The Correlation Between The Expression Of ACKR2 In Preeclampsia Placentas And Trophoblast Apoptosis

BackgroundPreeclampsia(PE)is a multi-systemic pregnancy syndrome and one of the main causes of increased perinatal morbidity and mortality,the prevalence rate is about 5?8%of pregnant women.It is defined as newly developed hypertension and proteinuria after 20 weeks of pregnancy,or without proteinuria,but combined with any of the following:thrombocytopenia;liver function impairment;renal function impairment;pulmonary edema;newly occurring central nervous system disorder or visual impairment.Although there are many hypotheses about the pathogenesis of preeclampsia,the exact mechanism is still unclear.However,according to the gestational weeks when the preeclampsia patients are diagnosed,with 34 weeks as the boundary,preeclampsia can be divided into two subtypes:early-onset preeclampsia and late-onset preeclampsia.Early onset of preeclampsia occurs early,speedly development,and the clinical symptoms are more severe,which often leads to serious maternal and fetal complications,while late-onset preeclampsia occurs late,slowly development,and the clinical symptoms are milder than early-onset preeclampsia,the prognosis of the mother and child is acceptable.It has been reported that early-onset preeclampsia and late-onset preeclampsia may have different pathogenesis.Currently the vast majority of research suggests that the excessive activation of inflammatory immunity is a very important pathological feature in preeclampsia,and ACKR2,as an atypical chemokine receptor,has a high affinity for CC-type inflammatory chemokines.It can be combined with chemokines,internalization and degradation,and then to control inflammation and regulate immunity.ACKR2 is mainly expressed in lymphatic endothelial cells,and some studies have found that ACKR2 can also be expressed in tissues derived from trophoblasts and has the effect of preventing excessive infiltration of placental leukocytes.However,there are still few studies about ACKR2 in the placental tissue of preeclampsia,so this study take this as point,and by analyzing the expression changes of ACKR2 in the placental tissue of preeclampsia,to preliminarily explore the role of ACKR2 in the pathogenesis of preeclampsia.PurposeTo further explore the expression changes of ACKR2 in placental tissues from different types of preeclampsia patients,and analyze the relationship between this expression changes and gestational weeks.By investigating the effect of hypoxia on the expression of ACKR2 in trophoblast cell line JAR and the effect of ACKR2 silencing on the expression of chemokine CCL2 and cell apoptosis and proliferation,the role of ACKR2 in the pathogenesis of preeclampsia was analyzed and verified.Experimental method1.Selection of research objects:A total of 120 placental tissue samples collected by the staff of the biological sample bank of the Scientific Research Center of the Third Affiliated Hospital of Zhengzhou University from February 2017 to October 2018 were divided into four groups:normal group(N,n=30),preterm birth group(PB,n=30),early-onset preeclampsia group(EOPE,n=30)and late-onset preeclampsia group(LOPE,n=30).In order to avoid the influence of different delivery methods on the experimental results,pregnant women in each group were selected for cesarean section delivery,and hypothyroidism,gestational diabetes,chronic hypertension,maternal autoimmune disease,and twin pregnancy and assisted reproduction were excluded.2.Specimen collection:Within half an hour after the placenta was delivered,two pieces of 1 � 1 � 1cm tissues were taken from the placental mother surface,and washed with normal saline.One piece was placed in liquid nitrogen as backup,and the other was fixed in formalin solution for subsequent experiments.3.The mRNA expression of ACKR2 in the four groups of placental tissues was detected by RT-PCR,and the data analysis was performed by 2-??Ct relative quantitative method.4.Immunohistochemical method was used to detect the protein expression of ACKR2 in four groups of placental tissues,and relevant semi-quantitative analysis was performed using image analysis software.5.Western blot was used to detect the protein expression of ACKR2 in four groups of placenta tissues,and image analysis software was used for quantitative analysis.6.Cobalt chloride was used to simulate hypoxia conditions,and MTT method was used to detect the effect of cobalt chloride on cell proliferation activity after 24h,36h,48h and 72h of treatment,respectively.Western blot was used to detect the effect of cobalt chloride at different concentrations on the expression of ACKR2 protein in the trophoblast-derived human choriocarcinoma cell line JAR.7.JAR cells were transfected with ACKR2 small interfering RNA,and the silencing efficiency of ACKR2 in the cells was detected by RT-PCR and Western blot.8.After ACKR2 in JAR cells was silenced,flow cytometry was used to detect the effect on cell apoptosis,MTT assay was used to detect the effect on cell proliferation,and RT-PCR and Western blot were used to detect the effect on the expression of chemokine CCL2.9.Statistical analysis:The statistical software SPSS24.0 was used to analyze the data.All data were expressed by mean�standard error.The quantitative data of two groups were compared using t test,the quantitative data of multiple groups were compared using one way ANOVA,P<0.05 indicated statistical significance.Result1.Comparison of general clinical data from the four classification groupsThe average systolic blood pressures of the normal group,PB group,EOPE group and LOPE group were 117.70 � 1.83mmHg,111.62�2.25mmHg,166.63�3.50mmHg and 157.67�3.94mmHg,respectively,the average diastolic blood pressures were 86.80�3.18mmHg,70.38�1.59mmHg,105.17�1.74mmHg and 97.93�2.21mmHg,respectively.In terms of blood pressures(systolic and diastolic blood pressures),the blood pressures in the EOPE group were higher than that in the normal and PB groups,and the blood pressures in the LOPE group were higher than in the normal group,and the differences were statistically significant(P<0.05).The average weight of the newborns in the normal group,PB group,EOPE group and LOPE group were 3.41� 0.05kg,1.98�0.07kg,1.47�0.07kg and 2.67 � 0.13kg,respectively.In terms of neonatal weight,the normal group was higher than the PB group,the EOPE group,and the LOPE group,while the EOPE group was lower than the PB group,and the differences were statistically significant(P<0.05).The average gestational weeks of the normal group,PB group,EOPE group and LOPE group were 39.00�0.06W,32.77�0.28W,31.90�0.28W and 37.01�0.30W,respectively.In terms of gestational weeks,compared with the normal group and PB group,the normal group and EOPE group,the normal group and LOPE group,and the PB group and EOPE group respectively,the differences were statistically significant(P<0.05).The average placental weight of the normal group,PB group,EOPE group and L OPE group were 0.61�0.02Kg,0.51�0.02Kg,0.47�0.02Kg and 0.52�0.02Kg,respectively.In terms of placental weight,the PB group,EOPE group and LOPE group were lower than the normal group,the differences were statistically significant(P<0.05),while the difference between the PB and EOPE groups was not statistically significant(P>0.05).The average pre-pregnancy BMI of the normal group,PB group,EOPE group and LOPE group were 23.25�0.75Kg/m2,24.26�0.77Kg/m2,24.93�0.85Kg/m2 and 23.93 � 0.56Kg/m2,respectively.There were no significant differences about pre-pregnancy BMI between the normal group and PB group,the normal group and EOPE group,the normal group and LOPE group,the PB group and EOPE group(P>0.05).The average ages of the normal group,PB group,EOPE group and LOPE group were 33.17�0.81 years old,32.07�0.83 years old,32.63�0.93 years old and 30.87�0.88 years old,respectively.In terms of the ages of the pregnant women,the differences between the normal and PB groups,the EOPE and LOPE groups,and the PB and EOPE groups were not statistically significant(P>0.05).2.Comparison of the relative mRNA and protein expression levels of ACKR2 in placental tissues between four groupsRT-PCR showed that compared with the normal group,the mRNA expression level of placental ACKR2 in the EOPE group and PB group were decreased,and the differences were statistically significant(P<0.05),while the difference between the normal and LOPE groups,the PB and EOPE groups were not statistically significant(P>0.05).Western blot and immunohistochemistry showed that the protein expression level of placental ACKR2 in the EOPE group and PB group were lower than that in the normal group(P<0.05),while the differences in ACKR2 protein levels between the EOPE and PB groups,and the LOPE and normal groups were not statistically significant(P>0.05).3.Effects of cobalt chloride on ACKR2 in a trophoblast-derived human chorionic carcinoma cell line JARAs the concentration of cobalt chloride increased,the proliferative activity of JAR cells was gradually decreased.When the concentration of cobalt chloride was 50?mol/L and 100?mol/L,and compared with the control group,the difference in ACKR2 protein expression in the cells was not statistically significant(P>0.05).But when the concentration of cobalt chloride was 150?mol/L,200?mol/L and 250?mol/L,compared with the control group,the expression level of ACKR2 protein in JAR cells was decreased,the difference was statistically significant(P<0.05).4.Effects of down-regulation of ACKR2 on cell apoptosis,proliferative activity and the expression of chemokine CCL2The results of RT-PCR showed that the the silencing efficiency of ACKR2 in cells was 73.9%,and the results of Western blot showed that the protein expression of ACKR2 in the cells had a significant silencing effect;after ACKR2 was silenced in the cells,the apoptosis level of JAR cells increased,and the proliferation activity decreased,the mRNA and protein expression levels of chemokine CCL2 increased.Conclusion1.The expression of ACKR2 in placental tissues of patients from different types of preeclampsia is different,but this difference may also be associated with gestational weeks.2.Hypoxia can inhibit ACKR2 protein expression.3.The down-regulation of ACKR2 can affect the apoptosis and proliferation of trophoblast cells and the expression of placental chemokine CCL2.