NETs Stimulate The Onset Of Preeclampsia By Upregulating Placental Inflammatory Response Through High Mobility Group Box 1 Protein

Background and ObjectivesPreeclampsia(PE)is a pregnancy-specific disorder characterized by elevated blood pressure and end-organ dysfunction with or without proteinuria after 20 weeks of gestation.Although PE onset is influenced by various factors,including the placenta,fetus,and mother,the precise mechanisms underlying its pathogenesis remain unclear.Recent studies have suggested that metabolites produced by the placenta of PE patients can promote the aggregation and activation of maternal neutrophils,exacerbate inflammatory responses,impair endothelial cells,and disrupt placental vascular remodeling,while also exerting negative effects on trophoblast cell invasion and migration.Therefore,investigating the excessive inflammatory response induced by neutrophils and its impact on trophoblast cell function represents a crucial aspect in unraveling the pathophysiology of PE.Neutrophil extracellular traps(NETs)are a highly effective neutrophil function that has gained attention from researchers due to its unique ability to trap and eliminate pathogens via the formation of an antimicrobial meshwork,rather than through traditional phagocytosis or degranulation mechanisms.However,excessive production of pathological NETs can elicit direct damage to cells and tissues via the induction of an exaggerated inflammatory response.High mobility group box 1 protein(HMGB1),an essential damage-associated molecule,contributes to the late-stage inflammatory response by binding to various cell surface receptors.As a component of NETs,HMGB1 plays a crucial role in the initiation of several diseases,including acute lung injury,tumors,systemic lupus erythematosus,among others.Nevertheless,whether HMGB1 is involved in preeclampsia remains an unresolved question that warrants further in-depth investigation.Collectively,our hypothesis suggests that NETs may contribute to the damage of trophoblast cells by releasing HMGB1,which subsequently promotes inflammatory responses in the placenta,leading to the release of inflammatory factors and consequent placental dysfunction,thus participating in the pathogenesis of PE.The objective of this study was to assess the expression of NETs and HMGB1 in vivo among patients with preeclampsia,and to explore whether NETs can impair the biological function of trophoblast cells and induce the release of inflammatory factors through in vitro cell experiments.Furthermore,we investigated whether the release of HMGB1 by NETs promotes the release of inflammatory factors from trophoblast cells,by administering the HMGB1 inhibitor glycyrrhizic acid.Our findings provide significant insights into the potential pathogenesis of PE.Methods1.Sample collection:Peripheral blood serum and placenta samples were collected from 20 individuals each with preeclampsia and normal pregnancy.The concentrations of myeloperoxidase(MPO)and neutrophil elastase(NE)in the serum were determined by ELISA,and the concentration of NETs was measured using the Quant-iTTM PicoGreen dsDNA kit.Differences in the transcriptome and protein expression levels of HMGB1 between placentas from the two groups were detected using quantitative real-time PCR(qRT-PCR)and Western blot analysis.Immunofluorescence colocalization was performed to verify that a portion of HMGB1 in the placenta was derived from NETs.2.To induce the release of NETs,human neutrophils were extracted using density gradient centrifugation.The purity of the cells was determined by flow cytometry analysis of cd15b expression and Giemsa staining.In vitro,the generation of NETs was stimulated using phorbol esters,and the concentration was optimized accordingly.3.To investigate the impact of NETs on HTR-8/SVneo cells,the cells were treated with NETs,NETs+DNase I,and a control group.The cell migration and invasion abilities were evaluated using cell scratch assay and Transwell migration and invasion assay.Apoptosis levels in each group were determined using flow cytometric apoptosis analysis.Changes in cellular reactive oxygen species(ROS)levels were measured using a ROS detection kit.4.NETs influence the release of inflammatory factors by inducing HMGB1 release from HTR-8/SVneo cells.To investigate this,HTR-8/SVneo cells were treated with NETs,NETs+Glycyrrhizic acid(An HMGB 1 inhibitor),and a control group.Cell migration and invasion abilities were assessed using cell scratch assay and Transwell migration and invasion assay.Apoptosis levels of each group were examined using flow cytometric apoptosis analysis.Changes in cellular ROS levels were measured using a ROS detection kit.qRT-PCR and western blot analysis were utilized to determine the levels of HMGB1 and the inflammatory cytokine IL-1 β,as well as expression levels of IL-6,IL-8,and TNF-α.Results1.The concentrations of NETs markers in the serum of patients with PE were significantly higher than those in the normal pregnancy group.2.HMGB1 expression in the placentas of patients with preeclampsia was significantly higher than that in the placentas of the normal pregnancy group.Immunofluorescence analysis confirmed the colocalization of MPO-HMGB1 within the placenta,supporting the hypothesis that HMGB1 may have originated from NETs.3.Stimulation of HTR-8/SVneo cells with NETs led to increased apoptosis of HTR-8/SVneo cells,decreased invasion and migration,as well as increased release of ROS.4.NETs impact the biological functions of HTR-8/SVneo cells through the release of HMGB1,promoting the expression of inflammatory factors in these cells.ConclusionIn this study,we observed a significant increase in the release of neutrophil extracellular traps(NETs)and expression level of high-mobility group box 1(HMGB1)in preeclampsia patients compared to normal pregnancy subjects.We confirmed that NETs can affect the biological function of HTR-8/SVneo cells through cellular assays.The addition of glycyrrhizic acid validated that NETs can inhibit HTR-8/SVneo cell invasion and migration,increase ROS release and cell apoptosis by releasing HMGB1.Furthermore,elevated expression of inflammatory factors was observed in HTR-8/SVneo cells,implying their involvement in the occurrence and development of PE.