The Interaction Between Hydrogen Peroxide And Human Leukocyte Antigen G Expressed By Trophoblasts In Maternal Placenta And Serum During Preeclampsia

Objective: Preeclampsia （PE） is a major contributor to maternal and neonatalmortality and morbidity and the precise etiology of PE is still open to debate. Inparticular, oxidative stress and immune maladaptation seem to attract people’sattention in the development of PE. The aim of this study was to investigate apossible association between Hydrogen peroxide （H₂O₂） and leukocyte antigen-G（HLA–G） expressed by trophoblasts in maternal placenta and serum duringpreeclampsia.Methods:1.（1）Sixty pregnant women, delivered through cesarean section in theDepartment of Obstetrics of and Gynecology, the First Affiliated Hospital ofNanjing Medical University from October2008to October2009, wereenrolled, including30women with preeclampsia and30healthy gravidas（control group）. Colorimetry was applied to determine the level of H₂O₂ofplacental tissues. Immunohistochemistry and western blot were used todetermine the relative expression of HLA-G protein in placental tissues. Thecorrelation between H₂O₂level and HLA-G protein expression was analyzed.（2）Immunofluorese and western blot were used to investigate the expressionof HLA-G protein in HTR-8/SVneo cells after incubation with H₂O₂for0h、 24h、48h.2.（1）Blood samplings of15women diagnosed early-onset preeclampsia,15women diagnosed later-onset preeclampsia and15healthy women （controlgroup） at term in the Department of Obstetrics of and Gynecology, the FirstAffiliated Hospital of Nanjing Medical University from June2010toDecember2010were collected. Colorimetry and ELISA were applied,respectively, to determine the level of H₂O₂and the expression of sHLA-Gprotein in maternal serum and the correlation between them were analyzed.（2）ELISA was used to investigate the expression of sHLA-G protein inHTR-8/SVneo cell supernatant after incubation with H₂O₂for0h、24h、48h.3.（1）HTR-8/SVneo cells were transfected with HLA-G siRNA and controlsiRNA oligofectamine and the effect was analyzed by RT-PCR and Westernblot.（2） Effects of HLA-G on the proliferation, apoptosis and invasion ofHTR-8/SVneo cells when exposed to H₂O₂were evaluated via MTT assay,Flow cytometric mothod, Transwell assay and Gelatin enzyme, respectively.Results:1. Compared with control group, higher level of H₂O₂and lower relativeexpression of HLA-G protein were found in preeclamptic placentas and therewas an inverse correlation between HLA-G and H₂O₂levels in the twogroups.（2） Compared to the relative expression of HLA-G protein in thecontrol group, the relative expression of HLA-G in HTR-8/SVneo cells afterexposure to H₂O₂was reduced with time-dependent. Reduced expression ofHLA-G was also observed in HTR-8/SVneo cells by fluorescence microscopein the intervention group compared to the control group.2. Compared with control goup, the concentration of H₂O₂was higher inearly-onset preeclampsia and later-onset preeclampsia serum. The concentration of serum H₂O₂was higher in early-onset preeclampsia thanlater-onset preeclampsia. Compared with control goup, the expression ofsHLA-G was decreased in early-onset preeclampsia and later-onsetpreeclampsia serum. The expression of serum sHLA-G was decreased inearly-onset preeclampsia than later-onset preeclampsia. There was an inversecorrelation between HLA-G and H₂O₂at term in the maternal serum.（2）Compared with control group, the expression of sHLA-G in the supernatantof cells exposed to H₂O₂was reduced with time-dependent.3. Compared with HLA-G knockdown HTR-8/SVneo, increased proliferationinhibition, higher apoptosis and decreased cell invasion were found in the cellexpressing HLA-G when exposed to H₂O₂.Conclusion：Our findings highlight that high levels of H₂O₂can down-regulateHLA-G protein （including sHLA-G） expression in trophoblasts during preeclampsiaand trophoblasts expressing HLA-G are vulnerable to oxidative stress.