姬松茸多糖对过氧化氢诱导的心肌细胞损伤的保护作用

Protective effect of Agaricus blazei polysaccharide on the injury of myocardial cells induced by H_(2)O_(2)

目的考察姬松茸多糖(Agaricus blazei polysaccharide,ABPS)对过氧化氢(H_(2)O_(2))诱导的心肌细胞(H9C2)损伤的影响及潜在机制。方法分别用H_(2)O_(2)(200μmol·L^(-1))、ABPS(0.5、1.0、2.0 g·L^(-1))、LY294002(10μmol·L^(-1))处理心肌细胞H9C2,将H9C2细胞分为NC组、H_(2)O_(2)组、H_(2)O_(2)+ABPS 0.5 g·L^(-1)组、H_(2)O_(2)+ABPS 1.0 g·L^(-1)组、H_(2)O_(2)+ABPS 2.0 g·L^(-1)组、H_(2)O_(2)+ABPS+PBS组及H_(2)O_(2)+ABPS+LY294002组,CCK8实验和流式细胞术分别检测细胞存活率和凋亡率;Western blot分别检测细胞相关蛋白P21、细胞周期蛋白D1(CyclinD1)、Cleaved含半胱氨酸的天冬氨酸蛋白水解酶3(Cysteinyl aspartate specific proteinase 3,caspase-3)、Cleaved含半胱氨酸的天冬氨酸蛋白水解酶9(Cysteinyl aspartate specific proteinase 9,Caspase-9)、p-蛋白激酶B(Protein kinase B,AKT)和AKT的表达水平,试剂盒检测H9C2细胞中丙二醛(malondialdehyde,MDA)、活性氧(reactive oxygen species,ROS)和谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)的水平。结果与对照NC组相比,H_(2)O_(2)组心肌细胞H9C2的凋亡率、P21、Cleaved caspase-3、Cleaved caspase-9、p-AKT蛋白、ROS和MDA含量均升高,Cyclin D1、GSH-Px含量和细胞存活率均降低,差异具有统计学意义(P<0.05);0.5、1.0、2.0 g·L^(-1)ABPS均可逆转H_(2)O_(2)对心肌细胞H9C2的作用,呈剂量依赖趋势,且2.0 g·L^(-1)ABPS效果最好;经LY294002阻断AKT信号后,与H_(2)O_(2)+ABPS+PBS组相比,H_(2)O_(2)+ABPS+LY294002组细胞凋亡率、P21、Cleaved caspase-3、Cleaved caspase-9、p-AKT蛋白、ROS和MDA含量均升高,Cyclin D1、GSH-Px含量和细胞存活率均降低,差异具有统计学意义(P<0.05)。结论姬松茸多糖通过AKT信号通路保护心肌细胞H9C2免受H_(2)O_(2)诱导的氧化应激损伤和凋亡。

Objective This study investigated the effects of Agaricus blazei polysaccharide(ABPS)on injury of myocardial cells induced by H_(2)O_(2)and its potential mechanism.Myocardial cells H9C2 were treated with H_(2)O_(2)(200μmol·L^(-1)),ABPS(0.5,1.0,2.0 mg·mL^(-1)),LY294002(10μmol·L^(-1)),respectively.H9C2 cells were divided into NC group,H_(2)O_(2)group,H_(2)O_(2)+ABPS 0.5 mg·mL^(-1)group,H_(2)O_(2)+ABPS 1.0 mg·mL^(-1)group,H_(2)O_(2)+ABPS 2.0 g·L^(-1)group,H_(2)O_(2)+ABPS+PBS group and H_(2)O_(2)+ABPS+LY294002 group.Cell survival rate and apoptosis rate were detected by CCK8 assay and flow cytometry,respectively.Western blot was performed to detect the expression levels of proteins P21,CyclinD1,Cleaved Cysteinyl aspartate specific proteinase 3(caspase-3),Cleaved Cysteinyl aspartate specific proteinase 9(caspase-9),p-Protein kinase B(AKT)and AKT respectively.The kits were used to detecte the levels of malondialdehyde(MDA),reactive oxygen species(ROS)and glutathione peroxidase(GSH-Px)in H9C2 cells.It was found that compared with the NC group,the apoptosis rate,P21,Cleaved caspase-3,Cleaved caspase-9,p-AKT protein,ROS and MDA contents of H9C2 cells in H_(2)O_(2)group were all increased,and the levels of Cyclin D1,GSH-Px and cell survival rate were all decreased,with statistically significant difference(P<0.05).0.5,1.0 and 2.0 mg·mL^(-1)ABPS all reversed the effect of H_(2)O_(2)on H9C2 cells in a dose-dependent manner,and 2.0 mg·mL^(-1)ABPS had the best effect.After blocking AKT signaling by LY294002,compared with H_(2)O_(2)+ABPS+PBS group,in H_(2)O_(2)+ABPS+LY294002 group the apoptosis rate,P21,Cleaved caspase-3,Cleaved caspase-9,p-AKT protein,ROS and MDA levels were all increased,and the levels of Cyclin D1,GSH-Px and cell survival rate were all decreased,with statistically significant difference(P<0.05).These results show that ABPS protects myocardial cell H9C2 from oxidative stress injury and apoptosis induced by H_(2)O_(2)through the AKT signaling pathway.