胃饥饿素在妊娠糖尿病患者外周血与脐带血中的表达及其对高糖所致胰岛B细胞凋亡及功能损伤的影响

Expression of ghrelin in peripheral blood and umbilical cord blood of patients with gestational diabetesmellitus and its effect on apoptosis and function damage of islet β cells induced by high glucose

目的分析妊娠糖尿病(GDM)患者外周血及脐带血中的胃饥饿素水平,并初步探讨胃饥饿素对高糖所致胰岛B细胞凋亡及功能损伤的影响及其作用机制。方法(1)选取50例GDM患者(GDM组)与50例正常孕产妇(对照组),检测其外周血及脐带血胃饥饿素水平。分析两组外周血与脐带血胃饥饿素水平的相关性,以及GDM组外周血和脐带血胃饥饿素水平与血糖、胰岛素代谢指标的相关性。(2)将体外培养的胰岛B细胞MIN6分为对照组(无干预)、阴性对照慢病毒载体(LV-NC)组、胃饥饿素过表达慢病毒载体(LV-胃饥饿素)组、高糖组、高糖+LV-NC组、高糖+LV-胃饥饿素组、高糖+磷脂酰肌醇3-激酶(PI3K)抑制剂(LY294002)组进行实验。给予慢病毒载体转染或药物干预后,各高糖干预组细胞采用含20.0 mmol/L葡萄糖的培养基进行培养,其余组采用含5.0 mmol/L葡萄糖的培养基进行正常培养。检测对照组、LV-NC组、LV-胃饥饿素组细胞的胃饥饿素表达水平以了解转染情况。检测对照组、各高糖干预组细胞增殖和细胞凋亡情况(除高糖+LY294002组外)、PI3K/蛋白激酶B(Akt)通路相关蛋白表达水平;除高糖+LY294002组外,检测各组细胞的胰岛素含量。结果(1)GDM组外周血及脐带血中胃饥饿素水平均低于对照组,且两组外周血胃饥饿素水平与脐带血胃饥饿素水平均呈正相关(均P<0.05)。GDM患者外周血及脐带血中的胃饥饿素水平均与其空腹血糖、空腹胰岛素水平及胰岛素抵抗指数呈负相关(均P<0.05)。(2)LV-胃饥饿素组胃饥饿素mRNA及蛋白的表达水平高于其他两组(均P<0.05)。与对照组比较,高糖组培养48 h时的细胞增殖能力、磷酸化Akt/Akt蛋白比值降低,而细胞凋亡率、B细胞淋巴瘤-2相关X蛋白(Bax)和活化型含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved-Caspase-3)蛋白表达水平均升高(均P<0.05);与高糖组、高糖+LV-NC组比较,高糖+LV-胃饥饿素组的细胞增殖能力增高而细胞凋亡率降低;与高糖组、高糖+LV-NC组、高糖+LY294002组相比,高糖+LV-胃饥饿素组的磷酸化Akt/Akt蛋白比值升高,而Bax及Cleaved-Caspase-3蛋白表达水平均降低(均P<0.05)。在低糖或高糖条件下,LV-胃饥饿素组的胰岛素释放能力均较低糖组或高糖组明显增加(P<0.05)。结论GDM患者外周血及脐带血中胃饥饿素水平均降低,且其与血糖、胰岛素代谢密切相关。过表达胃饥饿素能够通过促进PI3K/Akt信号通路的活性来改善高糖所致的胰岛B细胞损伤及功能障碍。

Objective To analyze the levels of ghrelin in peripheral blood and umbilical cord blood of patients with gestational diabetes mellitus(GDM),and to preliminarily explore the effect of ghrelin on isletβcell apoptosis and function damage induced by high glucose as well as its action mechanism.Methods(1)Fifty GDM patients(GDM group)and 50 normal pregnant women(control group)were selected to detect the levels of ghrelin in theiRperipheral blood and umbilical cord blood.An analysis was performed on the correlation between peripheral blood and umbilical cord blood ghrelin levels in both groups,as well as the correlation of peripheral blood and umbilical cord blood ghrelin levels with the indices of blood glucose and insulin metabolism in the GDM group.(2)Isletβcells MIN6 cultured in vitro were divided into control group(without intervention),negative control lentivirus vector(LV-NC)group,ghrelin and overexpressed lentivirus vector(LV-ghrelin)group,high glucose group,high glucose and LV-NC group,high glucose and LV-ghrelin group,and high glucose and phosphoinositide 3-kinase(PI3K)inhibitor(LY294002)group foRthe experiment.AfteRlentivirus vectoRtransfection oRmedicine intervention,cells in the high glucose intervention groups were cultured by using the culture medium containing 20.0 mmol/L glucose,the remaining groups received normal culture using the culture medium containing 5.0 mmol/L glucose.The expression level of ghrelin was detected in the control,LV-NC and LV-ghrelin groups to investigate the condition of transfection.Cell proliferation and cell apoptosis(except the high glucose and LY294002 group),and expression levels of PI3K/protein kinase B(Akt)pathway-related proteins were detected in the control group and all high glucose intervention groups;except the high glucose and LY294002 group,the content of insulin of the cells was detected in all groups.Results(1)The levels of ghrelin in peripheral blood and umbilical cord blood in the GDM group were loweRthan those in the control group,and there was a positive correlation between peripheral blood and umbilical cord blood ghrelin levels(all P<0.05).In GDM patients peripheral blood and umbilical cord blood ghrelin levels negatively correlated with FBG level,fasting insulin level,and insulin resistance index(all P<0.05).(2)The LV-ghrelin group reported higheRexpression levels of ghrelin mRNA and protein than the otheRtwo groups(all P<0.05).Compared with the control group,the high glucose group exhibited decreased cell proliferation capacity and loweRratio of phosphorylated Akt to Akt protein at 48 hours of culture,and yielded increased cell apoptosis rate,higheRexpression levels of B-cell lymphoma-2 associated X protein(Bax)and cleaved cysteinyl aspartate specific proteinase 3(Cleaved-Caspase-3)proteins(all P<0.05);the high glucose and LV-ghrelin group exhibited increased cell proliferation capacity and decreased cell apoptosis rate in contrast with the high glucose group oRthe high glucose and LV-NC group;and the high glucose and LV-ghrelin group yielded elevated ratio of phosphorylated Akt to Akt protein and loweRexpression levels of Bax and Cleaved-Caspase-3 proteins in contrast with the high glucose group,the high glucose and LV-NC group,oRthe high glucose and LY294002 group(all P<0.05).UndeRthe condition of low oRhigh glucose,the LV-ghrelin group yielded a significantly higheRinsulin release capacity than the low and high glucose groups(P<0.05).Conclusion The levels of ghrelin in peripheral blood and umbilical cord blood are decreased in patients with GDM,and closely correlate with blood glucose and insulin metabolism.Overexpression of ghrelin can ameliorate high glucose-induced damage and dysfunction of isletβcells by promoting the activity of PI3K/Akt signaling pathway.