内质网应激对妊娠期糖尿病滋养层细胞的影响

Impact of endoplasmic reticulum stress on trophoblast cells in gestational diabetes mellitus

目的探讨内质网应激(ERS)对妊娠期糖尿病(GDM)滋养层细胞的影响。方法选取GDM产妇22例(GDM组)和健康产妇22例(健康组),电镜和TUNEL分别观察胎盘组织滋养细胞内质网、凋亡情况,免疫印迹检测GRP-78、CHO、Caspase-3、Bax和Bcl-2蛋白表达。衣霉素(0、0.5、1、2μg/ml)处理人HTR-8/SVneo细胞并检测GRP-78、CHOP蛋白表达,流式细胞法检测细胞凋亡,酶联免疫法检测TNF-α、IL-6和IL-1β水平;2-NBDG法检测胰岛素刺激下人HTR-8/SVneo细胞的葡萄糖摄取情况并分析GLUT4和相关途径蛋白表达情况。结果GDM组胎盘滋养细胞内质网体积增大,胎盘滋养细胞凋亡指数和CHOP、GRP78、cleaved-caspase-3/caspase-3、Bax/Bcl-2蛋白表达高于健康组(P<0.05)。与衣霉素(0、0.5μg/ml)组相比,衣霉素(1、2μg/ml)组细胞凋亡率、TNF-ɑ、IL-6、IL-1β水平和CHOP、GRP78蛋白表达均升高(P<0.05),有剂量效应。与对照组相比,ERS组葡萄糖摄取率和GLUT4、p-PI3K/PI3K、p-Akt/Akt蛋白表达减低(P<0.05),p-IRS-1蛋白表达升高(P<0.05),胰岛素刺激组上述指标趋势相反,在ERS基础上增加胰岛素刺激可以部分逆转ERS效果。结论ERS可促进滋养细胞凋亡,提高炎症反应,降低细胞葡萄糖摄取,增强胰岛素抵抗。

Objective To investigate the impact of endoplasmic reticulum stress(ERS)on trophoblast cells in gestational diabetes mellitus(GDM).Methods Included in this study were 22 GDM parturients(GDM group)and 22 healthy parturients(healthy group).Electron microscopy and TUNEL assay were used to observe the morphology and apoptosis of endoplasmic reticulum in placental trophoblast cells.Western blotting was used to detect the expression of GRP-78,CHO,Caspase-3,Bax and Bcl-2 proteins.Human HTR-8/svneo cells were treated with tunicamycin(at doses of 0,0.5,1,2μg/ml)and then,detected for GRP-78 and CHOP protein expression by Western blotting,cell apoptosis by flow cytometry,and TNF-α,IL-6 and IL-1βlevels by enzyme-linked immunosorbent assay.The human HTR-8/SVneo cells were also examined for glucose uptake under insulin stimulation by 2-NBDG method and analyzed for expression of GLUT4 and other pathway proteins.Results GDM group had larger size of endoplasmic reticulum in placental trophoblasts,higher apoptotic index of placental trophoblasts and upregulated protein expression of CHOP,GRP78,cleaved-caspase-3/caspase-3 and Bax/Bcl-2,than those in the healthy group(P<0.05).Compared with those treated with 0 or 0.5μg/ml tunicamycin,placental trophoblasts treated with 1 or 2μg/ml tunicamycin showed higher apoptosis rate,higher levels of TNF-ɑ,IL-6 and IL-1β,and upregulated expression of CHOP and GRP78 proteins(P<0.05),with a dose-effect relationship.Compared with controls,placental trophoblasts with ERS had lower glucose uptake,down-regulated protein expression levels of GLUT4,p-PI3K/PI3K,and p-Akt/Akt(P<0.05),and upregulated protein expression level of p-IRS-1(P<0.05).Control cells with insulin stimulation group showed an inverse trend in the changes of above indicators.Insulin stimulation added on ERS-evoked cells seemed to partially reverse the effect of ERS.Conclusion ERS may promote trophoblast cell apoptosis,increase inflammation,reduce cellular glucose uptake,and enhance insulin resistance.