1.involvment Of NLRP3 Inflammasome In The Initiation Of Parturition 2.Correlation Between Hydrogen Sulfide Hynthetases And Contraction Associated Proteins In Human Myometrium

Preterm birth is the major cause of perinatal mortality and morbidity,accounting for 70-80% of all infant deaths.It lacks effective clinical management approach to delay preterm labor.This fail is largely due to our ignorance of the fundamental mechanisms underlying uterine transformation from quiescent state into contractive state during pregnancy.Recently,accumulating evidences indicate that parturition is closely associated with inflammation within pregnancy related tissues.NOD-Like Receptors,especially the pyrin domain-containing 3?NLRP3?inflammasome,which is composed of NLRP3 ? ASC and caspase-1,play a key role in mediating inflammatory responses by regulating production of the Pro-inflammatory cytokines IL-1??IL-18 in various tissues.Our previous study has reported that during the later of pregnancy,there secrete a large number of inflammatory cytokines,including IL-1??IL-6?TNF-? which contribute to the onset of labor.Therefore in the first part of the thesis we investigate whether NLRP3 plays a crucial role in the onset of the term and preterm labor.Hydrogen sulfide?H₂S?,which is now known as one of the new gas signal molecules,has been proven to serve as an inflammatory mediator in many inflammatory responses.Our previous study has reported that human myometrium and chorionic membrance can produce H₂S through its synthetase cystathionine-g-lyase?CSE?and cystathionine-b-synthetase?CBS?.The switch from pregnancy to delivery involves various changes in expression of contraction associated proteins.Increasing body of evidence indicates that human labor is an inflammatory event which results in the activation of nuclear factor kappa B?NF-?B?in uterus.Our aim was to analyze the correlation between the level of CBS or CSE and the expression of contraction related proteins including oxytocin receptor?OTR?,cyclooxygenase-2?COX-2?,connexin-43 and 15-hydroxyprosglandin dehydrogenase PGDH in pregnant human myometrium and chorionic membrance at term.Main Results: The results of first part1.The myometrial tissues?chorionic ?amniotic membranes were obtained from pregnant women who were in preterm labor?PTL??labor?TL?or not in labor?TNL?at term.Western blotting was used to determine the expression of NLRP3,ASC,ELISA was used to measure the levels of cytokines,and the caspase-1 enzyme activity was determined by the kit.The expression of NLRP3,ASC and the production of interleukin?IL?-1?,IL-18 were significantly up-regulated in TL?or PTL?groups compared with TNL group?P < 0.01?.The level of activated caspase-1 was significantly higher in TL?or PTL?tissues than that in TNL biopsies?P < 0.01?.2.The myometrial tissues were obtained from pregnant mouses which were in a series of pregnancy time points :GD15?GD17?GD19?term labor and labor after treated with inhibitor on GD18.5.Western blotting was used to determine the expression of NLRP3?ASC,ELISA was used to measure the levels of cytokines,and the caspase-1 enzyme activity was determined by the kit.The expression of NLRP3,ASC and the production of interleukin?IL?-1?,IL-18 were significantly up-regulated in TL groups compared with TNL group?P < 0.01?.The level of activated caspase-1 was significantly higher in TL tissues than that in TNL biopsies?P < 0.01?,However the expression of NLRP3,ASC and the production of interleukin?IL?-1?,IL-18 were significantly down-regulated in inhibitor treated groups compared with TL group?P < 0.01?.The level of activated caspase-1 was significantly lower in inhibitor treated tissues than that in TL biopsies?P < 0.01?.3.The myometrial tissues were obtained from preterm labor mouses which were treated with three differents drugs on GD15 : RU38486?RU38486 and inhibitor ?LPS.Western blotting was used to determine the expression of NLRP3,ASC,ELISA was used to measure the levels of cytokines,and the caspase-1 enzyme activity was determined by the kit.The expression of NLRP3,ASC and the production of interleukin?IL?-1?,IL-18 were significantly up-regulated in PTL groups?treated which RU38486 or LPS?compared with TNL group?P < 0.01?.The level of activated caspase-1 was significantly higher in PTL tissues?treated with RU38486 or LPS?than that in TNL biopsies?P < 0.01?,However the expression of NLRP3,ASC and the production of interleukin?IL?-1?,IL-18 were significantly down-regulated in inhibitor treated groups compared with PTL group?treated with RU38486??P < 0.01?.The level of activated caspase-1 was significantly lower in inhibitor treated tissues than that in PTL biopsies?treated with RU38486??P < 0.01?.The results of second part1.The myometrial tissues were obtained from pregnant women who were in labor?TL?or not in labor?TNL?at term.Western blotting was used to determine the expression of CSE?CBS and OTR?COX2?CX43 in pregnant myometrium.The expression of OTR,COX-2,was significantly up-regulated in TL group compared with TNL group?P < 0.01?.The expression of CBS and CSE was significantly lower in TL tissues than that in TNL biopsies?P < 0.01?.Significant negative correlations were found between the expression of CBS?CSE and the expression of CX43?P < 0.05?,OTR?P < 0.05?,COX-2?P < 0.05?.Upregulation of UAPs such as OTR,COX-2,CX43 is correlated to the H₂S in pregnant human myometrium at term.2.The myometrial tissues were obtained from pregnant women who were in labor?TL?or not in labor?TNL?at term.Western blotting was used to determine the level of activated NF-?B and the expression of OTR,COX-2 in pregnant myometrium.The expression of OTR,COX-2,was significantly up-regulated in TL group compared with TNL group?P < 0.01?.The level of activated NF-?B was significantly higher in TL tissues than that in TNL biopsies?P < 0.01?.Significant positive correlations were found between the level of activated NF-?B and the expression of OTR?P < 0.05?,COX-2?P < 0.05?.Upregulation of UAPs such as OTR,COX-2 is correlated to the activation of NF-?B in pregnant human myometrium at term.What is more,significant negative correlations were also found between the level of activated NF-?B and the expression of CBS and CSE,Downregulation of CBS and CSE is correlated to the activation of NF-?B in pregnant human myometrium at term.3.The chorionic tissues were obtained from pregnant women who were in labor?TL?preterm labor?PTL?or not in labor?TNL?at term.Western blotting was used to determine the expression of CSE?CBS and PGDH in pregnant chorionic.The expression of PGDH was significantly down-regulated in TL group compared with TNL group?P < 0.01?.The expression of CBS and CSE was significantly lower in TL tissues than that in TNL biopsies?P < 0.01?.Significant positive correlations were found between the expression of CBS?CSE and the expression of PGDH?P < 0.05?,Upregulation of PGDH is correlated to the H₂S in pregnant human chorionic tissues at term.Conclusions:1,NLRP3 inflammasome is activated in intrauterine tissues including chorionic,amniotic membrane in full-term birth and in preterm birth.NLRP3 inflammasome is also activated the animal model of full-term birth and preterm birth.Inflammatory cytokines and chemokines mRNA levels are increased significantly.After administration of NF-?B inhibitors,inflammatory cytokines and chemokines mRNA level was significantly decreased,and the activation of NLRP3 inflammasome was suppressed.It suggests that NLRP3 inflammasome may play an important role at the start of labor by regulating the inflammatory response.2,In the uterine and chorionic membranes,the correlation analysis showed that the level of CSE and CBS inversely correlated to the expression of CAPs,like:OTR,CX43,COX2 and PGDH,and the correlation between H₂S and the level of actived NF-?B both prove that during parturition,H₂S may by inhibiting the contraction related proteins expression via inhibit the inflammation,therefore inhibit the uterine contraction to maintain the resting;...