The Mechanisms Of AOPPs Modulating The Progression Of Endometriosis Through Epithelial-mesenchymal Transition And Fibrosis

BackgroundEndometriosis(EMs)is a disease in which functional endometrial glands and stroma are implanted outside the uterine cavity,which can widely accumulate the female reproductive system,including uterus,ovary,fallopian tube,pelvic organs and so on.At present,its pathogenesis is not clear.The main theories include:ectopic implantation theory(also known as menstrual blood countercurrent theory),eutopic endometrium determinism,body cavity metaplasia theory and induction theory,stem cell theory,genetic factors,immune factors and environmental factors and lifestyle.As a common gynecological disease,EMs can cause dysmenorrhea,sexual intercourse pain,infertility and chronic pelvic pain,which has a serious adverse impact on the reproductive health of women of childbearing age.It is reported that 40%of EMs is infertile,and about 25%of infertility is EMs,.Therefore,in 2000,foreign scholars put forward the new concept of "endometriosis-associated infertility"for the first time,that is,"endometriosis infertility".The clinical treatment of the disease includes drug and surgical treatment and assisted reproductive technology,but there are many problems,such as high cost,low pregnancy rate and so on,which seriously reduce the quality of life of patients and affect their family life.In recent years,the incidence of EMs has shown an upward trend,which has become a major public health problem of global concern.The fundamental reason is that the mechanism of EMs infertility is not clear,it is difficult to fundamentally solve the reproductive health problems of such patients.Epithelial mesenchymal transformation(EMT)refers to the transformation of epithelial cells into stromal cells under specific physiological and pathological conditions.in this process,the cells lose cell polarity,adhesion and cell bridging after cytoskeleton remodeling.The characteristics of interstitial cells,such as migration and invasiveness,were obtained.Previous studies have found that EMT and fibrosis play an important role in the occurrence of endometriosis,which is manifested in that EMT of endometrial epithelial cells promotes local inflammatory injury,repair,fibrosis,cell invasion and metastasis in endometriosis.At the same time,epithelial cells obtain interstitial cell-like invasion and motility,so that endometriosis is a benign disease but has the characteristics of malignant tumor invasion and metastasis.Advanced oxidation protein product(AOPPs)is the end-stage product of protein oxidation in vivo,which can induce EMT and fibrosis.it is the pathogenesis of many chronic diseases.AOPPs play an important role in inflammatory injury,repair,cell invasion and metastasis of endometrial epithelial cells in endometriosis.Previous studies by our group have shown that abnormally high AOPPs,as an endogenous pathogenic substance,induce proliferation,migration,oxidative stress and inflammatory injury of endometrial epithelial cells in and out of the conductor model.It is suggested that AOPPs are related to the dysfunction of endometrial cells,but it is not clear whether AOPPs are involved in the process of EMT and fibrosis of endometrial cells.Therefore,through the in-depth study of EMT and fibrosis of endometrial epithelial cells induced by AOPPs,new influencing factors and their mechanisms in the process of cell dysfunction were explored.To elucidate the pathogenesis of AOPPs-induced endometriosis to provide a new theoretical basis and develop new ways of intervention.The purpose of this study was to detect the expression of AOPPs in EMs,to analyze the correlation between AOPPs expression and EMT and fibrosis indexes,and to study the role and related molecular mechanism of AOPPs in the process of EMT and fibrosis of EMs in vitro and in vivo.It provides a new theoretical basis for further elucidating the pathogenesis of EMs induced by AOPPs and provides a new idea for finding targets for intervention in the pathogenesis of EMs.Materials and methods1.Study on the relationship between AOPPs and EMT and fibrosis in endometriosis.The specimens of ectopic endometrium and eutopic endometrium from 20 patients with clinically diagnosed EMs and 20 cases of normal endometrium were collected.immunohistochemical(IHC)was used to detect the presence of endometriosis in the state of endometriosis.The expression of AOPPs,EMT and fibrosis in ectopic ovarian cysts and eutopic endometrium was compared with that in normal women.2.Effect of AOPPs on EMT and fibrosis of endometrial epithelial cells and its mechanism.Preparation and concentration determination of AOPPs.Rat serum albumin(RSA)was incubated with sodium hypochlorite to prepare AOPPs,and then the concentration of AOPPs,the quantity of protein and the content of endotoxin were determined.Extraction,identification and culture of primary SD rat endometrial epithelial cells.The endometrium of 8-week-old SD female rats was scraped,and endometrial epithelial cells were isolated by digestion,filtration,cleavage and filtration.Immunofluorescence assay was used to identify the type and purity of the extracted cells,and cell culture was carried out.After exogenous supplementation and endogenous inhibition of AOPPs,the changes of EMT and fibrosis indexes of endometrial epithelial cells were detected by immunofluorescence and Western blot techniques.After exogenous supplementation and endogenous inhibition of AOPPs,the expression of ERK1/2 and P38 in endometrial epithelial cells was detected by Western blot,and the signal pathway blocking experiment was carried out to analyze the mechanism of AOPPs regulating EMT and fibrosis in endometrial epithelial cells.3.The role of AOPPs in EMT and fibrosis of endometrial epithelial cells was verified by animal experiments.After 4 weeks,8 weeks,12 weeks and 16 weeks of experimental treatment,the rats were killed by intraperitoneal injection of AOPPs,respectively.the endometrial tissue was taken out and EMT and fibrosis indexes were detected.In order to further verify that AOPPs mediates the process of EMT and fibrosis of endometrial epithelial cells.Results1.Expression and Identification of AOPPs,EMT and Fibrosis Indexes in EMs and normal female tissues.The results of IHC showed that the expression of AOPPs was high in EMs,suggesting that AOPPs was involved in the pathogenesis of EMs,while the expression of E-cadherin protein was down-regulated and the expression of Vimentin protein was up-regulated in EMs.The expression of Collagen I protein was up-regulated and the deposition of collagen fibers in EMs was increased,suggesting that AOPPs in EMT is related to the occurrence of fibrosis.The results of IHC showed that AOPPs was positively correlated with interstitial cell markers,negatively correlated with epithelial cell markers and positively correlated with fibrosis markers,suggesting that AOPPs may affect EMs-related fibrosis and disease progression through EMT.2.Effect of AOPPs on EMT and fibrosis of endometrial epithelial cells and its mechanism.The extracted cells were identified by immunofluorescence that keratin was expressed on the cell membrane and no vimentin was expressed,which could be recognized as endometrial epithelial cells.The results of immunofluorescence assay and Western blot showed that AOPPs down-regulated the expression of iconic protein in epithelial cells in a concentration-and time-dependent manner compared with the blank control group.However,the expression of interstitial cell marker protein and fibrosis marker protein was up-regulated in a concentration and time manner.These results suggest that AOPPs can induce EMT and fibrosis in endometrial epithelial cells.3.AOPPs induces EMT by activating oxidative stress in endometrial epithelial cells.The results of Western blot showed that in epithelial cells the expression of landmark protein was up-regulated compared with that in AOPPs group,while the expression of landmark protein and fibrosis marker protein in interstitial cells was down-regulated after pretreatment with oxidation inhibitor and stimulated by AOPPs.These results suggest that oxidation inhibitors can inhibit the occurrence of EMT and fibrosis in endometrial epithelial cells induced by AOPPs,which indicates that oxidative stress response is involved in the process of EMT and fibrosis induced by AOPPs in endometrial epithelial cells.4.AOPPs induces EMT and fibrosis in endometrial epithelial cells by activating MAPK pathway.Western blot results showed that compared with AOPPs group,the mRNA and protein expression of epithelial cell marker protein in AOPPs+MAPK pathway blocker group were up-regulated,while the expression of interstitial cell marker protein and fibrosis marker protein were down-regulated.These results suggest that MAPK blocker can inhibit the occurrence of EMT and fibrosis in endometrial epithelial cells induced by AOPPs,which indicates that MAPK pathway mediates the process of EMT and fibrosis induced by AOPPs in endometrial epithelial cells.5.The role of AOPPs in EMT and fibrosis of endometrial epithelial cells was verified by animal experiments.The results of IHC assay showed that the expression of epithelial cell marker protein in AOPPs treated group was up-regulated compared with the control group,while the expression of interstitial cell marker protein and fibrosis marker protein was down-regulated.It is suggested that AOPPs can promote the process of EMT and fibrosis in rat endometrial epithelium.ConclusionAOPPs is closely related to EMT and fibrosis of endometriosis.AOPPs can activate the expression of p-ERK and p-P38 in MAPK pathway,and then make rat endometrial epithelial cells transform into epithelial stroma,thus promoting the occurrence and development of endometriosis.