/L)group.Oil red O staining was used to detect the intra-cellular lipid content to determine whether PA(150μmol/L)was lipid toxic to cardiomyocytes(n=3).According to different PA concen-trations,the H9C2 cells was divided into 6 groups(CON,PA1,PA2,PA3,PA4 and PA5)(n=3),and different lira concentrations were divided into CON group,PA,CON+Lira3(1000 nmol/L)group,PA+Lira1(100 nmol/L)group,PA+Lira2(500 nmol/L)group and PA+Lira3(1000 nmol/L)group(n=3).The expression of apoptosis-related proteins and their relationship with PA and lira concentrations were determined by Western blotting(n=3).Results:CCK8 was used to detect and finally determine that 150μmol/L PA and stimulat-ed for 12 h,the lipotoxic damage of H9C2 cells was greater and the number of cell survival was the highest.Compared with CON group,the cell activity decreased and lipid droplets increased significantly in the PA stimulation group(150μmol/L)(t=34.53,P<0.05),and the lipid droplets decreased after lira pretreatmen(t t=19.07,P<0.05).Compared with the CON group,with the increase of PA concentra-tion,the expression of Bax and Caspase-3 proteins increased significantly(F=12.32,3.307,both P<0.05),and the expression of Bcl-2 protein decreased significantly(F=7.618,P<0.05).Compared with the PA stimulation group,the expression of Bcl-2 protein was signif-icantly increased by lira pretreatmen(F=7.104,P<0.05),and the expression of Bax and Caspase-3 proteins was significantly decreased(F=12.32,7.104,both P<0.05).Conclusion:Stimulating H9C2 cardiomyocytes with a concentration of 150μmol/L PA for 12 h can in-duce a model of myocardial cell lipotoxic damage.Lira can improve or even reverse the lipotoxic damage in H9C2 cardiomyocytes in-duced by PA,which may be related to the inhibition of inflammation and apoptosis.