人参皂苷Rg1介导PI3K/AKT/FOXO3通路缓解高糖诱导的肾小球系膜细胞氧化应激反应

Ginsenoside Rg1 alleviates the oxidative stress in mesangial cells induced by high glucose through PI3K/AKT/FOXO3 pathway

目的探究人参皂苷Rg1通过PI3K/AKT/FOXO3通路对高糖诱导的大鼠肾小球系膜细胞氧化应激反应的调控作用。方法首先将细胞分为对照组、HG组、低浓度Rg1组(HG+20 ng/ml Rg1)、中浓度Rg1组(HG+50 ng/ml Rg1)、高浓度Rg1组(HG+100 ng/ml Rg1),通过MTT法、Hoechst 33258染色和Western blot分别检测Rg1对高糖作用下HBZY-1细胞存活率、凋亡率和凋亡相关蛋白Bcl-2、Bax、caspase-3、caspase-9表达的调控作用。其次,通过试剂盒检测Rg1对高糖作用下HBZY-1细胞中氧化应激标志物ROS、MDA、SOD、Gpx浓度的影响。并通过Western blot检测在Rg1作用下,PI3K/AKT/FOXO3通路中各蛋白的磷酸化及表达情况。最后,验证在AKT被抑制的情况下Rg1对高糖作用下HBZY-1细胞的调控作用。结果与对照组相比,HG组中细胞存活率、Bcl-2表达量、SOD、Gpx含量显著下降(P<0.05),而凋亡率、Bax、caspase-3、caspase-9的表达量、ROS、MDA含量显著上升(P<0.05);与HG组相比,低、中、高浓度Rg1组中细胞存活率、Bcl-2表达量、SOD、Gpx含量显著上升(P<0.05),而凋亡率、Bax、caspase-3、caspase-9的表达量、ROS、MDA含量显著下降(P<0.05)。其次,与HG组相比,低、中、高浓度Rg1组细胞中p-PI3K/PI3K、p-AKT/AKT比值显著升高(P<0.05),而FOXO3蛋白表达量显著降低(P<0.05)。而在加入AKT抑制剂之后,与HG组相比,HG+MK-2206组中p-PI3K/PI3K、p-AKT/AKT比值显著下降(P<0.05),而FOXO3蛋白表达量显著升高(P<0.05)。最后,与HG+MK-2206组相比,HG+Rg1+MK-2206组中p-PI3K/PI3K、p-AKT/AKT比值显著升高(P<0.05),而FOXO3蛋白表达量显著降低(P<0.05)。结论人参皂苷Rg1可以通过活化PI3K/AKT/FOXO3通路缓解高糖对HBZY-1细胞的诱导作用,减少HBZY-1细胞的凋亡,并缓解氧化应激反应。

This study was performed to investigate the regulation of ginsenoside Rg1 on the oxidative stress induced by high glucose(HG)in rat mesangial cells through PI3 K/AKT/FOXO3 pathway.Mesangial cells were divided into control group,HG group,low concentration Rg1 group(HG+20 ng/ml Rg1),medium concentration Rg1 group(HG+50 ng/ml Rg1)and high concentration Rg1 group(HG+100 ng/ml Rg1).MTT,Hoechst 33258 staining and Western blot methods were used to detect the regulatory effects of Rg1 on the viability,apoptosis and expression of apoptosis-related proteins Bcl-2,Bax,caspase-3 and caspase-9 in HBZY-1 cells exposed to high glucose.Then,the effect of Rg1 on the concentration of oxidative stress markers ROS,MDA,SOD and Gpx in HBZY-1 cells under high glucose was detected by kits;the phosphorylation and expression of each protein in PI3K/AKT/FOXO3 pathway were detected by Western blotting under the action of Rg1.Finally,we verified the regulatory effect of Rg1 on HBZY-1 cells under high glucose when AKT was inhibited.Data showed that the cell viability,Bcl-2 expression,SOD and Gpx content decreased significantly in HG group compared with the control group(P<0.05),while the apoptosis rate,Bax,caspase-3,caspase-9 expression,ROS and MDA content increased significantly(P<0.05).Compared with HG group,the cell viability,Bcl-2 expression,SOD and Gpx content in all Rg1 groups increased significantly(P<0.05),while the apoptosis rate,Bax,caspase-3,caspase-9 expression,ROS and MDA content decreased significantly(P<0.05).Furthermore,compared with HG group,the ratio of p-PI3K/PI3K and p-AKT/AKT in cells of all Rg1 groups increased significantly(P<0.05),while the expression of FOXO3 protein decreased significantly(P<0.05).AKT inhibitor MK-2206 could down-regulate the ratio of p-PI3K/PI3K and p-AKT/AKT(P<0.05),while up-regulate the expression of FOXO3 protein(P<0.05)in HG group.Finally,compared with HG+MK-2206 group,the ratio of pPI3K/PI3K and p-AKT/AKT in HG+Rg1+MK-2206 group increased significantly(P<0.05),while the expression of FOXO3 protein decreased significantly(P<0.05).In conclusion,Ginsenoside Rg1 can alleviate the induction of high glucose on HBZY-1 cells through PI3K/AKT/FOXO3 pathway,reduce the apoptosis of HBZY-1 cells and alleviate the oxidative stress.