The Role Of Elabela In The Pathogenesis Of Preeclampsia And Its Potential Regulation Mechanism On Blood Pressure

Objective:Preeclampsia(PE)is the most important cardiovascular risk exposure during pregnancy.PE has endothelial dysfunction,oxidative stress,inflammation and other common physiological mechanisms with cardiovascular disease(CVD).Early screening and intervention of preeclampsia should be the beginning of early prevention of cardiovascular disease.Elabela,a newly discovered endogenous ligand for APJ receptors,has a positive effect on embryonic development,placental development,promotion of angiogenesis,inhibition of renal remodeling,inhibition of fibrosis,and regulation of fluid balance.In recent years,more and more animal experiments show that Elabela can promote the migration and invasion of trophoblast cells in the placental development,which is conducive to the remodeling of spiral arteries,and may also play a role in relaxing blood vessels and regulating blood pressure.At present,researchs about the role of Elabela on placental development and PE is less.Its role in the development of human pregnancy and PE needs to be further explored.Therefore,this study was going to observe the expression of Elabela in human placenta,and on the other hand,we used Elabela protein to intervene human chorionic trophoblast cells to explore its effect on human trophoblast cell function and its potential blood pressure regulation mechanism.Explore the potential clinical application value of Elabela in PE prevention and treatment.Methods:(1)Thirty-four pre-eclampsia pregnant women who were delivered in the obstetrics of the Chinese People's Armed Police Force Special Medical Center from December 2016 to April 2018 were enrolled as observation groups,and 40 normal pregnant women were collected as control group.RT-PCR was used to detect the relative gene expression levels of Elabela,APJ receptor and ERK1/2 in placenta.Immunohistochemistry was used to to detect the protein levels of Elabela and APJ receptors and their specific expression sites in placental structures.Western blot was used to detect the phosphorylation activation level of ERK1/2 in placenta.Finally we analyzed the correlation of Elabela expression level,APJ receptor expression level and ERK1/2 phosphorylation activation level with pregnant women's blood pressure level by the Pearson correlation.(2)The human chorionic trophoblast cells were cultured with the serum of normal pregnant women and pregnant women with PE.The conventional fetal bovine serum was used as a blank control,and Elabela protein was added for intervention.The gradient drug concentration intervention experiment was used to found the optimum concentration of Elabela protein acting on human chorionic trophoblast cells.The proliferation of each group was detected by CCK-8 technique.The invasive ability of each group was detected by Transwell technique.Finally,the change of ERK1/2 activation level in each group was detected by Western blot.Results:(1)There was no significant difference in the age and the gestational age between the control group and the preeclampsia group(P>0.05).Compared to the control group,the systolic blood pressure,diastolic blood pressure,heart rate,BMI,and weight gain during pregnancy were increased in the preeclampsia group(P<0.05).S/D,PI,and RI were both higher than the control group(P<0.05).RT-PCR results showed that the relative expression levels of Elabela and APJ receptor genes were lower than the control group,the difference was statistically significant(P<0.05).There was no statistically significant in ERK1/2 gene expression between the control group and the preeclampsia group(P>0.05).The results of immunohistochemistry showed that the expression sites of Elabela and APJ receptors in the placenta were basically the same,which were mainly expressed in the trophoblast cells and decidual cells,and were scattered in vascular endothelial cells.The main expression sites were cytoplasm and cell membrane.In terms of expression intensity,Elabela and APJ receptors were decreased in the preeclampsia group compared with the control group(12.13±1.93 vs.15.95±2.08,P=0.002;9.22±1.56 vs.14.57±4.167,P=0.009 respectively).Western blot analysis showed that the molecular mass of T-ERK1/2 and P-ERK1/2 was 42/44 KD,and the molecular mass of ?-actin was 42 KD.There was no statistical difference in the expression of total protein of ERK1/2 between the control group and the preeclamptic group(1.18 ± 0.18 vs.0.85 ± 0.10,P = 0.09),whereas ERK1/2 activation level was elevated in the preeclampsia group compared with the control group(1.10 ± 0.17 vs.0.47 ± 0.11,P = 0.03).Pearson correlation analysis showed that Elabela was negatively correlated with systolic blood pressure(r=-0.46,P=0.01),and negatively correlated with diastolic blood pressure(r=-0.44,P=0.01);APJ receptor was negatively correlated with systolic blood pressure(r=-0.47,P=0.006),there was a negative correlation with diastolic blood pressure(r=-0.45,P=0.01);there was a positive correlation between ERK1/2 activation level and systolic blood pressure(r=0.45,P=0.04),and there was no correlation between ERK1/2 activation level and diastolic blood pressure(r=0.31,P=0.17).(2)The gradient drug concentration experiment showed that when the concentration of Elabela was 1 ug/ml,the OD value detected by CCK-8 was the highest after 2 hours and 4 hours,so 1 ug/ml was the best concentration of Elabela on HTR-8/SVneo cell line.The results of CCK-8 showed that the OD values of the drug-added groups were significantly higher than those of the non-medicated group(FBS+Ela group vs FBS group,N+Ela group vs N group,PE+Ela group vsPE group,P<0.05).In the nonmedicated group,the OD value of the FBS group was higher than the N group and the PE group(P<0.05),and the OD value of the N group was higher than the PE group(P<0.05).In each of the drug-added groups,The OD value of the FBS group was also higher than the N group and the PE group(P<0.05),and the OD value of N group was also higher than that of PE group(P<0.05).Transwell results showed that the number of cells in the FBS+Ela group was significantly higher than that in the FBS group(10.61±3.62 vs.5.41±0.62,P<0.05).The number of cells in the N+Ela group was significantly higher than that in the N group(11.34±3.82 vs.6.34±0.73,P<0.05),and the number of cells in PE+Ela group was significantly higher than that in PE group(11.93+3.09 vs 5.77±0.47,P<0.05).The results of Western blot showed that the molecular masses of T-ERK1/2,P-ERK1/2 and ?-actin were 42/44 KD,42/44 KD and 42 KD,respectively.There was no significant difference in total protein levels of ERK1/2 between the drug-added groups and the corresponding non-medicated group(P>0.05),which were FBS+Ela group(1.06±0.11)vs FBS group(0.92±0.15),N+Ela group(0.97±0.63)vs N group(1.01±0.12),and PE+Ela group(0.55±0.22)vs PE group(0.78±0.16).However,compared with the non-medicated group,the ERK1/2 activation level of each corresponding drug-added group was significantly lower(P<0.05),which was FBS group(1.82±0.23)vs FBS+Ela group(1.19±0.15),N group.(0.91±0.11)vs N+Ela group(037±0.11),PE group(1.76±0.47)vs PE+Ela group(0.97±0.13).Conclusions:The Elabela/APJ pathway may play an important role in human pregnancy.Not only can strengthen the proliferation and invasion of placental trophoblast cells and subsequent uterine spiral arterial remodeling,but also relax the blood vessels and lower blood pressure by inhibiting the activation level of ERK1/2.It suggests that Elabela may provide a new idea for the treatment of preeclampsia and the initial prevention of cardiovascular disease.