The Role And Mechanism Of Down-regulating The Nrf-2/ARE Pathway Of Trophoblasts Induced By Advanced Oxidation Protein Products In Preeclampsia

BackgroundPreeclampsia(PE),a systemic disease associated with pregnancy,is characterized by maternal hypertension,proteinuria,and edema,which causes maternal and perinatal morbidity and mortality.However,the pathogenesis of preeclampsia has not been elucidated,as well as the methods for preventing preeclampsia are limited.Therefore,seeking an effective prevention method for preeclampsia is the key to improve maternal and perinatal outcomes,which has always been a major problem in the field of Obstetrics.Most scholars now believe that preeclampsia is a disease that originated in the placenta.Oxidative stress promotes the development and progression of preeclampsia.In the first trimester,excessive oxidative stress will lead to trophoblast apoptosis and dysfunction,resulting in abnormal development of the placenta,which further enhances preeclampsia.Advanced oxidation protein products(AOPPs)are the dityrosine-containing and cross-linking protein products formed during oxidative stress.Scholars have found that AOPP is not only a new class of protein markers of oxidative stress but also can induce the generation of ROS and activation of MAPK and NF-?B pathways through the combination with RAGE expressed by a variety of cells.Thus,AOPPs can cause apoptosis,dysfunction,and oxidative damage of cells.Our previous studies have shown a strong correlation between AOPPs and preeclampsia.Nuclear factor E2-related factor 2(Nrf-2)is a key transcription factor that regulates many cytoprotective genes in response to oxidative and electrophilic stress.Evidence suggests that the Nrf-2/ARE pathway is involved in defending the placenta against oxidative stress.Impairment of placental Nrf-2 signaling may be associated with preeclampsia.Heme oxygenase-1 is the antioxidant enzyme regulated by Nrf-2,which can decompose heme into biliverdin,Fe2+,and carbon monoxide.Nrf-2 can be regulated at the transcription level,translation level,post-translational modification,and nuclear translocation.Some proteins(such as p62,p21)and transcription factors(such as NF-?B)are also involved in the regulation of Nrf-2 gene expression,which regulates the ability of cells to resist oxidative stress.Some studies have shown that advanced glycation proteins(AGEs),which are highly similar to AOPPs in structure and physiological effects,can cause cell damage by downregulating the expression of Nrf-2 in cells.Therefore,we wonder whether AOPPs could regulate the antioxidant capacity of cells by regulating the Nrf-2 pathway of trophoblast cells?Is NF-?B involved in the regulation?What is the role of the Nrf-2/ARE pathway in defending the apoptosis induced by AOPPs?In the first two chapters,experimental exploratory researches in vitro at the HTR-8/SVneo cell lines were used to explore the above questions.The effective prevention and treatment of preeclampsia have always been a major problem in the clinical work of Obstetricians.At present,evidence-based medicine confirmed that taking low-dose aspirin in high-risk groups of preeclampsia has a certain role in the prevention of preeclampsia.With the deepening of research,some scholars found that aspirin has an antioxidant effect.Studies have shown that aspirin can relieve oxidative damage via activating the Nrf-2/ARE pathway in melanocytes.In previous studies,we have confirmed that the accumulation of AOPPs can cause trophoblast oxidative damage and dysfunction.However,whether aspirin can reduce the oxidative damage caused by AOPPs in trophoblasts via activating the Nrf-2 pathway is still unknown.We will discuss this problem in the third chapter of this study.Chapter One.AOPPs regulate the Nrf-2/ARE pathway in trophoblasts[Objective]The effects of AOPPs on the expression and regulation of Nrf-2/ARE/HO-1 pathway,NF-?B pathway,and apoptotic proteins in HTR-8/SVneo cells were observed to explore the potential mechanism of AOPP-induced trophoblasts damage.[Method]AOPP-HSA was prepared in vitro according to the methods reported by Witko-Sarsat.HTR-8/SVneo cells were incubated with 200?g/ml AOPP-HSA for 0,3,6,12,24,and 48 h.In another treatment group,HTR-8/SVneo cells were incubated with increasing concentrations(0,50,100,200 ?g/ml)of AOPP-HSA,AOPP-HSA(200?g/ml)+ROS scavenger NAC(10?M),or native HSA alone(200?g/ml)for 24 h.The viability of HTR-8/SVneo cells following exposure to AOPPs was measured by CCK-8.The mRNA expression of Nrf-2 and HO-1 after AOPPs stimulation was analyzed by q-PCR.The protein expression of Nrf-2,HO-1 and apoptotic proteins following exposure to AOPPs was determined by western blot analysis.The transcription of Nrf-2 after AOPPs stimulation was detected by immunofluorescence assay.The transcriptional activity of Nrf-2 after AOPPs stimulation was measured by dual-luciferase reporter gene assay.The effect of AOPPs on potential antioxidant activities in trophoblasts was detected by a total antioxidant capacity kit.The expression of the NF-?B pathway after AOPPs stimulation,and the expression of Nrf-2,HO-1 and apoptotic proteins after blocking NF-?B were measured by western blot analysis.Each cell experiment was repeated at least three times.All experimental data are expressed as mean ± standard deviation(SD)of at least three independent replicates analyzed using SPSS 13.0 software.Multiple samples were compared using One-Way ANOVA,variance pairwise comparisons using LSD test,using Dunnett's T3 test when heterogeneity of variance,P<0.05 was considered statistically significant.[Results](1)AOPPs treatment led to reduced trophoblasts activity in a dose-and time-dependent manner.Co-treatment with NAC(ROS scavenger)or treatment with native HSA elicited no differences in cell viability.(2)The trophoblasts were incubated with increasing concentrations(0,50,100,200 ?g/ml)of AOPP-HSA,AOPP-HSA(200 ?g/ml)+NAC,or native HSA alone for 24 h.The mRNA expression of Nrf-2 and HO-1,as well as the protein expression of total and nuclear Nrf-2 and HO-1,decreased when the AOPPs concentration exceeded 100 ?g/ml,whereas this decrease was not observed in the groups co-treated with NAC or treated with native HSA alone.(3)Following exposure to 200 ?g/ml AOPP-HSA for 0,3,6,12,24,or 48 h,mRNA expression levels of Nrf-2 and HO-1 in HTR-8/SVneo cells were increased from 0-6 h and 0-12 h,respectively,after which the expression then gradually declined,as evidenced by qPCR.Western blot analysis revealed that at 3-6 h,the protein expression of total and nuclear Nrf-2 was significantly higher than those at 0 h but decreased during the next 12-48 h.After 3-12 h of AOPPs treatment,HO-1 protein expression was significantly higher than that at 0 h but decreased in the next 24-48 h.(4)AOPPs reduced Nrf-2 nuclear transcription and transcription activity.(5)AOPPs treatment led to cell apoptosis in a dose-and time-dependent manner.Co-treatment with ROS scavenger NAC or treatment with native HSA did not affect cell apoptosis.(6)AOPPs promoted the activation of NF-?B in trophoblasts,resulting in a decreased Nrf-2 nucleation.[Conclusion](1)The expression of Nrf-2 and Nrf-2-dependent gene product HO-1 decreased following long-term exposure to high doses of AOPPs in trophoblasts.(2)AOPPs promote the activation of NF-?B in trophoblasts in a dose-and time-dependent manner,which may be involved in the regulation of Nrf-2 expression.(3)AOPPs trigger apoptosis in trophoblasts in vitro by activating a p53-induced caspase cascade.Chapter Two.The role of Nrf-2 on protecting trophoblasts from AOPP-induced oxidative damage[Objective]Targeted knockdown and overexpression techniques were used to regulate the expression of the Nrf-2 gene in trophoblasts,and to explore the role of Nrf-2 in AOPP-induced oxidative damage of trophoblasts.[Method]AOPP-HSA was prepared in vitro according to the methods reported by Witko-Sarsat.HTR-8/SVneo cells were transfected with Nrf-2-specific siRNA or Nrf-2 overexpression plasmid(GV141-Nrf-2)before exposure to AOPP-HSA for 24 h.siRNA interference technology were used to targeted silence Nrf-2 expression in trophoblast cells.Over-expression plasmids were used to targeted over-express Nrf-2 expression in trophoblasts.The viability of HTR-8/SVneo cells following treatment was measured by CCK-8.The pro-apoptotic effect of AOPPs on HTR-8/SVneo cells was assessed by staining cells with Hoechst33342.The apoptotic rate of each treatment group was determined by Annexin V-FITC/PI staining and analyzed by flow cytometry.The protein expression of p53,cleaved caspase-9,cleaved caspase-3,and cleaved PARP following exposure to AOPPs was determined by western blot analysis.The expression of HO-1 protein after regulating Nrf-2 and the effect of HO-1 on protecting trophoblasts were also determined by western blot analysis.The statistical method is the same as Chapter One.[Results](1)The Nrf-2 gene knockdown and overexpression cell model were successfully constructed.(2)Nrf-2 knockdown aggravated AOPP-induced cell apoptosis in vitro by triggering p53 and caspase activation,in turn,Nrf-2 overexpression attenuated AOPP-induced cell apoptosis in vitro by inhibiting p53 and caspase activation.(3)Nrf-2 knockdown reduces the expression of HO-1 protein in trophoblast cells,in turn,Nrf-2 overexpression increase the expression of HO-1 protein.(4)AOPP-induced cell apoptosis was markedly enhanced by pretreatment with the HO-1 inhibitor ZnPP,but was suppressed by the HO-1 inducer CoPP.[Conclusion]These data indicated that Nrf-2 played an important role in AOPP-triggered cell damage.Nrf-2 deficiency promotes AOPP-triggered apoptosis in trophoblasts.Nrf-2 can provide protection to trophoblasts against AOPP-mediated toxicity by increasing HO-1.Chapter Three.Aspirin inhibits AOPP-induced oxidative damage in trophoblasts via inducing HO-1 expression through activating the Nrf-2 pathway[Objective]To investigate the role and mechanism of aspirin on reducing AOPP-induced damage of trophoblasts.[Method]AOPP-HSA was prepared according to the methods reported by Witko-Sarsat.The effects of different concentrations of aspirin on the trophoblasts activity were detected by CCK-8.The expression of Nrf-2,HO-1,and apoptotic proteins in the trophoblasts was detected by Western blot.CCK-8 and LDH experiments were used to detect cell activity.The statistical method is the same as Chapter One.[Results](1)Aspirin within the experimental concentration had no effect on the activity of trophoblasts.(2)Aspirin reduced the cell apoptosis and damage caused by AOPPs,which can be partially blocked by the siNrf-2 and the HO-1 inhibitor.[Conclusion]Aspirin effectively relieves the oxidative damage and apoptosis of trophoblasts induced by AOPPs via activation of the Nrf-2/ARE/HO-l pathway.It may be one of the mechanisms involved in the preventive effect of low-dose aspirin on preeclampsia.