Shh Signaling Suppressing Autophagy Of Endometrial Stromal Cell Is Involved In Intrauterine Adhesion Pathogenesis

Background:Intrauterine adhesion（IUA）is the major cause of secondary infertility in women and can lead to menstrual abnormalities and a variety of adverse pregnancy outcomes.IUA most commonly causes by medical trauma to the gravid uterus,and its appropriate treatment of transcervical resection of adhesion（TCRA）can restore uterine anatomical form,while it can’t restore endometrial physiological function.Besides,there is a high IUA reformation rate of moderate-severe IUA after TCRA.It is therefore essential to clarify the IUA occurrence and development mechanisms,which will be the basis of new therapy exploitation.Endometrial fibrosis is the major histopathological change of IUA,and also is considered to assess the severity.Sonic hedgehog pathway is an evolutionaryly conservative morphology pathway,and autophagy is a cellular protein degradation system that responds adaptively to stress,and they all have important effects during tissue repair,but it is not yet known how they play a role in endometrial pathological repair.Study content and method:Firstly,this study explores whether the status of shh signaling and autophagy pathway change in the IUA lesion by detecting shh pathway molecules and autophagic related molecules.Endometrial fibrosis is the underlying pathological change of uterine adhesion,and endometrial stromal cells are the most promising ECM-producing cells,and are therefore the focus in this study.Then,based on T-HESCs,this study use the overexpression of SHH full-length protein,SMO activator,Glis inhibitor and a variety of autophagy-related methods to study whether the shh pathway regulates autophagy,and how shh pathway regulates autophagy.Further,we explores whether autophagy is involved in shh-induced fibrosis in-vitro using autophagy blockers or silencing autophagy key molecules.Moreover,based on murine uterine IUA model,this study use a combination of GANT61 and Chloroquine（CQ）to investigate whether autophagy contributes to shh-induced fibrosis in vivo.At last,applying endometrial stroma-specific genetically modified mice to establish IUA model and treated with autophagic drugs,including Amhr2^cre/+R26-Smo M2^+/-（AM2）combined with autophagy activator,and Amhr2^cre/+Smo^flox/flox（AFF）combined with autophagy inhibitors to explore whether autophagy is involved in shh-mediated endometrial fibrosis in endometrial stromal cells.Results:Firstly,we find SHH ligand expression increased significantly in both human and murine IUA tissue and autophagy-related molecules（LC3B,p62）expressions vary in distinct regions in the endometrial stroma of human IUA samples and are related to ECM expression.These indicate both shh signaling and autophagy have a role in endometrial regeneration and fibrosis progression.Then,considering PTCH1 receptor expresses in IUA endometrial stroma of human and mouse,we use T-HESCs as in-vitro systems,and find SHH-fl overexpression and SMO activators reduce the expression of LC3B-II,while Glis inhibitor（GANT61）increases the expression of LC3B-II in a graded manner.Subsequent Ad-m Cherry-GFP-LC3B and electroscopic experiments confirme that GANT61 induces the production of autophagosomes and autolysosomes.And using p-luci-p62 transfection,we infer that GANT61 promotes the degradation of exogenous p62.These results indicate shh pathway regulates negatively on autophagy initiation.As for the regulatory mechanism,we find GANT61 causes the gradient drop of p-AKT Ser473,p-p70 S6k accompanied with the gradient increase of LC3B-II,while AKT activator could reverse the GANT61-induced LC3B-II partially.In the exploration of downstream GLIs effect factor,we find that GANT61-induced autophagy could be reversed by overexpressing GLI2 activating form（GLI2A）or knocking down GLI3inhibiting form（GLI3R）.Moreover,we find collagen I is sensitive to autophagy degradation in human endometrial stromal cells after a simple screening,and autophagic modifications can reverse the shh-mediated collagen I change.In addition,based on murine IUA model,GANT61 treatment can relieve endometrial fibrosis in a dose-dependent manner,while combined with CQ counteract GANT61s effects partially.Importantly,characteristics of AM2 mice indicate sustained activation of the shh pathway in the endometrial stroma autonomously drives fibrotic features and is associated with decreased autophagy.And AM2 exacerbates fibrosis in the IUA model,which can be alleviated by the autophagy inducer rapamycin.On the other hand,using the newly establishment of severe IUA model,AFF mice significantly decrease the adhesion stenosis rate and fibrosis degree compaing to wild type,while the combined with CQ treatment reverses those obviously.Conclusion:In summary,defective autophagy is involved in shh signaling-driven endometrial fibrosis,suggesting a potential novel molecular target for IUA treatment.In the regulatory axis,shh pathway regulates autophagy initiation mainly through transcription factor GLI2A and GLI3R,and classical p AKT-m TORC1 autophagic regulatory mechanism also works.