利拉鲁肽通过磷酸肌醇3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白通路对高糖诱导的人足细胞自噬及凋亡影响的研究

Liraglutide in fluences human podocyte autophagy and apoptosis induced by high glucose through PI3K/Akt/mTOR pathway

目的 探讨利拉鲁肽对高糖诱导的人足细胞自噬及凋亡的影响及作用机制。方法 体外培养人足细胞分为正常对照(NC)组、高糖组(HG组)、25 nmol/L利拉鲁肽组(HG+Lir25组)、50 nmol/L利拉鲁肽组(HG+Lir50组)、利拉鲁肽+LY294002组(HG+Lir+LY294002组)和利拉鲁肽+3-MA组(HG+Lir+3-MA组)。CCK-8检测足细胞活力;流式细胞仪、Hoechst33342染色分别检测足细胞凋亡率和凋亡形态学;透射电镜、免疫荧光染色分别观察足细胞中自噬小体和自噬标志物LC3蛋白表达;Western blot法检测足细胞中凋亡、自噬和磷酸肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)通路相关蛋白表达。结果 与NC组比较,HG组足细胞活力及Bcl-2、Bcl-2/Bax、LC3Ⅱ/LC3Ⅰ、p-PI3K/PI3K、p-Akt/Akt蛋白表达降低(P<0.05),凋亡率及Bax、p62、p-mTOR/mTOR蛋白表达升高(P<0.05),可见较多胞核固缩的足细胞,自噬小体数量和LC3蛋白的绿色荧光斑点数量减少。与HG组比较,HG+Lir25、HG+Lir50组足细胞活力、Bcl-2、Bcl-2/Bax、LC3Ⅱ/LC3Ⅰ、p-PI3K/PI3K、p-Akt/Akt蛋白表达升高(P<0.05),细胞凋亡率、Bax、p62、p-mTOR/mTOR蛋白表达降低(P<0.05),减少胞核固缩的足细胞数量,增加自噬小体数量和LC3蛋白的绿色荧光斑点数量,且HG+Lir 50组上述指标变化更明显。与HG+Lir50组比较,HG+Lir+LY294002、HG+Lir+3-MA组可调节PI3K/Akt/mTOR通路,且能减弱利拉鲁肽对高糖诱导的足细胞活力、凋亡和自噬的改善作用。结论 利拉鲁肽可能通过PI3K/Akt/mTOR通路促进高糖诱导的人足细胞自噬并抑制其凋亡。

Objective To investigate the impact and mechanism of Liraglutide on autophagy and apoptosis of human podocyte induced by high glucose.Methods Human podocytes were cultured in vitro,and grouped into normal control group(NC group),high glucose group(HG group),25 nmol/L Liraglutide group(HG+Lir 25 group),50 nmol/L Liraglutide group(HG+Lir 50 group),Liraglutide+LY294002 group(HG+Lir+LY294002 group),and Liraglutide+3-MA group(HG+Lir+3-MA group).The podocyte activity was detected by CCK-8.The apoptosis rate and morphology of podocytes were detected by flow cytometry and Hoechst 33342 staining.The expression of autophagic body and autophagic marker LC3 protein in podocyteswas observed by transmission electron microscopy and immunofluorescence staining.Western blot was applied to detect the expression of apoptosis,autophagy and phosphoinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR) pathway related proteins in podocytes.Results Compared with NC group,the activity of podocytes and the expression of Bcl-2,Bcl-2/Bax,LC3 Ⅱ/LC3 Ⅰ,p-PI3K/PI3K,p-Akt/Akt proteins in HG group were decreased(P<0.05),while the apoptosis rate and the expression of Bax,p62,p-mTOR/mTOR proteins were increased in HG group(P<0.05).There were many podocytes with pyknotic nuclei,the number of autophagic bodies and the number of green fluorescent spots of LC3 protein were decreased in HG group.Compared with HG group,the activity of podocyte increased,and the expression of Bcl-2,Bcl-2/Bax,LC3 Ⅱ/LC3 Ⅰ,p-PI3K/PI3K,p-Akt/Akt protein increased(P<0.05),while the apoptosis rate and the expression of Bax,p62,p-mTOR/mTOR protein decreased(P<0.05) in HG+Lir 25 group and HG+Lir 50 group.The number of podocytes with karyopyknosis was reduced,the number of autophagosomes and the number of green fluorescent spots of LC3 protein were increased in HG+Lir 25 group and HG+Lir 50 group,and the above changes indexes were more obvious in the HG+Lir 50 group group.Compared with HG+Lir 50group,PI3K/Akt/mTOR pathway could be regulated,and reduce the improvement of Liraglutide on podocyte viability,apoptosis and autophagy induced by high glucose in HG+Lir+LY294002 group and HG+Lir+3-MA group.Conclusion Liraglutide may promote the autophagy of human podocyte induced by high glucose and inhibit its apoptosis through PI3K/Akt/mTOR pathway.