The Study On Relationship Between Nrf2 And NQO1 In Preeclampsia And Effect Of NQO1 On HTR-8/SVneo Cell Invasion

Objective To explore the relationship between the changes of Nuclear factor erythroid2-related factor 2?Nrf2?and NAD?P?H quinone oxidoreductase 1?NQO1?protein in preeclampsia?PE?by detecting the levels of them in plasma of patients with preeclampsia and normal pregnancy.Simultaneously,the HTR-8/SVneo cell oxidative stress system model was established to further explore whether Nrf2 regulates NQO1 in the pathogenesis of PE and whether the change of NQO1protein expression affects the invasion ability of HTR-8/SVneo cells under oxidative stress.To provide clues for the pathogenesis and prevention of PE.Methods1.The relationship betwen Nrf2 and NQO1 in plasma of preeclampsia patients?1?We collected medical record information of 1471 pregnant women?264 patients with severe PE,397 patients with mild PE and 810 healthy pregnant women?who gave birth in the obstetrics department of the Third People's Hospital of Dongguan from January 1,2014 to December 31,2016 and analyzed complications during pregnancy of three groups.?2?Plasma samples from 30 patients with severe PE and 30 healthy pregnant women were collected on the day before cesarean section.The activity of superoxide dismutase?SOD?,the contents of malondialdehyde?MDA?and glutathion?GSH?in plasma were determined by spectrophotometry.The levels of Nrf2 and NQO1 protein in plasma were determined by ELISA,and the correlation between them was analyzed by Pearson correlation.2.The effect of NQO1 on HTR-8/SVneo cell invasion.?1?Pretreatment of HTR-8/SVneo cells with Nrf2 inducer?tBHQ?,Nrf2 inhibitor?ML385?,NQO1 inducer?2-HBA?and NQO1 inhibitor?Dicoumarol?for 30 min and subsequently,250?mol/L H₂O₂ or blank treatment for 4 h.?2?Analysis of cell viability in HTR-8/SVneo cell oxidative stress model was carried out by MTT assay.?3?The ROS expression level in the oxidative stress model of HTR-8/SVneo cells was detected by chemiluminescence method.?4?The expression levels of Nrf2 and NQO1 protein and NQO1 mRNA in each group of HTR-8/SVneo cells were detected by Western Blot and qRT-PCR respectively.?5?Immunofluorescence was used to localize the expressions of Nrf2 and NQO1 in HTR-8/SVneo cells.?6?Transwell invasion assay was used to detect the number of matrigel cells in each group of the oxidative stress model of HTR-8/SVneo cells.Results1.The relationship betwen Nrf2 and NQO1 in plasma of preeclampsia patients?1?There was a significant difference in the incidence of gestational complications among the control group,the mild PE group and the severe PE group?P<0.05?.The incidence of hypoproteinemia,placental abruption and premature rupture of membranes were higher in the mild PE group than that in the control group?P<0.05?.Compared with the control group,the incidence of hypoproteinemia,ascites,HELLP syndrome,placental abruption,disseminated intravascular coagulation?DIC?,postpartum hemorrhage and premature rupture of membranes were higher in the severe PE group?P<0.05?.The incidence of hypoproteinemia,ascites,HELLP syndrome,placental abruption,DIC and premature rupture of membranes in the severe PE group was higher than that in the mild PE group?P<0.05?.The incidence of complications in the severe PE group from high to low was:premature rupture of membranes,hypoproteinemia,HELLP syndrome,placental abruption,DIC,postpartum hemorrhage,ascites and heart failure.The incidence of complications in the mild PE group from high to low was:premature rupture of membranes,hypoproteinemia,placental abruption,postpartum hemorrhage and DIC.?2?The content of GSH in the severe PE group was lower than that in the control group?P<0.05?.The activity of SOD and the content of MDA in the severe PE group were higher than those in the control group?P<0.05?.?3?Compared with the control group,the expressions of Nrf2 and NQO1in the plasma of severe PE group were decreased?P<0.05?and the expression of Nrf2 in plasma was positively correlated with the expression of NQO1?r=0.917,P<0.05?.2.The effect of NQO1 on HTR-8/SVneo cell invasion.?1?The cell survival rate of H₂O₂ group was 84.37%.?2?The mean fluorescence intensity?MFI?of H₂O₂ group was significantly higher than that in the control group?P<0.05?.?3?Compared with the control group,the expression levels of Nrf2 and NQO1 protein and NQO1mRNA in the cells were as follows,H₂O₂ group was increase?P<0.05?,tBHQ group was increase?P<0.05?,no significant change in ML385 group?P>0.05?.Compared with the H₂O₂group,Nrf2 and NQO1 protein and NQO1 mRNA expression were decreased in H₂O₂+ML385group?P<0.05?.?4?The expression positions of Nrf2 and NQO1 in cells were as follows,Nrf2was mainly expressed in the nucleus,while NQO1 was expressed in the cytoplasm of H₂O₂ group and tBHQ group.Nrf2 and NQO1 were less expressed and mainly distributed in the cytoplasm of ML385 group and H₂O₂+ML385 group.?5?Compared with the H₂O₂ group,the NQO1 protein expression level in the cells were changed as follows,2-HBA group was increased?P<0.05?,while Dicoumarol group was decreased?P<0.05?.?6?The number of cells in the H₂O₂ group that penetrated Matrigel were increased compared with the control group?P<0.05?,the number of cells in the H₂O₂+2-HBA group penetrated Matrigel were increased compared with the H₂O₂group?P<0.05?.Compared with the H₂O₂ group,the number of cells in the H₂O₂+Dicoumarol group penetrated Matrigel were decreased?P<0.05?.Conclusions?1?The incidence of various complications during pregnancy in patients with severe PE was increased.?2?Low expressions of Nrf2 and NQO1 in plasma of patients with severe PE weaken the body's antioxidant capacity.?3?Under oxidative stress,Nrf2 activated NQO1 in HTR-8/SVneo cells,which in turn increases cell invasiveness.