Phosphatidylinositol 3-Kinase/Akt Signal Pathway Resists The Apoptosis And Inflammation In Human Extravillous Trophoblasts Induced By Porphyromonas Gingivalis

Preterm birth(PTB),defined as birth before 37 gestational weeks and accompanied by low-birth-weight(PTLBW)defined as a birth weight<2,500 g according to the World Health Organization(WHO),is the most common reason of perinatal and infant mortality,which is a heavy burden to the society and family.Among many factors,bacterial infection either ascending from the urogenital tract or occurring transplacentally after bacteremia is a major cause of preterm deliveries.Epidemiological studies suggested that periodontal disease,as a series of common chronic infectious oral disease,is a risk factor for pregnancy complications including PTB.Increasing evidence indicates that Porphyromonas gingivalis,a vital periodontal pathogen found in amniotic fluid and intact membranes of women who deliver PTLBW infants,is thought to contribute to preterm labor.How periodontal bacteria induces PTLBW is not clear and may involve apoptosis at the matemal-fetal interface,leading to subsequent uterine contraction and PTLBW.Alternatively,periodontal pathogens may unbalance the inflammatory response with systemic consequences that trigger preterm labor.In this study,we planned to co-culture P.gingivalis and extravillous trophoblasts(HTR-8 cells)to understand whether P.gingivalis infection could contribute to PTLBW by inducing apoptosis and inflammation in EVT,and studied the potential pathway involved.Objective:The study was designed to study the effect of P.gingivalis on human extravillous trophoblast cell line and the role of p-Akt in this process.Methods:HTR-8 cells were infected with P.gingivalis ATCC 33277 at different multiplicity of infections(MOIs).We chose LY294002 to inactivate PI3K,inverted microscope and confocal microscope to record morphological changes of HTR-8 cells,flow cytometry to detect the apoptosis of HTR-8 cells after infection with P.gingivalis,Western Blot to test the amount of target protein,ELISA to detect the amount of target cytokine.Results:When HTR-8 cells were infected with P.gingivalis,a significant increase in the percentage of apoptotic cells was observed following 24 h and 48 h of P.gingivalis infection in a time and dose dependent manner compared with uninfected cells.A dose-dependent increase of IL-8 and production IFN-γ was induced in HTR-8 cells following 6 and 12 h of P.gingivalis stimulation and significant differences were observed at MOIs as indicated in the figure compared with non-stimulated controls.When HTR-8 cells were pretreated with LY294002 and then co-cultured with P.gingivalis(LY 200 Group),the apoptosis rate of LY 200 Group was greater-than 35%,which was significantly higher than that of MOI200 Group(P<0.001);and there was no statistical difference of apoptosis rate between MOI200 Group and D 200 Group(D 200 group denoted cells which were pre-treated with DMSO)(P>0.05).Pre-treatment of the cells with LY294002 for 2 h resulted in a significant increase in the of IL-8 and IFN-y after P.gingivalis stimulation at the different time points.Conclusion:Porphyromonas gingivalis induced apoptosis and inflammation in human extravillous trophoblast cells and p-Akt was activated in this process.In addition,when Akt activation was inhibited,apoptosis and inflammation was significantly increased.Thus,P.gingivalis infection contributes to PTLBW by triggering excessive inflammation and increasing apoptosis in trophoblasts and that the Phosphatidylinositol 3-Kinase/Akt(PI3K/Akt)signaling pathway is involved in the regulation of Pg-induced apoptosis and inflammation.