妊娠期糖尿病胎盘滋养细胞铁死亡超微结构及铁死亡相关基因ACSL4、GPX4的表达

Ultrastructure of ferroptosis and expression of ferroptosis⁃related ACSL4 and GPX4 genes in placental trophoblasts of gestational diabetes mellitus

目的:观察妊娠期糖尿病(gestational diabetes mellitus,GDM)孕妇胎盘滋养细胞超微结构,探讨铁死亡在GDM发生发展中的作用。方法:选取2020年9月—2022年12月在常州妇幼保健院分娩的孕妇,孕24~28周行口服葡萄糖耐量试验。以确诊GDM的孕产妇为研究对象,分为通过饮食治疗控制血糖满意组(G1组)、口服二甲双胍控制血糖组(G2组)、胰岛素控制血糖组(G3组)、血糖控制不满意但拒用药组(G4组)各30例,正常健康孕妇为对照组(N组)。通过透射电镜观察各组胎盘滋养细胞超微结构;普鲁士蓝染色检查胎盘铁沉积情况;免疫组化检测胎盘长链脂酰辅酶A合成酶4(acyl-CoA synthetase long chain family member 4,ACSL4)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)在合体滋养细胞层的定位和表达;Western blot和q RT-PCR分别检测胎盘ACSL4、GPX4蛋白和mRNA转录水平。结果:透射电镜下G1、G2、G3、G4组GDM产妇胎盘均可见不同程度的线粒体形态学改变,其中G1、G2、G3组线粒体改变较轻,G4组见典型的铁死亡萎缩线粒体。普鲁士蓝染色显示5组胎盘均见含铁颗粒,G1、G2、G3、G4组胎盘中含铁颗粒较N组明显增多,G1、G2、G3组胎盘中的含铁颗粒较G4组明显减少。Western blot结果显示,与N组相比,G1、G2、G3、G4组产妇胎盘中ACSL4蛋白表达增加而GPX4蛋白表达下降,qRT-PCR结果显示各组间ACSL4、GPX4的mRNA转录水平无显著性差异。结论:GDM孕妇胎盘滋养细胞中存在铁死亡,且与病情严重程度和用药相关。GDM孕妇胎盘中ACSL4、GPX4表达异常,二甲双胍和胰岛素有改善胎盘滋养细胞铁死亡的作用。

Objective:To observe the ultrastructure of placental trophoblast cells of pregnant women with gestational diabetes mellitus(GDM),and to investigate the role of ferroptosis in the occurrence and development of GDM.Methods:Pregnant women who delivered at the Changzhou Maternal and Child Health Hospital from September 2020 to December 2022 were enrolled and underwent an oral glucose tolerance test at 24-28 weeks of gestation.Pregnant women diagnosed with GDM were selected as the research subjects and divided into four groups:diet-controlled glucose satisfaction group(G1 group),oral metformin-controlled glucose group(G2 group),insulin-controlled glucose group(G3 group),and unsatisfactory glucose control but refused to use medication group(G4 group),each with 30 cases.Normal healthy pregnant women were included as the control group(N group).The ultrastructure of placental trophoblast cells in each group was observed by transmission electron microscopy.The placental iron deposition was detected by Prussian blue staining.The localizations and expressions of Acyl-CoA synthetase long chain family member 4(ACSL4)and glutathione peroxidase 4(GPX4)in the syncytiotrophoblast layer were detected by immunohistochemistry.The protein expressions and the mRNA transcription levels of ACSL4 and GPX4 in the placenta were detected by Western blot and qRT-PCR,respectively.Results:Under transmission electron microscopy,varying degrees of mitochondrial morphological changes were observed in the placentas of GDM women in the G1,G2,G3,and G4 groups,with G1,G2,and G3 groups showing milder mitochondrial changes,and the G4 group exhibiting typical ferroptosis with shrunken mitochondria.Prussian blue staining demonstrated iron particles in all five groups,with a significant increase in iron particles in the G1,G2,G3,and G4 groups compared to the N group and a significant decrease in iron particles in the G1,G2 and G3 groups compared to the G4 group.Western blot results showed an increase in ACSL4 protein expression and a decrease in GPX4 protein expression in the placenta of G1,G2,G3,and G4 groups compared to the N group,while qRT-PCR results showed no significant difference in ACSL4 and GPX4 mRNA transcription levels between the groups.Conclusion:Ferroptosis exists in placental trophoblast cells of pregnant women with GDM,and it is correlated with the severity of the disease and medication.Abnormal expression of ACSL4 and GPX4 in the placenta of GDM pregnant women.Metformin and insulin have a role in improving ferroptosis of placental trophoblast cells.