The Role Of COX-2/PGs-HIF-1? On Endometrial Disintegration And Exfoliation And A Preliminary Study On The Physiological Repair Of Endometrial Using A Rat Pseudopregnancy Menstrual-like Model

Menstrual cycle is an important physiological character in primate,in which endometrium undergoes periodic change of proliferation,differentiation and endometrial breakdown under the finely regulation of estrogen and progesterone.The repair and regeneration process is well ready for pregnancy.Here,we were focus on the mouse/rat menstrual-like model,and study the mechanism of endometrial breakdown shedding and the following repair.It included two aspects:the study on COX-2/PGs regulated HIF-1? in endometrial breakdown and shedding,and study on the endometrium physiological repair by rat pseudopregnancy menstrual-like model.1.The study on COX-2/PGs regulated HIF-1 ? in endometrial breakdown and shedding.The typical menstruation hypothesis is that progesterone withdrawal increased prostaglandin level,then cause the spiral artery contraction,which led to ischemia and hypoxia,finally endometrial breakdown,shedding and bleeding.Cyclooxygenase is the rate limiting synthetase of prostaglandin,and its' subtype COX-2 is important in female reproduction.In our previous study,COX-2 mRNA and protein increased in endometrial breakdown and shedding.In our study,COX-2 inhibitor Dup-697 was administrated to mouse menstrual-like model as treatment group,and the vehicle was as the control group.The mice were killed at 0 h-24 h after progesterone withdrawal,the uteri were collected and endometrial breakdown and shedding was observed by H&E.Differential gene expressions were explored at 16 h after progesterone withdrawal by expression microarray.The expressions of HIF-1 signal member including Il-6,PHD3,VGEFA,HIF-l?,NOS-2,Eno and Filt-lwere verified by qPCR.The HIF-1? protein location was explored after progesterone withdrawal 16h?24 h by immunohistochemistry.And the expression of HIF-1? protein was detected after progesterone withdrawal 0 h-24 h by western blot.Moreover,HIF-la inhibitor 2ME was administrated to mouse menstrual-like model and endometrial breakdown and shedding was assayed by H&E.The result showed that comparing to the control group,Dup-697 treatment could obviously decrease the endometrial breakdown and shedding,and the difference in the two groups increased with the breakdown proceeding.Comparing to the control group,there were 1873 upregulated genes and 2017 down-regulated genes in Dup-697 treatment group.Moreover,the members of HIF-1 signal differentiated obviously by expression microarray.Il-6?PHD3 and VGEFA mRNA were significantly decreased at 0h-24 h after progesterone withdrawal(P<0.05),and HIF-1? mRNA was significantly decreased at 0 h~16 h after progesterone withdrawal(P<0.05),significantly increased at 24 h after progesterone withdrawal by qPCR.These genes all increased at 0-16 h after progesterone withdrawal,which was consistent with trend of endometrial breakdown proceeding.So it indicated that COX-2 regulated HIF-1?signal members Il-6,PHD3,VGEFA and HIF-1? in endometrial breakdown and shedding.Further,comparing to control group,COX-2 inhibitor could inhibit HIF-1A action,and 2ME significantly could also inhibit endometrial breakdown and shedding.In all,COX-2/PGs activated HIF-1A to breakdown endometrium,in which,HIF-1? was not potentially easy to degradation in cytoplasm by PHD3 ubiquitylation being inhibited,and HIF-1? was activated by Il-6 in vascular endothelial cell.2?The study on the endometrium physiological repair by rat pseudopregnancy menstrual-like model.After breaking down and shedding,the endometrium functional layer underwent rapidly physiological repair,whereas,the molecular mechanism was not clear.The clinical endometrium sample was not easy to available and individual difference.Moreover,mouse,as the mammal model,was not easy to operate in the small uterus.Here,the rat pseudopregnancy menstrual-like model was explored and the endometrial repair process was also studied.The female rats were mated with castrated male rats,the vaginal plug was observed as the pseudopregnancy 0 d,and decidualization was artificially induced at 4 d and 5 d,the ovariectomy was operated from 6 d~8 d,the time of decidualization beginning and duration were explored.After decidualization optimization,the rats were killed after ovariectomy 32 h,40 h,48 h,56 h and 64 h.The uteri were collected,the endometrial repair was observed by H&E.Moreover,the repair changes in the rat menstrual-like model were compared to mouse menstrual-like model.The results showed that decidualization optimization was that began at pseudopregnancy 4 d,and ended at pseudopregnancy 7 d,which led to successful decidualization and endometrial breakdown and bleeding.Further,the physiological repair began at 32 h after ovariectomy,and ended 64 h after ovariectomy,which lasted 32 h.The functional layer cells and epithelial cells in the uterine cavity were gradually regenerated,proliferated and differentiated during the repair process.Moreover,the endometrial repair in the rat began late 8 h,and was longer 8 h than in the mouse.