利拉鲁肽对高糖诱导足细胞损伤后相关蛋白mRNA和蛋白表达的影响及其机制

Effect of liraglutide on expressions of mRNA and protein of related proteins after podocyte injury induced by high glucose and its mechanism

目的:观察胰高血糖素样肽1(GLP-1)受体激动剂利拉鲁肽对高糖诱导足细胞损伤后相关蛋白mRNA及蛋白表达的影响,并探讨其作用机制。方法:将分化成熟的足细胞随机分为对照组、高糖组、甘露醇对照组、利拉鲁肽组和高糖+利拉鲁肽组。提取各组细胞RNA,应用Real-time PCR(RT-PCR)法检测各组足细胞中Nephrin、Podocin和ZO-1 mRNA表达水平,Western blotting法检测各组细胞中Nephrin、Podocin、ZO-1、Bcl-2和caspase-3蛋白表达水平,免疫荧光法观察足细胞中骨架蛋白Synaptopodin表达情况,流式细胞术检测细胞凋亡率。将分化成熟的足细胞随机分为对照组、高糖组、高糖+利拉鲁肽组、高糖+利拉鲁肽+LY294002组。采用Western blotting法检测磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(PI3K/Akt)信号通路转导蛋白、Bcl-2和caspase-3表达水平。结果:与对照组比较,高糖组足细胞中标志蛋白Nephrin、Podocin和ZO-1 mRNA及蛋白表达水平降低(P<0.05),Bcl-2蛋白表达水平降低(P<0.05),caspase-3蛋白表达水平升高(P<0.05),足细胞特异性骨架蛋白Synaptopodin荧光强度明显降低(P<0.05),足细胞凋亡率明显升高(P<0.05)。与高糖组比较,高糖+利拉鲁肽组足细胞中Nephrin、Podocin和ZO-1 mRNA及蛋白表达水平升高(P<0.05),Bcl-2蛋白表达水平升高(P<0.05),caspase-3蛋白表达水平降低(P<0.05),足细胞特异性骨架蛋白Synaptopodin荧光强度明显升高(P<0.05),足细胞凋亡率明显降低(P<0.05)。与对照组比较,高糖组足细胞中PI3K和pAkt蛋白表达水平明显降低(P<0.05),Bcl-2蛋白表达水平降低(P<0.05),caspase-3蛋白表达水平升高(P<0.05);与高糖组比较,高糖+利拉鲁肽组足细胞内PI3K和磷酸化Akt(p-Akt)蛋白表达水平明显升高(P<0.05),凋亡相关蛋白Bcl-2蛋白表达水平升高(P<0.05),caspase-3蛋白表达水平降低(P<0.05),与高糖+利拉鲁肽组比较,高糖+利拉鲁肽+LY294002组足细胞中PI3K和磷酸化Akt(p-Akt)蛋白表达水平降低(P<0.05),Bcl-2蛋白表达水平降低(P<0.05),caspase-3蛋白表达水平升高(P<0.05)。结论:利拉鲁肽通过激活PI3K/Akt信号通路抑制足细胞凋亡,减轻高糖诱导的足细胞损伤。

Objective:To observe the effect of glucagon like peptide-1(GLP-1)receptor agonist liraglutide on the expressions of the mRNA and protein of related proteins after podocyte injury induced by high glucose,and to explore its mechanism.Methods:The differentiated mature podocytes were randomly divided into control group,high glucose group,mannitol control group,liraglutide group,high glucose+liraglutide group.The RNA was extracted from cells in each group.Real-time PCR(RT-PCR)was used to detect the expression levels of Nephrin,Podocin and ZO-1 mRNA in the podocytes in various groups.Western blotting method was used to detect the expression levels of Nephrin,Podocin,ZO-1,Bcl-2 and caspase-3 proteins in the podocytes in various groups.The Synaptopodin expression in the podocytes was observed by immunofluorescence method.The apoptotic rate was measured by flow cytometry.The differentiated mature podocytes were randomly divided into control group,high glucose group,high glucose+liraglutide group,high glucose+liraglutide+LY294002 group.The expression levels of the phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)signaling pathway proteins were observed by Western blotting method.Results:Compared with control group,the expression levels of mRNA and protein of the podocytes marker proteins Nephrin,Podocin and ZO-1 in the podocytes in high glucose group were reduced(P<0.05),the expression level of Bcl-2 proein was reduced(P<0.05),the expression level of caspase-3 protein was increased(P<0.05),the fluorescence intensity of specific skeleton protein Synaptopodin was decreased significantly(P<0.05),and the apoptotic rate of podocytes was increased significantly(P<0.05).Compared with high glucose group,the expression levels of mRNA and protein of Nephrin,Podocin and ZO-1 in the podocytes in high glucose+liraglutide group were increased(P<0.05),the expression level of Bcl-2 proein was increased(P<0.05),the expression level of caspase-3 protein was decreased(P<0.05),the fluorescence intensity of podocyte specific skeleton protein Synaptopodin was increased significantly(P<0.05),and the apoptotic rate of podocytes was decreased significantly(P<0.05).Compared with control group,the expression levels of PI3K and phosphorylated(p-Akt)proteins in the podocytes in high glucose group were decreased significantly(P<0.05),the expression level of Bcl-2 protein was decreased(P<0.05),and the expression level of caspase-3 protein was increased(P<0.05).Compared with high glucose group,the expression levels of PI3 K and p-Akt proeins in high glucose+liraglutide group were increased significantly(P<0.05),the expression level of Bcl-2 proein was increased(P<0.05),and the expression level of caspase-3 protein was decreased significantly(P<0.05).Compared with high glucose+liraglutide group,the expression levels of PI3K and p-Akt proteins in the podocytes in high glucose+liraglutide+LY294002 group were decreased(P<0.05),the expression level of Bcl-2 protein was decreased(P<0.05),and the expression level of caspase-3 protein was increased(P<0.05).Conclusion:Liraglutidecan inhibit the apoptosis of podocytes by activating the PI3K/Akt signaling pathway and alleviates the podocyte injury induced by high glucose.