Effect of oxidative stress on human Wiskott-Aldrich syndrome related protein 2 expression in placental trophoblasts in preeclampsia / 中华围产医学杂志

ABSTRACT

Objective To explore the effect of oxidative stress on human Wiskott-Aldrich syndrome related protein 2 (WAVE2) expression in placental trophoblasts in women with preeclampsia.Methods (1) Twenty women with preeclampsia and twenty-three normal term pregnant women,delivered from August 15,2011 to February 23,2012 in the First Affiliated Hospital of Chongqing Medical University,were recruited and divided into preeclampsia group and control group.Placenta samples were collected after cesarean section.The localization and distribution of WAVE2 in placenta was studied by immunohistochemistry.Quantitative real-time polymerase chain reaction and Western blot were employed to assay the WAVE2 mRNA and protein levels.Tissue homogenates was applied to determine the levels of reactive oxygen species (ROS).The correlation between ROS levels and WAVE2 was also analyzed.(2) An in vitro hypoxia/reoxygenation (H/R) model was utilized to simulate ischemia/reperfusion injury to placental trophoblasts.The HTR-8/ SVneo cells (immortalized human first trimester extravillous trophoblast cells) were pre-incubated overnight,after exposure to H/R or normoxic conditions for 48 hours.Flow cytometry was employed to analyze intracellular ROS level.Meanwhile,Transwell assay was utilized to analyze the invasion and migration of HTR-8/SVneo cells.The location and expression of WAVE2 in trophoblasts was evaluated by cell immunofluorescence and Western blot.Statistical differences between the two groups were evaluated by independent t-tests.Pearson's correlation coefficient test was used for correlation analysis.Results (1)Compared with the control group,preeclampsia group had significantly higher 24-hour proteinuria [(1.96±0.24) g vs (0.08±0.05) g,t=19.436,P＜0.05],systolic blood pressure [(154 ± 13) mm Hg vs (98 ±11) mm Hg,t=11.324,P＜0.05] and diastolic blood pressure [(105±14) mm Hgvs (69±8) mm Hg,t=9.612,P＜0.05].In addition,the placental weight and birth weight of infants in preeclampsia group were significantly reduced as compared to the control group [(432±53) g vs (536±67) g,(2446± 187) g vs (3207± 233) g,t=14.562 and 16.307,allP＜0.05)].The WAVE2 mRNA level (0.28±0.07 vs 1.01±0.02,t=12.747,P＜0.05) and the WAVE2 protein levels (0.63±0.08 vs 1.34±0.19,t=11.648,P＜0.05) were also significantly decreased in preeclampsia groups.The level of ROS in placenta in the preeclampsia group was significantly higher than in control group [(144.22 ± 12.32) nmol/(mg · prot) vs (75.17 ± 8.71) nmol/(mg · prot),t=20.467,P＜0.05].There was significant negative correlation between ROS level and WAVE2 protein expression in preeclamptic placenta (r =-0.726,P =0.000).(2) In vitro study showed that,the levels of ROS in normoxia group and H/R group was (82.9±5.8)％ and (155.6±8.1)％,(t=12.747,P＜0.05).Compared with normoxia condition,decreased cell invasion and migration were found in HTR-8/SVneo cells in H/R group [(51.9 ± 3.3)％ and (58.4 ±4.2)％ respectively,t=11.034 and 13.839,P＜0.05].Results from the cell immunofluorescence showed that WAVE2 protein located in the cytoplasm of HTR-8/SVneo cells,and the expression of WAVE2 protein was significant decreased in HTR-8/SVneo cells after exposure to H/R for 48 h (0.37±0.05 vs 0.76±0.06,t=8.631,P＜0.05).Conclusions Excessive oxidative stress in preeclamptic placentas was correlated with the decreased expression of WAVE2.H/R-induced oxidative stress could decrease WAVE2 expression,which may contribute to impaired trophoblast invasion and migration in preeclampsia.