妊娠糖尿病抑制胎盘AKT-mTOR及SIRT1信号通路

Gestational diabetes mellitus impairs AKT-mTOR and SIRT1 signaling pathways in placenta

目的:探讨AKT-mTOR信号通路与SIRT1在妊娠期糖尿病(gestational diabetes mellitus,GDM)编程胎儿生长发育中的作用。方法:收集重庆医科大学附属第一医院2014年12月至2015年6月分娩的15例GDM和15例正常产妇的胎盘组织,应用Western blot检测胎盘中AKT-mTOR磷酸化水平与SIRT1的表达水平。以人绒毛外滋养细胞(HTR8/SVneo)体外培养,分为空白对照组、渗透对照组和高糖组。各组处理后使用Western blot检测AKT-mTOR总蛋白和磷酸化水平,以及SIRT1的表达水平,应用流式细胞术(flow cytometry)检测各组的凋亡率。db/+杂合雌鼠妊娠至18.5 d处死后,取其胎盘组织,对其进行基因型鉴定后,选取野生型子代胎盘为GDM组,C57雌鼠胎盘组织为正常对照组,每组各6只。然后应用Western blot检测胎盘组织中AKT-mTOR信号通路。结果:AKT及其下游mTOR磷酸化水平在GDM胎盘中的表达明显低于正常胎盘(0.347±0.031 vs.1.000±0.175,P=0.004;0.465±0.045 vs.1.000±0.098,P=0.000)。同样,GDM组的SIRT1的表达水平也明显低于正常组(0.682±0.055 vs.1.000±0.127,P=0.044);在细胞模型中,经高糖处理后,AKT-mTOR磷酸化水平明显降低(0.512±0.056 vs.1.103±0.111,P=0.023;0.262±0.091 vs.1.153±0.057,P=0.001),而SIRT1的表达同样明显降低(0.472±0.034 vs.1.013±0.098,P=0.040)。高糖组的细胞凋亡率明显升高(14.550±1.624 vs.9.547±0.685,P=0.032)。在动物模型中,GDM组的AKT-mTOR磷酸化水平明显降低(0.527±0.080 vs 1.000±0.055,P=0.003;0.418±0.059 vs.1.000±0.084,P=0.001)。结论:宫内高血糖环境可能通过抑制胎盘AKT-mTOR信号通路,从而编程子代的发育轨迹。

Objective：To explore the role of AKT-mTOR signaling pathway and SIRT1 in fetal growth and development programming of gestational diabetes mellitus（GDM）. Methods：Fifteen cases of GDM and 15 cases of normal maternal placental tissue were collected in the First Affiliated Hospital of Chongqing Medical University from December 2014 to June 2015. Western blot was used to detect the expression of AKT-mTOR and SIRT1 in human placenta. Human transformed primary extravillous trophoblast cell line（HTR8/SVneo）cells were used to establish the model of GDM,which were divided into normal group,osmotic control,and high glucose group.After the treatment,the expressions of AKT-mTOR and SIRT1 were detected by Western blot;Flow cytometry was used to detect the apoptosis rate in each group. On day 18.5 of gestation,db/＋ mice were anesthetized with pentobarbitone and a caesarean section was performed. The placentas were excised and frozen immediately for genotyping,and then the wild type placentas were selected for GDM group. C57 mice placentas were control group（normal）. Then the expressions of AKT-mTOR were detected by Western blot.Results：The phosphorylation of AKT and mTOR in placenta of GDM group was significantly lower than that of normal group（0.347±0.031 vs. 1.000±0.175,P=0.004;0.465±0.045 vs.1.000±0.098,P=0.000）. Similarly,the expression of SIRT1 was obviously decreased in GDM group than in normal group（0.682±0.055 vs. 1.000±0.127,P=0.044）. In the cell model,high glucose could decrease AKT and mTOR phosphorylation（0.512±0.056 vs. 1.103±0.111,P=0.023;0.262±0.091 vs. 1.153±0.057,P=0.001）and SIRT1 expression（0.472±0.034 vs. 1.013±0.098,P=0.040）. The apoptosis rate in high glucose group was significantly increased than that in normal group（14.550±1.624 vs.9.547±0.685,P=0.032）. In the animal model,the phosphorylation of AKT and mTOR also was significantly decreased in GDM group（0.527±0.080 vs. 1.000±0.055,P=0.003;0.418±0.059 vs. 1.000±0.084,P=0.001）. Conclusion：The intrauterine environment of high glucose may inhibit placental AKT-mTOR signaling pathways,thereby programming offspring development track.