Study On The Mechanism Of Keap1/Nrf2 Signaling Pathway In Regulatimg Preeclampsia Oxidative Stress

BackgroundsPreeclampsia(PE)is the clinical syndrome in the second trimesterwith incidence rate of 2-9.4%.It is characterized by 20 weeks after pregnancy,high blood pressure and proteinuria.PE is one of the main causes of maternal mortality and morbidity,particularly in developing countries.Little is known about pathological mechanisms leading to the development of PE.However,the development of this syndrome is considered to occur in two stages.The first stage is the trophoblast shallow invasion of the maternal uterine muscle layer,resulting in insufficient remodeling of the uterine spiral artery,leading to placental ischemia and hypoxia environment.This leads to the release of proinflammatory cytokines and angiogenic factors in placenta.The second stage of PEis systemic inflammation which causes the maternal vascular system of endothelial dysfunction and decreases renal function caused by increased blood pressure as the main features,and ultimately lead to maternal hypertension and proteinuria as the main performance of the clinical syndrome.The only effective treatment for reducing maternal symptoms is the delivery of the placenta and fetus.Previous studies have shown that PE patients with placental oxidative stress imbalance and placental local oxidative stress,can induce trophoblastic biological function damage,resulting in increased trophoblastic apoptosis and decreased invasive ability,causing maternal systemic vascular endothelial dysfunction and leukocyte activation.Keap1/Nrf2 signaling pathway plays a protective role in oxidative stress injury of PE,but its mechanism is not clear.Therefore,it is of great clinical significance to explore the mechanism of oxidative stress of PE and to seek effective therapeutic target,to alleviate the symptoms of PE patients,to prolong the time of fetal in the mother and reduce the mortality of PE patients.Oxidative stress(OS)is the body's stress response.Reactive oxygen species(ROS)and Reactive Nitrogen Species(RNS)are generated too much and eliminated too little,resulting in an imbalance of the oxidation/antioxidant system and causing tissue damage.During normal pregnancy the amount of reactive oxygen species increase comparedto non-pregnant state.With the maturation of the placenta and the formation of blood vessels,the placenta is changed into an oxygen-rich environment and the increase of reactive oxygen species stimulates the enhancement of antioxidant systems during pregnancy.The enhanced antioxidant system effectively compensates for the oxidation of OS.Compared to normal pregnancies,PE showed increased OS and reduced antioxidants,characterized by increased circulating and tissue levels of reactive oxygen species(ROS).Previous studies have reported that the Keapl/Nrf2 signaling pathway plays an important role in antioxidant and antiinflammation.In the absence of stress,Nrf2 interacts with its negative regulatory factor Keapl and forms an Nrf2/Keap1 complex.Most of the Nrf2 is catalyzed by Keapl-mediated ubiquitination,so that it is in a state of relative inhibition.However,in the stress statethe conformational change of Keapl or the phosphorylation of Nrf2promoted by a highly active oxide directly leads to dissociation and activation between Nrf2 and Keapl.Then activated Nrf2 is transferred into the nucleus,forming a chain reaction to next step,so itis combined with the antioxidant response element(ARE)to activate a variety of downstream antioxidant genes.Therefore,Keap 1/Nrf2 signal regulation pathway plays an important role in the pathogenesis of PE,and it needs to further study its regulatory mechanism.Purpose1.To confirm the changes of oxidative stress level of different groups and the inhibitory effect of Nrf2 on oxidative stress.By detecting the activities of CAT,GSH-Px and SOD in different groups of HTR8/SVneo cells,the mechanism of Nrf2 on oxidative stress-related active enzymeswas further clarified.2.Based on the previous experimental work,further studies were carried out from the cellular level to clinical level.Using Western Blot,qRT-PCR,SiRNA,ELISA and other related techniques to determine the influence of Keapl/Nrf2 pathway on the biological function of HTR8/SVneo in the oxidative stress model and the oxidative stress level in PE patients.MethodWe performed cell culture,resuscitation and passage of human trophoblast cell line HTR8/SVneo.The levels of HTR8/Svneo and placental tissue oxidative stress were determined by ROS and oxidative stress related enzymes CAT,GSH-Px and SOD activity.And through detecting plasma MMPs to predict the risk of preeclampsia.SiRNA-Nrf2 was designed by RNAi method and use the target gene silencing experiments to clarify the function of the target gene.The difference of transcription level between Keapl/Nrf2 pathway and oxidative stress-related molecules was confirmed by qRT-PCR.The expression of Keap1/Nrf2 pathway and oxidative stress-related molecular proteins were confirmed by Western blot.Using SPSS 21.0 statistical software,the measured data were expressed as Means±Standard Deviation(Means±SD).The comparison between the two groups used t test and the comparison between multiple groups used one-way ANOVA,P<0.05,P<0.01 or P<0.001 for the difference was statistically significant.Result1.The expression of Nrf2 mRNA in Si-Nrf2 group was significantly lower than that in Si-NC group(P<0.001).The expression of Nrf2 protein was detected by Western Blot.The results showed that Si Nrf2 group was significantly lower than Si-NC group,the difference was statistically significant(P<0.001).2.The level of ROS in HTR8/SVneo H/R group was significantly higher than that in non-treated group(p<0.01).The level of ROS in Nrf2 siRNA+H/R group was significantly higher than that in H/R group after Nrf2 gene was knoched down(p<0.01).3.The activity of oxidative stress-related enzymes(CAT,GSH-Px,SOD)in HTR8/SVneo cells was significantly lower in H/R group than that in no-treatedgroup(P<0.01).The activities of CAT,GSH-Px and SOD were significantly decreased in SiRNA + H/R group(P<0.01).4.After the process of hypoxia and reoxygenation the expression of Keapl mRNA in HTR8/SVneo cells was lower than that in normal controls gentlely(P<0.05).The expression of Nrf2 mRNA and HO-1 mRNA was significantly higher than that in non-treated group(P<0.001).5.The expression of Keapl protein in HTR8/SVneo cells after hypoxia-reoxygenation was lower than that in normal controls(P<0.05).The expression of Nrf2 and HO-1 protein in HTR8/SVneo cells was significantly higher after hypoxia-reoxygenation(P<0.001).6.Some clinical indicatorssuch as gestational age,systolic blood pressure,diastolic blood pressure and neonatal weight were statistically significant in normal pregnant women and severe preeclampsia.7.We observed that MMP-2 level of PE participants insecond trimesterwas elevated than that of normal participants,but the difference was found to be statistically insignificant.On the other hand,plasma level of MMP-9 of PE participants was found to be slightly lower,but statistically significant(P<0.05).When we found that the ratio of MMP-2/MMP-9 was markedly higher in PE group participants than those in normal pregnant group,and this difference was also statistically significant(P<0.001).The area under the ROC curve ofMMP-2for predicting PE was merely 0.563,with95%confidence interval(CI):0.497-0.629(P>0.05).On the other hand,in the analysis for plasma level of MMP-9 the area under the ROC curve for predicting PE was 0.587,with 95%CI:0.522-0.652,which was statistically significant(P<0.05).The area under the ROC curve using MMP-2/MMP-9 ratio to predict PE was much higher than either MMP-2 or MMP-9 alone,at 0.841,with 95%Cl:0.797-0.886(P<0.001).8.The activity of CAT(P<0.001),GSH-Px(P<0.01)and SOD(P<0.01)in placental tissue of patients with PE were lower than those of normal participants.9.The expression of Keap1 mRNA in placenta of patients with severe preeclampsia was slightly lower than that of normal placenta(P<0.05).The expression of Nrf2 mRNA and HO-1 mRNA in placenta of severe preeclampsia participants was significantly higher than that in normal participants(P<0.001).10.The expression level of Keapl protein in placenta of patients with severe preeclampsia was slightly lower than that of normal placenta(P<0.05).The expression of Nrf2 protein and HO-1 protein in placenta of severe preeclampsia was significantly higher than that in normal participants(P<0.01).ConclusionIn this study we investigated the activity of reactive oxygen species,CAT,GSH-Px and SOD in HTR8/SVneo cells.When it hasOxidative stress,the level of reactive oxygen species increased and the related enzyme activity decreased.The mechanism of action of Nrf2on oxidative stress-related active enzymes was further clarified.At the mRNA and protein levels,the expression level of Keap1/Nrf2 signaling pathway in hypoxia-reoxygenation was significantly changed.Suggesting that the level of oxidative stress can be suppressed by regulating the Keapl/Nrf2 signaling pathway.The plasma MMPs level can assess predictive significance for preeclampsia.Detection the oxidative stress-related active enzyme of different samplesdue to different clinical status showed there are significant differences between the two groups.Furthermore,the mRNA and protein expression of Keapl,Nrf2,HO-1 of placenta between severe preeclampsia participants and normal participants,suggestedthat the expression level of Keapl/Nrf2 signal pathway in the stage of severe preeclampsia was significantly increased.So regulation of the Keapl/Nrf2 signaling pathway may slow the onset of preeclampsia.