The Roles And Mechanisms Of H₂S On Early Pregnancy Via Interfering With Invasion Of Trophoblasts And Regulating TH1/TH2 At The Maternal-fetal Interface

Early abortion is a key factor affecting pregnancy.Recurrent spontaneous abortion(RSA)is a common pregnancy complication.It usually refers to more than two consecutive spontaneous abortions.Currently,RSA is a main fertility problem in the worldwide.Unexplained RSA(URSA)is often associated with immunological abnormalities at the maternal-infant interface,suggesting that immunological disorders are its predisposing factors.However,the exact mechanism of RSA is still unknown.Hydrogen sulfide(H2S)is primarily synthesized from L-cysteine(L-Cys)by cystathionine ?-synthase(CBS)and cystathionine ?-lyase(CSE).CBS and CSE are expressed in a variety of organs with a tissue/cell-specific pattern.In recent years,studies have shown that H2S exists in the human reproductive system,and participates in regulating various reproductive processes.Not only that,H2S is also involved in immune regulation and immune tolerance.However,the role of H2S in early pregnancy is unclear.Our goal was to clarify whether H2S affected embryonic development in early pregnancy and the mechanism by which it worked.Our research found that CBS/H2S signaling regulated the migration and invasion of trophoblast cells to affect early pregnancy by changing the levels of metal matrix proteinase 2(MMP2),vascular endothelial growth factor(VEGF).In the study of clinical samples,it was found that the CBS but not CSE protein in RSA women's villi was significantly reduced.And,CBS protein was obviously lower in the villous cytotrophoblast(CTB),but not syntiotrophoblast(STB).Next,using HTR8/SVneo and JEG3 cell lines for vitro experiments,we found that either exogenous H2S or endogenous over-expression of CBS could cause the up-regulation of MMP2 and VEGF to promote migration and invasion of trophoblast cells.In vivo experiments,by CBS deficiency mice and pharmacological intervention methods,we found that the deficiency or inhibition of CBS caused the mice embryo absorption in gestational day 12.5.The important thing was that the above embryo absorption could be significantly ameliorated after treatment with exogenous H2S donors.Meanwhile,by?CBA/J x?DBA/2 spontaneous abortion mice,we confirmed that H2S could rescue the embryo loss.On the other hand,our experimental results proved that CBS/H2S signal exerted immunomodulatory effects via interfering with NF-?B inflammation pathway and human chorionic gonadotropin(HCG),cyclooxygenase-2(COX2),prostaglandin 2(PGE2)and other cytokines,and ultimately affected the balance of TH1/TH2 by the changes of helper T cell type 1(Type 1 T helper,TH1)and helper T cell type 2(Type 2 T helper,TH2)cytokine at the maternal-fetal interface.The metabolic disorder of above-mentioned cytokines could lead to RSA,and CBS-derived H2S was essential for maintaining immune homeostasis.Vitro studies showed that the levels of Th2 cytokines(IL-4 and IL-6)in decidual tissues of aborted mice were significantly reduced,while Th1 cytokines(IFN-y and TNF-?)were not changed or increased.And,the low-dose H2S donor and CBS over-expression could reduce the level of HCG,but no change for IDO and TSLP.We performed RNA-seq analysis on the transcriptome of HTR8/SVneo cells after over expressing or knocking down CBS.We found that CBS/H2S targeted the NF-?B inflammation signaling pathway.Interleukin-1 receptor 1(IL-1R1),prostaglandin synthase 2(PTGS2/COX2)were rich in NF-?B signaling pathway and biological processes about inflammation.Moreover,CBS/H2S affected the levels of IL-1R1,COX2 and PGE2,and CBS overexpression could down-regulate IL-1R1,COX2 and PGE2.By Western blotting,the key proteins P-IKB and P-P65 in the NF-?B signaling pathway were significantly down-regulated when the CBS protein was over-expressed,but no difference for P65.In conclusion,we believed that CBS/H2S signaling played very important roles in maintaining early pregnancy,which might be achieved by interfering with trophoblast cells invasion and regulating maternal-fetal interface immune tolerance to affect the TH1/TH2 balance.Part I The role and mechanism of H2S on early pregnancy via interfering with invasion of trophoblastsObjective1.Investigate the characteristic expression of CBS/H2S at the mother-fetal interface2.Explore whether abnormal H2S causes abnormal pregnancy3.Study whether H2S affects the migration and invasion of trophoblast cells4.Explore how H2S affects the invasion and migration of trophoblastsMethods1.CBS and CSE proteins in human chorionic trophoblasts were detected by Western blot.Confirmed that CBS and CSE proteins in chorionic villi and decidua of trophoblast cells were expressed specifically by immunohistochemical technique.2.Using H2S synthesis-and release technology,we proved that human trophoblast cells HTR8/SVneo and JEG3 could produce H2S.3.Through virus transfection technology,the CBS proteins in human trophoblast cells were over expressed or knocked down.Trans well experimental technology was used to detect the role of H2S signaling on the migration and invasion of human trophoblast cells and choriocarcinoma cells.The effect of H2S signaling on the expression of MMP2 and VEGF in human trophoblast cells was further detected by western blot.4.Using CBS-deficient mice model and drug treatments,the effect of CBS on the absorption of mouse embryos was examined.Furthermore,it was showed that H2S could rescue embryo absorption.Results1.CBS and CSE proteins were detected in the first trimester placental villus of normal abortion women and URSA women,and the expression of CBS in URSA women was more significantly reduced.The level of CBS but not CSE protein was obviously lower in the villous cytotrophoblast(CTB),but not syntiotrophoblast(STB),in URSA vs.Normal pregnant women.2.CBS and CSE proteins were detected in HTR8/SVneo and JEG3 cell lines.Both of them could produce H2S,and the production of H2S could be significantly inhibited by the CBS inhibitor aminooxyacetic acid(AOAA)and the CSE inhibitor cystathinoine(PAG).3.Sodium hydrosulfide(NaHS)or L-Cys as H2S donor could enhance the migration and invasion of human trophoblast cells,and the abilities of migration and invasion depended on the concentration of H2S donor.Down-regulation of CBS could significantly inhibit the migration and invasion of HTR8/SVneo cells,while over-expression of CBS was opposite.4.H2S donors could promote the expression of MMP2 and VEGF in human trophoblast cells,and the expression level of MMP2 or VEGF depended on the concentration of the donor.Over-expression of CBS could up-regulate the levels of MMP2 and VEGF protein,while down-regulation of CBS was opposite.5.Vivo studies had shown that CBS-deficient mice had an obvious embryonic absorption.The treatment of AOAA or PAG could also lead to the fetal losses.Importantly,the treatment of H2S donor GYY4137 or NaHS could rescue the fetal losses caused by CBS deficiency or inhibition,and prevent immune abortions.Conclusions1.CBS proteins were reduced in the first trimester placental villus in URSA women,and they mainly occurred in the cytotrophoblast.2.H2S promoted the migration and invasion of human trophoblast cells.3.CBS/H2S promoted the expression of MMP2 and VEGF in human trophoblast cells.4.H2S donor could rescue embryonic absorption caused by CBS deficiency or inhibition.5.Both CBS deficiency and pharmacological blockade of H2S synthesis could cause obvious embryo absorption,which could be effectively improved by injecting H2S donors.Part IIThe role and mechanism of H2S on early pregnancy via regulating TH1/TH2 at the maternal-fetal interfaceObjective1.Study the effects of H2S signal on spontaneous abortion mouse model2.Study the change of cytokine concentration in the decidua tissue of aborted mice3.Study whether H2S signal mediates the synthesis and release of immune mediators of villus trophoblasts4.Study the target genes of CBS/H2S signaling in trophoblast cellsMethods1.Through RT-qPCR and ELISA technology,we detected whether CBS deficiency or inhibition affected the levels of Thl,Th2 factors in the decidua of aborted mice.2.Using ELISA technology to detect the changes of HCG,TSLP,IDO and PGE2.3.Analyzed the signal pathway of CBS/H2S targeting by RNA-seq technology.4.Detected the effects of H2S donor and up-regulation or down-regulation of CBS on IL-1R1 and COX2 in trophoblast cells by RT-qPCR.5.Detected the expression of key proteins in the NF-?B inflammation pathway by Western blot.Results1.The H2S donor(GYY4137 and NaHS)could reduce the embryonic absorption of spontaneous abortion mice and effectively improve the level of TGF-? in their decidua.2.Treatment with AOAA or PAG via i.p.injection resulted in significantly lower levels of Th2 cytokines:interleukin 4(IL-4)and interleukin 6(IL-6)in the decidua from resorbed fetuses,without significantly altering Th1 cytokines:interferon-gamma(IFN-y)and tumour necrosis factor alpha(TNF-?).Consistently,CBS deficiency also resulted in decreased Th2 cytokines and increased Th1 cytokines in the decidua of resorbed fetuses.3.Low-dose H2S could inhibit the secretion of HCG in human trophoblast cells,but little effect on TSLP and IDO.CBS over-expression could also reduce the level of HCG in human trophoblast cells,while CBS knockdown could promote it.4.CBS/H2S targeted the nuclear factor kappa-light-chain-enhancer of activated B cells(NF-?B)signaling pathway.Over-expression of CBS could reduce the levels of IL-1R1,COX2 and the secretion of PGE2 in the NF-?B signaling pathway and inflammatory response related biological process.But,down-regulation of CBS in HTR8/SVneo cells was opposite.In addition,up-regulation of CBS reduced the levels of key proteins P-P65 and P-1KB in the NF-?B inflammation signaling pathway.Conclusion1.H2S could improve the embryo absorption.2.The embryo absorption caused by CBS/H2S metabolic disorder might be related to Th1/Th2 imbalance in decidua.3.H2S might maintain immune tolerance during early pregnancy by affecting the releases of HCG and PGE2 at the maternal-fetal interface.4.CBS/H2S targeted the NF-?B signaling pathway.