The Expression Of Atrial Natriurectic Petide And Natriurectic Peptide Receptor A In Human Deciduas And Placenta And Its Relationship With The Development Of Preeclampsia

Background:Pre-eclampsia is characterized by new-onset hypertension and poteinuria at>20 weeks of gestation.In the absence of proteinuria,diagnosis requires the presence of hypertension together with evidence of systemic disease(such as thrombocytopenia,elevated levels of liver transaminases,renal insufficiency,pulmonary oedema and visual or cerebral disturbances).This gestation-specific syndrome affects 3-5%of all pregnancies,and is a leading cause of maternal and perinatal morbidity and mortality.In view of the high incidence,high fatality rate of preeclampsia,to explore pathogenesis and treatment of preeclampsia,looking for effective therapeutic targets,become a hot and difficult problem in the field of obstetrics and gynecology.Most scholars now believe that the incidence of preeclampsia,the causes and course of development may be related to several factors and theories related to:immune imbalance mechanism,shallow placental implantation,vascular endothelial cell injury and ischemic placental trophoblastic cells,insulin resistance,calcium balance disorders,genetic factors,nutritional deficiencies and so on.Atrial natriuretic peptide(ANP)is a hormone,produced mainly by cardiomyocytes,with a major role in cardiovascular homeostatic mechanisms such as natriuresis and vasodilation,which serve to regulate blood pressure.However,ANP also acts as an autocrine/paracrine factor on other targets such as kidney,lung,thymus,liver and the immune system.ANP participates in the regulation of cell growth and proliferation.Recently,Cui et al.found that ANP plays an important role in regulate trophoblast cell invasion and uterine spiral artery remodling and speculated that the abnormal function of ANP may be involved in the pathogenesis of PE.They find out that ANP-deficient pregnant mice developed high blood pressure and proteinuria,which are characteristics of pre-eclampsia.In these mice,trophoblast invasion and uterine spiral artery remodelling were markedly impaired.Consistent with this,the ANP potently stimulated BeWo cell and human primary trophoblasts in invading Matrigels.The cGMP leavel increased in those cell remarkably,indicated that ANP promoted the invasion of those cells by interated with receptor NPR-A.Previous studies found out that mRNA of ANP and NPR-A were ditected in human placenta and deciduas and the affinity of ANP and it’s receptors in placenta of preeclampsia was decreased significantly,above of which were indicated that ANP and NPR-A may play an important role in pregnancy and the abnormal of ANP and/or it’s receptors may be involved in the pathogenesis of PE.However,the expression and location of ANP and NPR-A in placenta and deciduas was still unclear and weather and how ANP and/or NPR-A participated in the development of preeclampsia were sill unknow.It is generally accepted that the presence of a placenta,and more specifically,the trophoblast cells,is a major cause of preeclampsia.The imparison of trophoblast cell invasion and uterine spiral artery remodlling is the pathological basis of the disease.Uterine decidua,especially basal decidua microenvironment plays an important role in regulating the invasion of trophoblast cells.Therefore,in the first part of this research immunohistochemical technology and Western blot technology were used to identify the distribution of ANP and its receptor NPR-A in basial decidu of first tri pregnancy and placenta of third tri pregnancy,and to compare the expression of ANP and NPR-A between normal placenta and severe preeclampsia placenta,so as to explore the correlation between ANP/NPR-A and pathogenesis of preeclampsia.In this part,we found out that trophoblast in basial decidu and placenta both expresse ANP and NPR-A,and the expression of NPR-A in severe preeclampsia placenta significantly decreased.So that we speculate that defection of NPR-A in trophoblast may impare its function,which may result in the development of preeclampsia.So in the second part of our reaserch,by using siRNA technology to knockdown the expression of NPR-A in HTR8/SVneo cytotrophoblast cell,we identify the effect of silencing of NPR-A on apotopsis rate,invasion ability and proliferation of HTR8/SVneo cell.A greater incidence of villous as well as extravillous trophoblast apoptosis has been observed in placentas from pregnancies complicated with PE suggesting that dysregulated trophoblast apoptosis may contribute to the pathogenesis of preeclampsia.The elevated trophoblast apoptosis observed in pregnancies complicated with PE is thought to be the result of placental oxidative stress which may,in part,be triggered by hypoxia.Oxidative stress refers to the increased formation of reactive oxygen(ROS)in tissues or cells,or antioxidant capacity were reduced leading to ROS accumulate in tissue or cell.Hypoxia is one of the inducing factors of oxidative stress.In preeclampsia placenta,antioxidant activity,such as superoxide dismutase SOD and some antioxidants such as glutathione expression was reduced.However,ROS generation,s protein peroxidation product,lipid peroxidation product,DNA fragments significantly increased.And it is pointed out that that reducing of antioxidant activity in the placenta during pregnancy is involved in the pathogenesis of preeclampsia process.Studies have shown that ANP,by interacting with the surface receptors NPR-A,play the role of resistance to oxidative stress.lt has been found out that ANP can inhibit the liver cell apoptosis induced by ischemia and reperfusion,by activaing NPR-A/cGMP system.ANP can improve the ability of liver cell against ROS5 because ANP can promote the expression of heat shock protein response,especially the expression of HSP70 and HSP32.ANP can activate the heat shock transcription factor,one for the regulation of heat shock protein synthesis is very important factor,it can inhibit the proinflammatory factor such as TNF alpha and or NO entrainment.Existing literature reports that ANP can inhibit cell injury mediated by Ca2+,which may be induced by NPR-A/cGMP system activation,the study of myocardial cells show that ANP can inhibit myocardial ROS production incucing by the H2O2,endothelin,angiotensin Ⅱ,so as to inhibit myocardial hypertrophy.Thus we speculated that in the human placenta,by interacting with NPR-A,ANP can protect trophoblast cell agaist injury mediated by ROS.Therefore,the third part of this study,cobalt chloride were used to induced HTR8/SVneo hypoxia modle,we investigate whether the ANP/NPR-A can inhibit the ROS induce aptoposis,and discusses its mechanism.PART ONE THE EXPRESSION OF ANP AND NPR-AIN EARLY PREGNACY DECIDUA AND PLACENTAOBJECTIVETo identify the distribution characteristics of ANP and NPR-A in basal decidu and villus of the first trimester pregnancy and the third trimester placenta.Comparing the expression of ANP and NPR-A between normal placenta and severe preeclapsia placenta,so as to exploring the correlation ANP and NPR-A with the pathology of preeclampsia.METHODS1、Sample collection:① First trimester decidual and villus samples were obtained from women(n=10)undergoing surgical elective termination of pregnancy between 8 to 13 weeks gestation.②Placentas were obtained from women with uncomplicated pregnancies who delivered in the third trimester(control group,n =10)and from those whose pregnancies were complicated by preeclamspsia(PE)(n = 10).2、Immunohistochemical Analysis were used to identify the expression pattern of ANP and NPR-A in basal decidua which were indetify by immunohistochemistry for human leukocyte antigen(HLA)-G,villus and placenta from third trimester pregnancy.Western blot were used to compared the expression of ANP and NPR-A between normal pregnancy placenta and placenta from patient developed severe preeclampsia.RESULTS1、The distribution of ANP and NPR-A in the fisrt trimester basal deciduas:Immunoreactivity for ANP and NPR-A was consistently found in decidual glandular epithelial cells,extravillous trophoblast and in decidual stromal cells.And in the decidual stromal,ANP(+)and NPR-A(+)cells mainly distributed in the area around the spiral arteries,in which area was aboundant with CD56 + NK cells and HLA-G positive extravillous trophoblast.The decidual serial section dyeing found that extravillous trophoblast were ANP+ and NPR-A+ cell.In first trimester villus and third trimester placenta,ANP and NPR-A were expressed in syncytiotrophoblasts and villouscytotrophoblas.2.、There was no significant difference in the expression of ANP in placenta between severe preeclampsia group and the control group.(0.6800±0.11301 vs.0.6519±0.07650;P=0.582).Howevre,the expression of NPR-A in severe preeclampsia placenta were significantly lower than normal group(0.9257± 0.13907 vs.0.5312±0.13884;P = 0.000).CONCLUSION1、ANP and NPR-A were expresse in extravillous trophoblast cell,glandular epithelium,decidual stromal cells of basial deciduas and syncytiotrophoblasts and villouscytotrophoblas of first trimester villis and term placenta.2、the expression of NPR-A in placenta of severe preeclampsia were significantly dcreased indicated that NPR-A signal in preeclampsia were abnormal,which may result in the development of preeclampsia.PART TWO THE IMPACT OF SIRNA SILIENCE OF NPR-A EXPRESSION IN HTR8/SVNEO TRAPHOBLAST CELLOBJECTIVEBy using siRNA technology to knockdown the expression of NPR-A in HTR8/SVneo trophoblast cell,we identify the effect of silencing of NPR-A on apotopsis rate,invasion ability and proliferation of HTR8/SVneo cell.METHODS siRNA interference technology were used to target silence NPR-A receptor expression in HTR8/SVneo cells,the methods of CCK8,transwell Chambers and Annexin V/PI were used to emplore the effect of targeted silencing NPR-A on proliferation,invasion,and apoptosis in HTR8/SVneo cell.SPSS 13.0 were used to analysis the results.Multiple samples were compared using One-Way ANOVA,variance Aceh pairwise comparisons using LSD,using Dunnett T3 law when heterogeneity of variance,P<0.05 was considered statistically significant.RESULTS1、targeted silencing NPR-A had no effect on proliferation function in HTR8/SVneo cell:Comparing with negative control,the there were no significantly different in OD value between negative control and NPR-A-siRNA group 24 h,48 h,72 h after the transfection.2、targeted silencing NPR-A had no effect on apoptosis in HTR8/SVneo cell:the there were no significantly different in apoptosis rate between negative control and NPR-A-siRNA group 48 h,72 h after the transfection,which suggesting that targeted silencing NPR-A had no effect on apoptosis in HTR8/SVneo cell3、targeted silencing NPR-A signicantly impared invasion ability of HTR8/SVneo cell:there were no significantly difference in the cell number invasing across the champer between negative control group and blank control group(41.16667±5.407626 vs 42.12500±5.276295,P = 0.666).However,comparing with negative control group,the cell number invasing across the champer after transfected with NPR-A-siRNA were significantly reduce(42.12500±5.276295 vs 25.83333±3.761850,P = 0.000),indicating that targeted silencing NPR-A signicantly impared invasion ability of HTR8/SVneo cell.CONCLUSIONTargeted knockout NPR-A receptor inhibits the invasion ability of HTR8/SVneo trophoblast cell,and the invasive ability reduce has nothing to do with proliferation and apoptosis.NPR-A receptor expression in trophoblast may be involved in the pathogenesis of preeclampsia.PART THREE THE INHIBITION AND MECHANISM OF of ANP ON TROPHOBLATIC CELLS APOPTOSIS INDUCED BY HYPOXIAOBJECTIVE To identify whether the ANP/NPR-A can inhibit the aptoposis induced by hpyoxia,and discusses its mechanism.METHODSCobalt chloride hexahydrate(cocl2.6H20)was used to construct hypoxia model.Experimental group:1.the blank control group:PBS as stimulus 2.chemical hypoxia group:simply add cocl2;3.ANP pretreatment groups:ANP pretreatment before hypoxia.4.NPR-A receptor inhibitor groups:the NPR-A receptor inhibitor preprocessing for 30 min before adding the ANP and cocl2.24 h after treatment,CCK8,Annexin V/PI staining,;DCFH-DA dyeing,Rhodanmin 123 dyeing were used to measure the polification ability,apoptosis rate,ROS levels.,and mitochondrial membrane potential,respectly;Westernblot method were used to detect the leavle of cytochrome c,cleaved caspase 9 and cleaved caspase 3.SPSS13.0 were used to analysis the results.Multiple samples were compared using One-Way ANOVA,variance Aceh pairwise comparisons using LSD,using Dunnett T3 law when heterogeneity of variance,P<0.05 was considered statistically significant.RESULTS1、Cobalt chloride can significantly reduce HTR8/SVneo cell activity,and this effect has a dose dependent manner,the greater the dose the more obvious inhibitory effect.By calculating,IC50 doses was 550 umol/L.So this experiment adopts 550umol/L as the concentration used in the following study.2、ANP suppress the trophoblast cells apoptosis induced by hypoxia:after hypoxia for24 hours induced by Cocl2,apoptosis rate of HTR8/SVneo cell significantly increased(4.63%±1.04%VS 16.36%±1.42%,P=0.000),comparing with blank control group.After pretreatment with ANP,apoptosis rate significantly reduced(16.36%±1.42%vs 6.87%±0.66%,P=0.000),rompted that ANP has antiapoptotic effect.The NPR-A receptor inhibitors inhibit antiapoptotic effect of ANP:compared with the ANP group,NPR-A inhibitor significantly increase apoptosis rate(11.27%±1.09%vs 6.87%±0.66%,P=0.00)3、ANP inhibiting the production of ROS induced by hypoxia:results from flow cytometry show that hypoxia can significantly increased the generation of ROS in HTR8/SVneo cells.Compared with the blank control group,the fluorescence intensity of intracellular ROS significantly increased 24 h after hypoxia(222.00±41.61 VS 1201.67±66.73,P=0.00).Pretreatment with ANP can significantly lower fluorescence intensity of ROS in HTR8/SVneo cells(1201.67±66.73 vs.588.00±69.54,P=0.00),comparing with hypoxia groups.NPR-A receptor inhibitor can weaken this effect of ANP:compared with the ANP group.NPR-A inhibitor significantly increase the level of ROS in HTR8/SVneo cells.4、ANP inhibit the mitochondrial membrane potential loose induced by hypoxia:compared with the blank control group,the average fluorescence intensity of Rhodamin 123 were significantly decreased 24h after hypoxia(548.00±49.1 vs 235.00±21.37,P = 0.000).Pretreatment with ANP can significantly increase the average fluorescence intensity of Rhodamin 123 in HTR8/SVneo cells(357.00±58.28 vs.235.00±21.37,P=0.008).However,The NPR-A inhibitors can partly suppress this effect of ANP:compared with the ANP pretreatment group,NPR-A receptor inhibitor can significantly reduced Rhodamin 123 fluorescence intensity(246.66±28.57 v s±58.28 246.66,P = 0.012).5、ANP can inhibit the release of cytochrome c,reduce the activation of Caspase 3 and Caspase 9 in HTR8/SVneo trophoblast cells:Compared with blank control group,hypoxia can significantly increased the content of cytochrome c(0.2423±0.01514 vs 0.5161±0.03099,P=0.00),and significantly increase the activation of Caspase 9(0.4533±0.04041 vs 0.8500±0.02000,P=0.00)and the expression of Caspase 3(0.3533±.04726 vs.6767±.05132,P=0.00)in HTR8/SVneo trphoblast cells.The ANP can significantly reduce the content of cytochrome c in the cytoplasm(0.5161±0.03099 vs 0.3463±0.03649,P=0.00)and cleaved caspase 9(0.8500±0.02000 vs 0.5400±0.02000,P=0.00),cleaved caspase 3(0.6767±0.05132 vs 0.4800±0.03000,P=0.00)in HTR8/SVneo trphoblast cells.And NPR-A receptor inhibitor can inhibit the effect of ANPCONCLUSION1、hypoxia can decrease the activity of trohpblast cells,increase the production of ROS,and activated mitochondrial apoptosis pathway which to induce HTR8/SVneo trophoblast cell apoptosis.2、By interacting with NPR-A,ANP can reduce the production of ROS,inhibit the mitochondrial apoptosis pathway,as a result to reduce the HTR8/SVneo apoptosis induced by hypoxia.