Mechanism Of Neutrophil Extracellular Traps Generation And Their Role In Trophoblasts Apoptosis In Gestational Diabetes Mellitus

Research Background and Purpose:Gestational diabetes mellitus(GDM)is a metabolic syndrome occurring in pregnant women and increases the risk of placental dysplasia.GDM patients often have hypoadiponectinemia and high levels of Neutrophils Extracellular Traps(NETs).adiponectin(APN)negatively regulates neutrophil function and suppresses neutrophil activation,phagocytosis,apoptosis,and NADPH oxidase.Activated neutrophils release NETs with a large number of ROS produced by the activation of NADPH oxidase,a process called NETosis.NETs are extracellular fibrous structures composed of chromatin and granular proteins,myeloperoxidase(MPO),Neutrophil Elastase(NE)and citH3.Althoμgh it has been proved that high glucose(HG)can induce NETosis,the reasons for the high level of NETs in GDM patients are complicated.Based on the above studies,we speculated that it may also be related to hypoadiponectinemia.Trophoblast apoptosis was significantly increased in GDM patients,and inhibition of trophoblast apoptosis could significantly reduce the incidence of abortion and preeclampsia.NETs are associated with placental dysplasia,but whether NETs promote trophoblast apoptosis is unclear.This study aims to reveal the mechanism of NETs generation and their role in trophoblast apoptosis in GDM.Materials and Methods:1.The present study was conducted on 20 pregnant women with GDM and 20 healthy control pregnant women(HC).The patients were recruited to the study cohort following GDM diagnosis based on an oral glucose tolerance test(OGTT)at 24-29 weeks of gestation at the Maternal and Child Health Care Hospital of Shandong Provincial;none of the study participants were receiving any other treatments.All participants in this study provided written informed consent.2.Quant-ItTMPicogreen dsDNA Test Kit were used for quantitative detection cell-free DNA in serum of GDM and HC.The concentrations of APN,NE,MPO and MPO-DNA complex in serum were analyzed by ELISA,and the levels of APN and NETs markers were compared between the two groups.3.Quantitative determination of cf-DNA in vitro,determination of NET%by live cell fluorescence staining,co-locating DNA/MPO/NE by cellular immunofluorescence,ELISA and Western blot were used to detect the level of NETs marker to evaluate human recombinant adiponectin(HAP)regulates the formation of NETs under HG condition.4.Reactive Oxygen Species Assay Kit was used to detect ROS,which revealed that HAP regulates the formation of NETs throμgh NADPH oxidase/ROS pathway.5.Western blot was used to explore the role of ERK1/2 and p38MAPK signaling pathways in the formation of HAP regulated NETs.6.Extraction of NETs:Neutrophils were induced to produce NETs by HG in vitro,and the NETs were extracted and the concentration was detected.7.After adding NETs into trophoblast cells pretreated with or without ROS scaver NAC for 24 h,The apoptotic effects of NETs on trophoblast cells and related mechanisms were evaluated by CCK-8 analysis of trophoblast cell viability,flow cytometry detection of cell apoptosis rate,western blotting evaluation of apoptotic protein expression related to mitochondrial pathway,and ROS quantification.Results:1.GDM patients exhibit lower APN levels and high NETs levels.Compared with healthy pregnant women of the same gestational age,the serum APN levels of GDM patients were decreased,and the levels of NETs markers:cf-DNA,MPO,NE,and MPO-DNA complexes were increased.2.NETs release is increased in patients with GDM.cf-DNA quantification and live cell fluorescence revealed a higher proportion of neutrophils releasing NETs in GDM patients compared with HC.3.HG induces neutrophils to produce NETs throμgh NADPH oxidase/ROS pathway.HG can activate neutrophils to generate a large number of ROS and NETs,and the level is similar to that of positive control PMA.After pretreatment of neutrophils with DPI(NADPH oxidase inhibitor),the production of HG induced ROS and NETs is significantly reduced.4.HAP blocks neutrophils from producing NETs by inhibiting NADPH oxidase/ROS pathway.After HAP pretreatment,ROS and NETs production of neutrophils decreased significantly under HG condition.5.HAP blocks HG-induced NETs formation by inhibiting ERK1/2 and p38MAPK signaling pathways.Western blot analysis showed that HG promoted the phosphorylation of neutrophil ERK1/2 and p38MAPK.HAP pretreatment of neutrophils can inhibit the expression of p-ERK 1/2 and p-p38MAPK.6.NETs promote trophoblast apoptosis throμgh ROS/mitochondrial pathway.NETs can promote HTR8/SVneo cells to produce a large number of ROS,damage cell viability,increase apoptosis rate,and up-regulate the expression of pro-apoptotic proteins in the mitochondrial pathway.NAC can significantly reduce ROS production,repair cell viability,inhibit apoptosis rate,and downregulate the expression of pro-apoptotic proteins.7.DNase I can antagonize the effect of NETs on promoting trophoblast apoptosis.8.NETs promote trophoblast apoptosis by inhibiting ERK1/2 signaling pathway.Western blot analysis showed that p-ERK1/2 expression in HTR8/SVneo cells was significantly decreased after NETs treatment.Conclusions:Hypoadiponectinemia in HG is responsible for the high level of NETs in GDM patients.NETs can induce oxidative stress and promote apoptosis of trophoblasts,and affect placental development,resulting in adverse pregnancy outcomes.Therefore,upregulated adiponectin levels may block the formation of NETs,which is expected to be an effective treatment for GDM.