The Association Between Nrf₂、TLR-1 And Premature Rupture Of Membranes With Histological Chorioamnionitis.

Premature rupture of membranes(premature rupture of membrane,PROM)is one of the most common obstetric complications.The study found that nearly 80% of premature rupture of membranes occurred in full-term pregnancy.79% of women in the spontaneous rupture of the film within 12 hours,and the spontaneous contraction of the uterus within 24 hours after the rupture of the membrane in 95%.The disease is closely associated with chorioamnionitis(histological chorioamnionitis)(HCA,also known as subclinical / chorioamnionitis).PROM and HCA had no obvious clinical symptoms,and clinical non sensitive index for the diagnosis and detection,prediction and diagnosis helps to take the most appropriate solutions to reduce the perinatal morbidity and mortality.It is found that amniotic fluid culture can be used for the diagnosis,but this method is time-consuming and false positive rate is high[1].The dynamic monitoring of white blood cell count and the expression level of C reactive protein is helpful to the diagnosis of HCA,but some studies have found that the specificity of HCA is not strong,the clinical application is limited[2].Therefore,it is important to search for specific and sensitive markers to predict and diagnose HCATranscription factor Nrf2(NF-E2 related factor 2)has been shown to play a central role in the regulation of inflammation.Nrf2 is isolated in the cytoplasm by Kelch like ECH associated protein 1(Keap1),which is inactive.A large number of cell protection and antioxidant proteins have been identified as Nrf2 target[3].In vitro,Nrf2 silencing increased tumor necrosis factor in human mononuclear cells(TNF-)induced by chronic inflammation,LPS enhanced proinflammatory cytokine gene expression of[4],inhibit skin fibroblasts after oxidative stress induced by proinflammatory cytokine gene expression of and vascular smooth muscle cell(VSMC)in platelet derived growth factor(PDGF)induced inflammation[3].Foreign studies have found that when PROM combined with HCA,the expression of Nrf2 m RNA in the membrane of the fetal membranes was increased,which was speculated to be related to the inflammation of the amniotic cavity.TLR-1,a member of the Toll like receptor(TLR)family,is a molecular model for identifying microbial pathogens and indicates the presence of invasive microbes in the host’s immune system.Toll like receptors can be located on the surface or inside of the cell,and can be expressed by various kinds of cells and tissues in the body,including white blood cells and[6].In response to inflammation,TLR is involved in the activation of monocytes,the secretion of proinflammatory cytokines and the upregulation of chemokine receptors[9].It has been found that the expression of TLR-1 in amniotic fluid is related to infection,but there is no relationship between the level of TLR-1 in blood and HCA.objectiveThe expression of Nrf2 and TLR-1 on the detection of PROM from peripheral blood of pregnant women and umbilical cord blood mononuclear cells and fetal tissues,and to investigate the relationship between Nrf2 and TLR-1 and chorioamnionitis,in order to find the diagnosis of premature rupture of membranes sensitive index with chorioamnionitis.Materials and methods 1 Research objects From November 2015 to February 2016 in the delivery of the Third Affiliated Hospital of Zhengzhou University of foot at the beginning of the month production of 60 pregnant women with PROM.According to the histopathological results(the number of inflammatory cells per high-power microscope),5 inflammatory cells weredivided into HCA group(HCA group,n = 21)and control group(no pregnant women with HCA,n = 39).All pregnant women are single births,no maternal disease,obstetric complications and fetal and fetal abnormalities.All pregnant women were delivered within 12 h after the break.The diagnostic standard of PROM,according to Xie Xing Gou Wenli editor of the eighth edition of "obstetrics and Gynecology".2 Experimental methods 2.1 Collection and treatment of fetal membrane tissue After delivery of the placenta,the rupture of membranes part as the center,take a 2cm ×3cm width of membranes,rolls were "jelly roll",and in 3,a formaldehyde fixed and paraffin embedded for histological examination.The remaining two were placed in a frozen tube for real-time fluorescent quantitative PCR and Western blotting.2.2 Collection of peripheral blood mononuclear cells All of pregnant women were admitted to hospital after without any treatment before 8ml cubital venous blood samples with EDTA tube,with immediate human lymphocyteseparation(Tianjin Hao Yang)isolated from peripheral blood mononuclear cells by density gradient centrifugation.Fetus after delivery of the placenta,umbilical venous blood before 8ml and subsequent treatment.2.3 Treatment of peripheral blood mononuclear cells The 5ml separation liquid added to the 15 ml centrifuge tube,draw 5ml blood samples with 2500r/min 28 min Straw,centrifugal,centrifugal visible after the centrifuge tube is divided into four layers from top to bottom,followed by plasma layer,white layer,ring lymphocyte separation liquid layer,red cell layer.From peripheral blood mononuclear cells for subsequent operations.2.4 Immunohistochemical method was used to detect the expression of Nrf2 and TLR-1 in fetal membranes.The expression of Nrf2 and TLR-1 in the fetal membrane under microscope.Each slice was taken from 10 fields of vision,and the expression of Nrf2 in each field of view was counted,and the sum of the ten visual fields was counted and averaged.Using PBS as a negative control.2.5 The expression of Nrf2 m RNA and TLR-1m RNA in human fetal membranes andperipheral blood mononuclear cells was detected by reverse transcription PCR.2.6 The expression of Nrf2 protein and TLR-1 protein in human fetal membranes and peripheral blood mononuclear cells was detected by Western blot analysis.3 Statistical methods Using SPSS17.0 software,The measurement data were compared with the standard deviation((?)±s),the use of single variable multi group repeated measurement GLM analysis of variance,the LSD test was used to compare the two groups..Taking a=0.05 as the test level.Result 1 HCA group and control group general situation comparison There were no significant differences in the gestational age,age and body temperature between the HCA group and the control group(P> 0.05).The white blood cell count and C-reactive protein in the HCA group were(13.31 ± 1.79)× 109 /(10.29 ± 2.08)mg / L,and the control group were(7.99 ± 1.01)× 109 / L and(4.02 ± 1.24)mg / L,respectively.There was significant difference between the HCA group and the control group(P <0.05).2 Immunohistochemical results of fetal membranes Nrf2 protein in the two groups of amniotic membrane,chorionic layer and the end of the decidua in the cytoplasm or nucleus can be seen in brown positive staining;TLR-1 protein two groups of fetal membranes amniotic membrane,chorionic layer and the end of the shed The cytoplasm in the membrane layer can be seen in brown positive staining.The expression levels of Nrf2 in the HCA group were(16.29 ± 2.81),(10.86 ± 2.41),(3.43 ± 0.98),respectively.In the control group,the expression of Nrf2 in the amniotic layer,chorionic layer and decidual layer were(26.29 ± 3.25),(15.00 ± 2.65),(7.00 ± 1.29),respectively.There was significant difference between the HCA group and the control group(P <0.05).The expression level was lowest in the basal decidua and highest in the amniotic layer.3 The expression of Nrf2 m RNA and TLR-1 m RNA in fetal membranes of HCA group and control group The levels of Nrf2 m RNA and TLR-1 m RNA in the fetal membranes of HCA group were(0.48 ± 0.09)and(1.72±0.23).The levels of Nrf2 m RNA and TLR-1 m RNA in the control group were(1.00 ± 0.12)and(0.96±0.18).The difference was statistically significant(P <0.05).4 The expression of Nrf2 protein and TLR-1 protein in fetal membranes of HCA group and control group The levels of Nrf2 protein and TLR-1 protein in HCA group were(0.10± 0.02)and(0.47±0.07)respectively.The level of TLR-1 protein in control group was(0.39±0.05)and(0.19±0.02).The difference between the two groups was statistically significant(P <0.05).5 Expression of Nrf2 m RNA and TLR-1 m RNA in peripheral blood mononuclear cells of HCA group and control group The level of Nrf2 m RNA in peripheral blood mononuclear cells of HCA group was(0.73 ± 0.11)and TLR-1 m RNA level was(3.47±0.62),the level of Nrf2 m RNA in control group was(1.08±0.17)and TLR-1 m RNA level was(1.01±0.08).The difference between the two groups was statistically significant(P <0.05).6 The expression of Nrf2 protein and TLR-1 protein in peripheral blood mononuclear cells of HCA group and control group The levels of Nrf2 protein and TLR-1 protein in HCA group were(0.29±0.05)and(0.80±0.10)respectively,and the levels of TLR-1 protein in the control group were(0.70 ± 0.09)and(0.32±0.05)(P <0.05).There was significant difference between the two groups(P <0.05).7 The expression levels of Nrf2 m RNA and TLR-1 m RNA in umbilical cord blood mononuclear cells of neonatal HCA group and control group HCA group Nrf2 m RNA human umbilical cord blood mononuclear cells in the level of(0.51±0.09)and TLR-1m RNA level(2.35±0.29),control group Nrf2 m RNA level(1.01±0.19)and TLR-1m RNA level(1.05±0.18),two groups of corresponding comparison,the differences were statistically significant(P<0.05).8 The levels of Nrf2 protein and TLR-1 protein in umbilical cord blood mononuclear cells of HCA group and control group Nrf2 protein of HCA group neonatal umbilical cord blood mononuclear cells inthe level of(0.18±0.03)and TLR-1 protein level(0.48±0.06),the control group of Nrf2 protein level(0.44±0.07)and TLR-1 protein level(0.22±0.03),two groups of corresponding comparison,the differences were there statistical significance(P<0.05).Conclusion 1 Nrf2 and TLR-1 may be involved in the occurrence of t PROM merger HCA;2 the expression level of Nrf2 and TLR-1 may be a new biomarker for the diagnosis of t PROM combined with HCA.