Expresssion Of NFAT5 And MCP-1 And Their Relationship With Macrophage Infiltration In Preeclampsia

Preeclampsia(PE)is a multisystem inflammatory syndrome resulting in hypertension and proteinuria and edema with multiorgan dysfunction.There is growing evidence that these effects persist after pregnancy.It affects 5% ~ 8% of all pregnancies,which is one of the main cause of the increased maternal and infant mortality.Although there are many theories about the pathogenesis of preeclampsia,the pathogenesis is still not well understand.Nowdays,PE is divided into mild and severe preeclampsia(SPE).Severe preeclampsia is generally divided into two major subgroups: early-and late-onset SPE.Early-onset SPE(EPE)is characterized by the rapid rise in blood pressure and proteinuria and more complications of the serious damage to the terminal organ than late-onset SPE(LPE),which is referred to as a placenta disease.Its placental function damage obviously,fetal intrauterine growth restriction and maternal and neonatal outcomes are poor.It has been the focus of obstetric field at home and abroad.LPE is characterized by the normal of the placenta morphology and associated with maternal metabolic disease and cardiovascular disease,which also known as the maternal disease.NFAT5(nuclear factor of activated T cells 5,NFAT5)is a member of the Rel/NFKB/NFAT family,extensively existing in the liver,kidneys and placenta.It involved in cellular adaptation to hypertonicity by activating osmoprotective genes transcription.Under various pathophysiological conditions NFAT5 is activated by local osmotic stress and stimulates the expression of proinflammatory cytokines.Therefore,penetrating environment has closely linked with the inflammatory state in the tissues.NFAT5 play an important role in the autoimmunity disease,cell stress and inflammatory diseases.MCP-1(monocyte chemoattractant protein-1,MCP-1)is a secretory protein,containing 76 amino acids.It triggers chemotaxis and transendothelial migration of monocytes to inflammatory lesions by interacting with the membrane CC chemokine receptor 2(CCR2)in monocyte.Sertoli cells and decidual cells can secrete MCP-1,which prompted blood mononuclear cells migrate to the deciduas,playing an important role in the process of spiral arery remodeling.It has been found that the promoter region of MCP-1 contains NFAT5 binding sites.NFAT5 is activated in response to high osmolalities in mesothelial cells and that this activation contributes to increased expression of MCP-1,which can activate the downstream signaling pathways.Macrophage is a major immune cells with removal of apoptosis composition,which attach great attention about its immune regulation in the uteroplacental bed.Macrophage infiltration was negative correlated with sertoli cell infiltration.There are a large number of macrophages infiltration in preeclampsia patients,through a variety of mechanisms regulating the sertoli cell invasive aability.MCP-1 can chemotaxis macrophage migration to decidual area,which play a role in the process of sertoli cell infiltration.Currently,the relationship between macrophages and preeclampsia has done a great deal of research,there is little about the study of NFAT5,MCP-1 and macrophages infiltration with the relevance of preeclampsia.Objective To explore the expression of MCP-1 in maternal serum,umbilical cord blood and placenta in the different types of severe preeclampsia and analyze the correlation,provide new targets for predicting the severity of preeclampsia;To test the expression of MCP-1 and NFAT5 and CD68+macrophages in placenta in different types of severe preeclampsia and correlation analysis of NFAT5 and MCP-1 and macrophage infiltration.Materials and Methods 1 Study population 30 control group and 31 EPE and 30 LPE patients from Zhengzhou University Third Hospital between January 2015 and January 2016 were selected as our study population.Preeclampsia were diagnosed according to the eighth edition of journal of obstetrics and gynecology.All the study subjects were singleton pregnancies.Women with risk factors for developing preeclampsia such as premature preterm rupture of membranes,autoimmune diseases,gestational diabetes,and chromosomal or structural anomalies were excluded from this study.2 Methods 1.Serum and placenta specimen collection:elbow venous blood 3ml came from research object and the placenta umbilical venous blood 3ml after placenta deliveried.3000r/min centrifuge 15 min,take out the serum,then saved at-80? refrigerator.As soon as possible after delivery,2 samples of parent flour of placenta,1cm x1 cm x1cm in size,were taken from the central area of placental tissues.We should avoid hemorrhage,infarction and calcification area.After 4? saline rinsing,one was set in 1.5ml centrifuge tube,immediately put into liquid nitrogen and then stored at-80?,the other was fixed in 10% formalin for IHC.2.Using the enzyme-linked immunoadsordent assay(ELISA)to test pregnant women MCP-1 levels in peripheral blood and umbilical cord blood.3.Using the real-time PCR to detect the placenta tissues NFAT5 m RNA,MCP-1 m RNA expression levels.We analyzed the data through 2-??CT relative quantitative method.4.Using the immunohistoche Illical method to detect the MCP-1and NFAT5 inplacental tissues and CD68+ macrophage in decidual tissues.We analyzed the three indicators by using computer image analysis.3 Statistics methods All statistical analyses were performed on the SPSS 21 platform.The clinical data and the results were expressed as the mean±SD,the methods of One-Way ANOVA analysis and LSD test is used,Pearson correlation were used in the test.For all tests performed,we considered the data is significant when P<0.05.Results 1 The comparison of the general clinical data among the three groups: There is no difference in age,termination of the gestational age and BMI before pregnancy between control group,EPE group and LPE group(P>0.05);We found that the neonatal birth weight,the placenta weight in the EPE group was significantly lower than in the control group and LPE group(P<0.05),but 24 h urine protein in EPE group patients was higher than the LPE group(P<0.05).2 The expression level of MCP-1 in the maternal blood,cord blood among the three groups The maternal blood MCP-1 level was higher in the EPE group than the LPE and control group(185.80±15.73)ng/L vs(164.93±6.24)ng/L and(154.78±9.61)ng/L,both P<0.05,and so was the cord blood MCP-1 level(223.49±41.68)ng/L vs(151.85±13.92)ng/L and(113.91±6.67)ng/L,both P<0.05.3 The NFAT5 and MCP-1 m RNA expression level in the placenta tissues among the three groups The NFAT5 m RNA level(1.07±0.03)was higher than the EPE group(0.92±0.02)and LPE(0.90±0.04)group(P<0.05),and so was the MCP-1 m RNA level(1.08±0.12)vs(1.02±0.08)vs(0.99±0.02)(P<0.05).4 The NFAT5 and MCP-1 and CD68+ macrophage protein expression level in the placenta among the three groups The NFAT5 protein level was higher than the EPE and LPE group(34.16±4.65 vs 11.39±3.77 and 10.65±1.85,both P<0.05),respectively,so was the MCP-1 protein level(38.47 ± 4.81 vs 19.29 ± 3.86 and 20.33 ± 3.92,both P<0.05),respectively;and so was the CD68+ macrophage protein level(80.60±10.47 vs 24.25±6.69 and 22.79±5.58,both P<0.05).5 The correlation analysis of NFAT5 and MCP-1 and CD68+macrophage infiltration 1.In the EPE and LPE group,the expression of MCP-1 in the cord serum was positively correlated with both the expression of MCP-1 in the maternal serum(r =0.587,P < 0.05;r=0.576,P < 0.05)and in the placental tissues(r =0.754,P < 0.05;r =0.791,P < 0.05).However,the level of MCP-1 in the cord blood was negative correlated with the neonatal birth weight only(r =-0.508,P < 0.05;-0.496,P < 0.05).2.In the EPE and LPE group,the expression of MCP-1 in the maternal serum was positively correlated with the expression of MCP-1 in the placental tissues(r =0.671,P < 0.05;r =0.676,P < 0.05).3.The expression of NFAT5 m RNA in the EPE group and LPE group was positively correlated with both the level of MCP-1 m RNA(r =0.724,P < 0.05;r =0.631,P < 0.05)and the expression of CD68+ macrophage(r=0.640,P < 0.05;r =0.608,P <0.05).At the same time,the expression of MCP-1 m RNA was positively correlated with the expression of CD68+ macrophage(r = 0.704,P < 0.05;r =0.686,P < 0.05).Conclusions 1.The expression of MCP-1 increased in maternal serum,cord blood and placental of patients in severe preeclampsia and it had positive correlation with the severity of illness,which suggest it may be used as an indicator of severity of disease.2.The expression of NFAT5 and MCP-1 and CD68+ macrophage is higher in the placenta of patients in the early-onset preeclampsia than control group,NFAT5 is consistent with an increase in the expression of CD68+ macrophage,so as the MCP-1 and CD68+ macrophage,indicating that the signal pathway of NFAT5,MCP-1and CD68+ macrophage may contribute to the pathogenesis of EPE.