Rapamycin Prevents The Formation Of Thin Endometrium By Regulating Autophagy

Background: Up to now,the definition of thin endometrium hasn’t reached a clear consensus.An appropriate thickness of the endometrium is a necessary condition for the successful intrauterine implantation of fertilized eggs.Relevant studies showed that under certain conditions,the pregnancy rate has no significant relationship with the age of patients and the quality of embryo,however,is positively correlated with the thickness of endometrium.Thin endometrium may be associated with spiral arteries and ovarian arteries.Insufficient perfusion of arterial blood flow not only affects the implantation of fertilized eggs,but also leads to local hypoxia unfavorable for embryonic growth.Thin endometrium not only affects the fertilized egg planting,but also increases the risk of biochemical pregnancy/natural abortion.It is also an important influencial factor for hypomenorrhea,amenorrhea and infertility.The causes of thin endometrium are still unknown.Systemic factors(age and endocrine),local factors(the history of uterine cavity operation and history of uterine diseases),external use of drugs and other underlying diseases,and concealment of unknown factors have been proposed as potential causes of thin endometrium.No system of any theory to explain the development of thin endometrium has been established and no treatment has been proven to be significantly effective.Therefore,it is necessary to explore the possible pathogenesis and treatment of thin endometrium.Purposes: 1.Rat thin endometrial models of bilateral uterine artery ligation intervened by rapamycin drugs were used to study the efficacy and possible molecular mechanisms of rapamycin on thin endometrium at animal level.2.Human endometrial primary cells in the inflammatory environment were treated with rapamycin and 3-MA,and the molecular mechanisms of rapamycin treatment for thin endometrium was explored at cellular level.Methods: 1.Uterine ischemic and hypoxic endometrium models with bilateral uterine artery ligation were established in female Sprague-Dawley rats aged 8-10 weeks,and detached uteri were sectioned after 4 weeks of rapamycin intervention.Staining and histomorphological analysis was performed;RT-PCR detection of endometrial phenotype protein and autophagy-related protein expression levels to preliminarily explore the effects of rapamycin on ischemia and hypoxia of rat endometrium and potential molecular mechanisms.2.Human endometrial epithelial cells(HEEC)and stromal cells(HESC)were isolated and cultured;primary cells were identified by immunofluorescence;HEEC and HESC were treated with IL-1β of different concentrations,and immunofluorescence and western blot were performed to detect the expression level of vimentin and Cytokeratin 8,autophagy-related proteins including Beclin-1,LC3 and p-mTOR.Rapamycin and 3-MA treated with IL-1β-induced HEEC and HESC,respectively.Fluorescence and Western blot were used to detect the expression of Vimentin,Cytokeratin 8,autophagy-related proteins Beclin-1,LC3 and p-mTOR.Results: 1.In the same period of estrous cycle,the thickness of endometrium measured in the surgical group was reduced compared with the sham group;the thickness of the endometrium measured in the surgical group was increased compared with the control group.The mRNA of the endometrial cell markers in the surgical group was decreased compared with the sham group,and the level of mRNA related to autophagy-related protein was lower than that of the sham group.The mRNA level of the endometrial cells in the surgical group was higher than that in the sham group,and the mRNA level of the autophagy-related protein was increased.Compared with the control group,the autophagy-related protein was positively correlated with the endometrial phenotypic protein.2.Screening after type II collagenase digestion could effectively extract and isolate HEEC and HESC efficiently.IL-1β inhibited the expression of two important phenotypic proteins in endometrium.When IL-1β concentration is 20 ng/ml,the phenotypic proteins were all significantly inhibited.20 ng/ml was recognized as the appropriate concentration for inducing the pathophysiological process of thin endometrium at the cellular level.3.IL-1β bidirectionally regulated the autophagy level of HESC and HEEC.The autophagy-related proteins were promoted by IL-1β of low concentration.However,IL-1β of high concentration,e.g.20ng/ml,suppressed the expression of autophagyrelated proteins obviously.4.The phenotypic proteins of HESC and HEEC were corelated with autophagy positively.IL-1β regulated the expression of phenotypic protein through biphasic regulation of autophagy of HESC and HEEC.Rapamycin could promote autophagy in HESC and HEEC to antagonize the inhibitory effect of high concentration of IL-1β on phenotypic proteins.Conclusion: 1.Rapamycin inhibits the formation of thin endometrium in rats by regulating the level of autophagy in endometrial cells.2.IL-1β affects the expression of phenotypic proteins of HESC and HEEC in a concentration-dependent manner.3.IL-1β regulates the autophagy level of HESC and HEEC bidirectionally in a concentration-dependent manner.4.Rapamycin antagonizes the inhibitory effect of IL-1β on phenotypic proteins of HESC and HEEC by regulating autophagy.