Exploration On The Mechanism Of Action Of Apelin Administration Improving The Clinical Manifestations And Adverse Pregnancy Outcomes Of RUPP Rats

Hypertensive disorders complicating pregnancy is a pregnancy-specific disease,affecting up to 10%of pregnant women in China.In recent years,hypertensive disorders complicating pregnancy has become the main causes of the increased maternal and perinatal morbidity and short and long term mortality.Its serious complications include organ dysfunction,placental abruption,diffuse intravascular coagulation,etc.It could result in fetal growth restriction,preterm delivery,hypoxic nerve damage,perinatal death and other long-term risks associated with premature delivery.Current treatments focus on close monitoring of changes in condition,and terminating pregnancy as appropriate.Safe and effective medicine for the prevention and treatment of preeclampsia has not been found yet,and delivery of the placenta remains the main effective treatment.Apelin,also named APLN,is a newly found small molecular peptide with various biological functions,mediated by orphan G protein-coupled receptors(APJ),which could participate in many kinds of biological effects such as regulating cardiovascular system steady state,dilating blood vessels,regulating water and salt metabolism,mediating immune regulation and so on.It has been confirmed that apelin have multiple isoforms,among which short peptides are more active than long peptides.They are distributed in different organizations,and apelin-36 and apelin-13 is most active in the cardiovascular system.Reduced uterine perfusion pressure(RUPP)rat model is the closest simulation of hypertension,oxidative stress and fetal growth restriction in preeclampsia,and could be successfully copied in other species,therefore serves as a powerful tool for the finding of preeclampsia etiology and potential therapeutic approach.So far,it is not reported that apelin affects RUPP rat model among the domestic and foreign studies.The purpose of this study is to improve the clinical manifestations of preeclampsia by exogenous apelinadministration.Choosing RUPP rat modal as the experimental subject,the research were carried out in the following three aspects:1)We detectied the serum levels of apelin in maternal blood and umbilical cord blood,and the levels of apelin in placenta tissues of preeclampsia.And we analyzed that the correlation between the serum levels of apelin and placenta weight,and the correlation between the serum levels of apelin and fetal weight;2)influence of apelinon RUPP rat model pregnancy outcome;3)the possible mechanism of alleviating adversed pregnancy outcomes in RUPP rat model by apelin administration.In this study,for the first time,we reported that exogenous apelin administration could improve the perinatal outcome of RUPP rat model.And we explore its possible molecular mechanism.Our outcome maybe provide an experimental evidence for apelin used in clinical prevention and treatment of preeclampsia someday.Part one The levels of apelin of patients with preeclampsia in serum and placentaObjective:Investigate the level of apelin from maternal serum,umbilical cord serum and placenta tissues,and the level of APJ expression in placenta tissues of patients with preeclampsia.And analyze the correlation between apelin and placental or fetal weight.And explore the possible role of apelin in preeclampsia.Methods:We collected maternal serum,umbilical cord serum and placenta tissue of patients with preeclampsia(PE group,28 cases)and normal pregnant women(N group,20 cases).We detected the levels of apelin,VEGF-A,sFlt-1 from maternal serum and the levels of apelin from umbilical cord serum with ELISA technique,the levels of T-AOC and MDA from maternal serum with chemical method,and the levels of apelin and APJ protein expression in placenta tissue with immunohistochemical technique.Results:1 The general situation of the two groupsThere were 20 cases in N group,the average age was 28.40±4.04 years old,body mass index(BMI)was 28.18±2.90kg/m2.28 cases in PE group,the average age was 27.46±4.63 years old,BMI was 28.90±4.17kg/m2.There was no statistical difference between the two groups(P>0.05),indicating that the two groups were comparable.1.1 The clinical indicators of the two groupsIn PE group,blood pressure of all patients were higher than 140/90mmHg.For example,systolic pressure(SP)was 166.25± 16.03 mmHg,diastolic pressure(DP)was 107.89±12.38mm Hg,and mean arterial pressure(MAP)was 127.35±11.18mmHg.In N group,blood pressure were normal.For example,SP was 117.75±11.84mmHg,DP was 76.10±8.44mmHg,MAP was 89.99±9.15mmHg.The difference between the two groups was statistically significant(P<0.05).The qualitative or quantitative tests of urine protein in PE group were expressed as +?+++ or more than 2g/24h,while the results were negative or trace in N group,indicating statistical significance(P<0.05).1.2 Neonatal gestational age,Neonatal birth weight and placental weightNo significant differences in gestational age were found between PE group and N group(35.82±2.10 vs.37.21±1.11 weeks,P>0.05).Neonatal birth weight in N group was significantly heavier than in PE group(3290.00±442.96 vs.1958.90±466.05g,P<0.05),indicating newborns were prone to intrauterine growth restriction or small for gestational age in PE group.The placental weight in N group was significantly heavier than in PE group(551.15±78.34g vs.321.89±98.12g,P<0.05),demonstrating placenta maldevelopment in PE group.2.The levels of apelin,VEGF-A,sFlt-1,MDA and T-AOC in maternal serum,the levels of apelin in umbilical cord serum and the levels of apelin and APJ in placenta tissues2.1 the levels of apelinThe levels of apelin from maternal serum was significantly higher in PE group than in N group(70.08±11.35 vs.46.27±7.40pg/ml,P<0.05).The levels of apelin from umbilical cord serum was significantly higher in PE group than in N group(71.20±7.90 vs.59.55±3.44pg/ml,P<0.05).The levels of apelin and APJ protein expression was decreased in placental trophoblastic cells through the immunohistochemical analysis.And the MOD of apelin and APJ in PE group were respectively lower than in N group(0.20±0.03 vs.0.28±0.04,P<0.05;0.22±0.04 vs.0.29±0.02,P<0.05).2.2 The levels of VEGF-AThe levels of VEGF-A from maternal serum was significantly lower in PE group than in N group(55.22±6.55 vs.72.29±11.86 pg/ml,P<0.05).2.3 The levels of sFlt-1The levels of sFlt-1 from maternal serum was significantly higher in PE group than in N group(236.15±47.44 vs.26.82±5.90pg/ml,P<0.05).2.4 The levels of MDAThe levels of MDA from maternal serum was significantly higher in PE group than in N group(3.73±0.66 vs.2.87±0.65umol/L,P<0.05).2.5 The levels of T-AOCThe levels of T-AOC from maternal serum was significantly lower in PE group than in N group(10.35±1.35 vs.13.49±1.71 U/ml,P<0.05).3 The correlation analysis of neonatal birth weight and placental weight with the levels of apelin in maternal serum,umbilical cord serum and placental tission.Neonatal birth weight was strongly negatively correlated with the levels of apelin from maternal serum and umbilical cord serum,and placental weight(Respectively,r=-0.560,P<0.05;r=-0.662,P<0.05;r=0.742,P<0.05),and positively correlated with the levels of apelin in placenta tissues(r=0.447,P<0.05);Placental weight was strongly positively correlated with the levels of apelin in placenta tissues(r=0.293,P<0.05),and negatively correlated with the levels of apelin from maternal serum(r=-0.469,P<0.05)?Summary:The increased apelin in maternal and umbilical cord serum and decreased apelin/APJ in placental tission of patients with preeclampsia,were associated with low birth weight and low placenta quality of preeclampsia.This suggested that apelin may be associated with the pathogenesis of preeclampsia.The levels of VEGF-A,sFlt-1,MDA and T-AOC in maternal serum of preeclampsia had obviously changed,compared with the normal pregnant women.And apelin may be play a role in preeclampsia by oxidative stress and vascular endothelial cell.Part two The effect of apelin administration on clinical manifestations and adverse pregnancy outcomes of RUPP rat modelObjective:By establishing RUPP rat model,this study investigated if apelin administration could improve clinical manifestations and adversed pregnancy outcomes of preeclampsia.Methods:Clean grade Sprague Dawley(SD)rats,weighing 180-200g(about three months old),were used to establish the RUPP rat model.The pregnant rats were divided randomly into 4 groups on day 14(200-250g):sham operation group(N,6 cases),pregnancy with RUPP operating group(PE,7 cases),sham operation group plus apelin-13 group(APLN,7 cases),PE group plus apelin-13 group(PE+APLN,8 cases).From day 14 of gesdation,the APLN group and PE+APLNgroup received respectively subcutaneous injection of apelin-13,100ug/Kg,Bid for 5 days.At day 18 of gesdation,carotid artery catheter was cannulated,and then they were placed respectively in metabolic cages to collect 24-hour urine.At day 19 of gesdation,blood pressure,cardiac function,and placental fetal weight were measured,blood specimen and placental tissues of rats were collected.We observed blood pressure,cardiac function,morphological changes of placenta,fetal survival,body weight,and the serum levels of VEGF-A and sFlt-1 in the pregnant rats.Result:1 Hemodynamics changesThe blood pressure in PE group(SP 149.00± 5.80mmHg,DP 103.14±3.24mmHg,MAP 118.36 ± 4.10mmHg)increased significantly,compared with that in N group(SP 124.00±7.59mmHg,DP74.83± 6.21mmHg,MAP 91.51±6.76mmHg).The difference between the two groups was statistically significant(P<0.05).LVESP and LV+dp/dtmax were lower in PE group than in N group(107.21 ± 12.95 vs.122.18±6.61mmHg;1829.50±513.74 vs.2641.67±314.23mmHg/s),but LVEDP was higher in PE group(2.55±2.48 vs.-4.899±1.72mmHg).The difference is statically significant(P<0.05).Hower,the blood pressure in PE+APLN group(SP 139.38±4.03mmHg,DP 88.50 ± 8.42mmHg,MAP 105.46 ± 6.44mmHg)decreased significantly,compared with that in PE group(P<0.05).LVESP and LV+dp/dtmax were significantly higher in PE+APLN group than in PE group(129.51±8.01 vs.107.21±12.95 mmHg,P<0.05;2568.12±393.70 vs.1829.50±513.74mmHg/s,P<0.05),and LVEDP decreased significantly in PE+APLN group((-0.66±1.37 vs.2.55±2.48mmHg,P<0.05).The difference of the above indices between APLN group and N group was not statistically significant(P>0.05).2 Quantitative analysis of urine proteinThe urinary protein quantity in 24 hours is 2.79±0.53mg in N group,4.79±0.75 mg in PE group.The difference between the two groups was statistically significant(P<0.05).The urinary protein was not negative in N group,not the same as human.The urinary protein quantity in 24 hours in PE+APLN group is lower than in PE group(4.03±0.88 vs.4.79±0.75mg,P<0.05).The urinary protein quantity in 24 hours in APLN group is lower than in N group(2.44±0.52 vs.2.799±0.53mg,P>0.05).3 Embryo survival rateThe embryo survival rate was 100%in N group,0.32±0.12 in PE group,and 0.46±0.17 in PE+APLN group.Compared with PE group,it was significantly increased in PE+APLN group(P<0.05).4 Survival embryo weightSurvival embryo weight was 2.17±0.21g in N group,1.96±0.29g in PE group,and 2.16±0.24g in PE+APLN group.Compared with N group,the average weight was significantly decreased in PE group(P<0.05);And compared with PE group,the average weight was significantly increased in PE+APLN group(P<0.05).5 Placenta weight of the survival embryoMean placental weight was 0.42±0.06g in N group,0.36±0.07g in PE group,and 0.40±0.05g in PE+APLN group.Compared with PE group,mean weight was significantly increased in PE+APLN group(P<0.05).6 Morphological changes of placentaThe rat placenta is constituted with four layers from the maternal surface to the fetal side.They are decidua,cytotrophoblast,sponge trophoblast,and labyrinthine trophoblast.The villi is embedded in the labyrinthine trophoblast,showing a tree branch like pattern,and usually covered with three layers of trophoblast cells.The axis of villi is composed of connective tissue rich in blood vessels.HE staining showed that the villi demonstrated the greater difference than other parts of the rat placenta among the groups.Compared with N group,PE group showed features of villous edema,irregularly arranged cells with variously sized,and hyperchromatic nucleus,while in PE+APLN group,the edema of the placental villi was alleviated,and the cells were arranged regularly with less hyperchromatic area.Observed by electron microscope,the endoplasmic reticulum expanded,mitochondrial cristae and membrane fused,and there were some glycogen in PE group;Endoplasmic reticulum also expanded,part of mitochondrial crest and membrane fused,and there are a lot of glycogenrich in PE + APLN group.7 The level of serum VEGF-A and sFlt-1The levels of serum VEGF-A decreased significantly in PE group,compared with that in N group(22.14±3.77 vs.29.51±5.27pg/ml,P<0.05),and that in PE+APLN group(15.57±3.90pg/ml)was lower than both PE group and N group.The levels of serum sFlt-1 in PE group was higher than in N group(4.37±0.92 vs.2.98±0.86pg/ml,P<0.05),and that in PE+APLN group demonstrated no significant difference when compared with that in PE group(4.52±0.97pg/ml,P>0.05).Summary:The main clinical manifestations of preeclampsia were successfully simulated with RUPP rat model in this study.And apelin administration could effectively improve the hemodynamic parameters,fetus survival rate placenta and fetus weight in PE group.Part three Exploration on the mechanism of action of apelin administration improving the clinical manifestations and adverse pregnancy outcomes of RUPP rat modelObjective:Exploring the effects of apelin administration on NO signaling pathway and oxidative stress state in RUPP rat model.Methods:We measured the serum levels of nitric oxide(NO),endothelial nitric oxide synthase(eNOS),total antioxidant capacity(T-AOC),and malondialdehyde(MDA)in the rats with chemical method.We measured the levels of eNOS mRNA expression in the placenta tissue with RT-PCR method,and measured eNOS Protein expression levels in the placenta tissue with Western-blot method.Results:1.The effect of apelin administration on the level of serum NO products,the activity of serum eNOS in the RUPP rat model..The levels of serum NO decreased significantly in PE group than in N group(36.02±5.89 vs.45.75±6.26umol/l,P<0.05);The levels of serum NO increased significantly in PE+APLN group than in PE group(44.01 ±5.15 vs.36.02±5.89 umol/1,P<0.05).2 The effect of apelin administration on the level of eNOS mRNA and protein expression of the placental tissue in the RUPP rat model..The levels of eNOS mRNA in the placental tissue decreased significantly in PE group than in N group(0.52±0.06 vs.0.66±0.05,P<0.05);the levels of eNOS mRNA in the placental tissue increased significantly in PE+APLN group than in PE group(0.73±0.09 vs.0.52±0.06,P<0.05).The levels of eNOS protein in the placenta tissue in PE group decreased,but increased in PE+APLN group.3.The effect of apelin on the levels of serum T-AOC and MDA of the RUPP rat model.The levels of serum T-AOC group decreased significantly in PE group than in PE group(7.20±0.88 vs.8.39±1.04U/ml,P<0.05).The levels of serum T-AOC group increased significantly in PE+APLN than in PE group(8.78±1.00 vs.7.20±0.88U/ml,P<0.05).The levels of serum MDA increased significantly in PE group than in N group(0.55±0.09 vs.0.41±0.05umol/l,P<0.05).the levels of serum MDA decreased significantly than in PE group(0.44±0.08 vs.0.55±0.09umol/l,P<0.05).Summary:Apelin administration improving states of preeclampsia increased the serum activity of eNOS,the serum levels of NO products and the serum levels of T-AOC and decreased the serum levels of MDA.This suggested that the effect of apelin administration,improving vascular endothelial function and oxidative stress state on RUPP rat model,may be the mechanism of action of apelin.Conclusion:1 The increased apelin in maternal and umbilical cord serum and decreased apelin/APJ in placental tission of patients with preeclampsia,were associated with low birth weight and low placenta quality of preeclampsia.This suggested that apelin may be associated with the pathogenesis of preeclampsia.2 In this study,the main clinical manifestations of preeclampsia were successfully simulated with RUPP rat model.And apelin administration could effectively improve the hemodynamic parameters,fetus survival rate and placental and fetal weight in PE group.3 Apelin administration improving states of preeclampsia increased the activity of eNOS,the levels of NO products and the levels of T-AOC and decreased the levels of MDA.This suggested that the effect of apelin administration,improving vascular endothelial function and oxidative stress state on RUPP rat model,may be the mechanism of action of apelin.