The Mechanism Of Preeclampsia O-GlcNAc Cylation In Iron Overload Induced Abnormal Function And Oxidative Stress Injury In Trophoblast Cells

Background and objective:Preeclampsia（PE）is a common disease in pregnant women,mainly manifested as clinical complications such as hypertension,proteinuria and systemic multi-organ failure after 20 weeks of pregnancy,and its pathogenesis is related to insufficient invasion of trophoblasts,abnormal syncytialization of trophoblasts,oxidative stress and other factors.Iron,as an important trace element in the human body,is involved in oxygen transport and various biological processes.Total iron requirements increase rapidly due to fetal development during pregnancy and maternal volume expansion.Appropriate iron supplementation can improve the iron deficiency status of pregnant women,but excessive iron will promote the production of reactive oxygen species（ROS）,which may lead to increased oxidative stress,inhibit trophoblast cell syncytialization,and prevent the normal development of the placenta.Studies have shown that women with preeclampsia have higher levels of iron than normal pregnant women,suggesting that iron overload is associated with preeclampsia.Ferroptosis is an iron-dependent,lipid peroxidation-mediated form of programmed cell death.Iron overload and lipid peroxidation work together to destroy the lipid bilayer of cell membranes,hinder cell growth and development,and induce ferroptosis in cells.O-Glc NAcylation is a protein post-translational glycosylation modification that attaches a single O-linked-D-Nacetylglucosamine to protein serine or threonine residues resulting from a hexosamine-6-phosphoamidotransferase-mediated hexosamine pathway mediated by glutamine fructose（GFAT）.Added and removed by O-Glc NAc transferase（OGT）and glycosidase O-Glc NAcase（OGA）.Studies have shown a link between O-Glc NAc modification and oxidative stress.The results of the previous laboratory showed that O-Glc NAc modification can promote the establishment of endometrial receptivity,and this study aimed to investigate the role and mechanism of O-Glc NAc modification in iron overload induced functional damage of trophoblasts.Methods:1.Detect Fe²⁺content in normal placental and preeclampsia placental tissue by using Fe²⁺test kit.Detection of Glutathione peroxidase 4（GPX4）,Acyl-Co A synthetase long chain family member 4（ACSL4）,Cytochrome c oxidase subunit II（COX2）,Ferritin heavy chain 1（FTH1）and Transferrin receptor（TFRC）the key molecules of ferroptosis in normal placenta and preeclampsia placental tissues by immunohistochemistry experiments.2.In vitro Ammonium iron（Ⅲ）citrate（FAC）was used to induce iron overload of trophoblasts（HTR-8/SVneo,Be Wo）,endometrial stromal cells（h ESC）,and endometrial cancer cells（Ishikawa）.Fe²⁺detection kit detects the change of Fe²⁺content in cells;Western Blot experiments detected the expression of GPX4,ACSL4,COX2,FTH1 and TFRC proteins,key molecules of ferroptosis.CCK-8 experiments to detect the effect of iron overload on cell viability;Inverted microscope to observe cell morphological changes.3.Detection of O-Glc NAc modification and OGA,OGT and GFAT expression in normal placental and preeclampsia placental tissues through Western Blot and Real-time PCR experiments.4.In vitro FAC was used to induce HTR-8/SVneo and Be Wo iron overload in trophoblast cells,and Western Blot detected the effect of iron overload on O-Glc NAc modification of trophoblast cells and changes in OGA,OGT and GFAT expression levels.5.In vitro FAC was used to induce HTR-8/SVneo iron overload in trophoblasts,and O-Glc NAc modification was upregulated by OGA inhibitor Thiamet G.Compared with iron overload,the CCK-8 experiment,Transwell experiment and Western Blot experiment investigated the effects of elevated O-Glc NAc modification on the survival,migration and invasion ability of HTR-8/SVneo cells,and the effects on the protein expression levels of invasion marker molecules（MMP2,MMP9）and EMT-related molecules（N-cadherin,E-cadherin）were analyzed.6.In vitro using the trophoblast cell syncytialization inducer Forskolin to induce trophoblast cell Be Wo syncytialization,Western Blot detected the syncytialization marker molecules Human chorionic gonadotrophin（HCG）,Syncytin（Syncytin1,Syncytin2）,Chorion-specific transcription factor（GCM1）and trophoblast O-Glc NAc modification,OGA,OGT,and GFAT protein expression;Immunofluorescence detects cell fusion during syncytialization.7.In vitro Forskolin was used to induce syncytialization and FAC to induce iron overload,and O-Glc NAc modification was upregulated by OGA inhibitor Thiamet G and transfected over OGT plasmid,and O-Glc NAc modification was downregulated with OGT inhibitor OSMI-1 and transfected sh OGT plasmid,respectively.Western Blot detected the effects of iron overload and regulation of O-Glc NAc modification on the expression of key molecular proteins in Be Wo cell syncytialization;Immunofluorescence detection of iron overload and regulation of O-Glc NAc modification on cell fusion during Be Wo cell syncytialization.8.In vitro Forskolin was used to induce syncytialization,FAC induced iron overload,ferroptosis inducer Erastin,and O-Glc NAc modification was upregulated by OGA inhibitor Thiamet G and transfected over OGT plasmid.Western Blot detected the effects of iron overload on the expression of key molecular proteins in apoptosis,autophagy and ferroptosis.Western Blot detected the effects of induction of syncytialization,ferroptosis,and elevated O-Glc NAc modification on the expression of key molecular proteins of syncytialization.Transmission electron microscopy observed the effects of iron overload and elevated O-Glc NAc modification on intracellular mitochondrial morphology,in order to explore whether iron overload affects Be Wo cell syncytialization through the ferroptosis pathway.The effects of induction of syncytialization,iron overload,ferroptosis and increased O-Glc NAc modification on the contents of GSH,SOD,MDA,and ROS in HTR-8/SVneo and Be Wo cells were detected by use glutathione（GSH）,anti-superoxide dismutase（SOD）,malondialdehyde（MDA）,and reactive oxygen species（ROS）.Results:1.Compared with normal placental tissue,the content of Fe²⁺in preeclampsia placental tissue is higher.Compared with normal placental tissue,the expression of GPX4,the key molecule of ferroptosis in preeclampsia placental tissue was reduced,and the expression of ACSL4,COX2,FTH1 and TFRC was increased.2.Compared with endometrial stromal cells and endometrial cancer cells,the morphology of cells was significantly changed,cell viability was significantly reduced,and the protein expression level of key molecules of ferroptosis changed significantly during iron overload in trophoblasts.3.Compared with normal placental tissue,the expression of O-Glc NAc modification in preeclampsia placental tissue is reduced.4.Iron overload inhibits the expression of O-Glc NAc modification proteins in trophoblast cells.5.Iron overload inhibits the survival,migration and invasion ability of trophoblast cells,and increases O-Glc NAc modification to reduce the damage of iron overload to the survival,migration and invasion ability of trophoblast cells.6.Induce trophoblast cell Be Wo syncytialization to promote O-Glc NAc modification expression.7.Increase O-Glc NAc modification to reduce the inhibition of iron overload on Be Wo cell syncytialization,and reduction of O-Glc NAc modification promotes the inhibition of iron overload on Be Wo cell syncytialization.8.Iron overload inhibits Be Wo cell syncytialization through the ferroptosis pathway,and increases O-Glc NAc modification to reduce oxidative stress damage of trophoblast cells induced by iron overload.Conclusions:1.Iron overload and ferroptosis are involved in the development of preeclampsia,and O-Glc NAc modification is reduced during the development of preeclampsia.2.Elevated O-Glc NAc modification reduces trophoblast cells damage induced by iron overload.