Molecular Mechanism Of Endometrial Serum And Glucocorticoid Regulated Kinase 1 In Early Pregnancy Loss

Part I The alteration of SGK1 expression in human deciduas during early pregnancy lossObjective:To determinate the expression of serum glucocorticoids regulated kinase 1(SGK1)in the decidual tissue of early pregnancy,compare the difference of SGK1 expression in the decidual tissue between early miscarriage and normal pregnancy and explore the biological significance of SGK1 in the microenvironment of the maternal-fetal interface during the first trimester of pregnancy.Materials and methods:1.88 individuals who had been diagnosed with unexplained early miscarriage composed as the research group,and in the same period,71 individuals of early normal pregnancy as the control group.2.The expression of decidual SGK1 in uterine tissues was detected by immune histochemical method and immunofluorescence technique.3.The relative mRNA expressions of SGK1 in the deciduas of the two groups were measured by Real-time quantitative PCR after reverse transcription.4.After cleavage of the decidual protein,BCA method was applied to detect SGK1 protein quantification,and the relative expressions of SGK1 protein in the decidual tissue of the two groups were detected by Western blot.Result:1.No statistically difference were found as regards age,gestational age,menarche age,menstrual cycle,menstrual days between the two groups.The level of serum estradiol(E2)of early miscarriage was lower when compared with normal pregnancy(P<0.001).2.Immune histochemical method and immunofluorescence showed that SGK1 was expressed in the decidual tissue of patients with early pregnancy loss and normal pregnancies.3.Real time PCR showed a significant decreased relative mRNA expression of SGK1 in the decidual tissue of early miscarriage when compared with early normal pregnancy(P<0.001).4.The total protein level of SGK1 in the decidual tissue of early miscarriage was significantly decreased as compared with the control group(P<0.001).The phosphorylated SGK1 protein in the decidual tissue of early miscarriage,as the same as the total SGK1 protein,was also significantly decreased as compared with the control group(P<0.001).5.The rate of phosphorylated to total SGK1 protein in the decidual tissue of early miscarriage was significantly decreased when compared with the control group(P<0.001).Conclusion:1.SGK1 was expressed abundantly in the decidual tissue during early pregnancy.2.The tanscription level of SGK1 was obviously decreased in the decidual tissue of early miscarriage.3.The total and phosphorylated protein level,and rate of phosphorylaed to total protein of SGK1 was obviously reduced in the decidual tissue of early miscarriage.Part ? The mechanism involved in the down-regulation of decidual SGK1 causing early pregnancy lossObjective:To explore the function and mechanisms of decidual SGK1 in early pregnancy loss.Materials and methods:1.Uterine decidual cells cultures were established and the purity of decidual cells was determined by immunofluorescence method.2.The MTT colorimetric test was used to investigate the proliferation of decidual cells.The apoptosis of decidual cells was measured by Annexin V-FITC/PI test.3.Specific SGK1 siRNA was applied to down-regulate the expression SGK1 in decidual cells,and the measurement of the apoptosis in decidual cell was followed.4.The cell model of early pregnancy loss in vitro was established by treating the decidual cell culture with Lipopolysaccharide(LPS)and function and molecular mechanisms of SGK1 in early pregnancy loss were explored.5.Using enzyme linked immunosorbent assay,the levels of cytokines in the supernatants of cultures of decidual cells were detected.Results:1.Specific SGK1 siRNA interference to decidual cells for 24 hours significantly increased the cell apoptosis as compared to the control group(P<0.01).2.The expression of SGK1 in the decidua cells was significantly up-regulated by estradiol administration(P<0.001).3.The reduced cell proliferation of decidual cells caused by lipopolysaccharide treatment was recovered by estradiol administration and SGK1 activation.The activation of SGK1 also decreased cell apoptosis of decidual cells caused by lipopolysaccharide(P<0.01).The expressions of anti-apoptosis genes BCL-2 and XIAP were increased by the activation of SGK1 in the decidual cells after the lipopolysaccharide treatment(P<0.01).4.The activation of SGK1 by estradiol treatment recovered the reduced secretion of IL-4 and IL-5 cytokine of decidual cells caused by lipopolysaccharide treatment(P<0.001 and P<0.01);whereas decreased the secretion of IFN-? of decidual cells(P<0.01).The ratio of IL-4/IFN-y was increased by the activation of SGK1 when compared with lipopolysaccharide treatment(P<0.001).5.Estradiol-activated SGK1 reduced the phosphorylation of NF-?B protein,therefore suppressed the activity of NF-?B(P<0.01).Conclusion:1.SGK1 actived by estradiol enhanced the cell proliferation and reduced the cell apoptosis of uterine decidual cells.2.By means of inhibiting the activity of NF-?B,estradiol activated SGK1 can reduce Thl secretion and increase Th2 secretion in uterine decidual cells to suppresse the progress of early pregnancy loss and support a normal pregnancy.