| BackgroundEndometriosis(EM)is a chronic inflammatory disease with high incidence in women of childbearing age,which is closely related to autoimmunity.EM usually occurs in pelvic and abdominal organs,such as ovary,peritoneum,and rectum.The most common type is ovarian Endometriosis.EM is often accompanied by severe clinical symptoms such as dysmenorrhea,chronic pelvic pain,infertility,and EM related gastrointestinal symptoms,which seriously affect women’s quality of life and fertility.At the same time,clinical diagnosis,and treatment of EM face problems such as difficulty in early diagnosis,high recurrence rate,and difficulty in radical treatment.Therefore,EM brings a huge burden to society and brings serious physical and mental pain to women.However,the pathogenesis of EM is still complex and unknown to this day.The widely accepted pathogenesis theories include the theory of menstrual reflux,the theory of epithelial metaplasia in the body cavity,and the theory of studying abroad with Mullerian duct disabilities.However,there is no specific molecular mechanism and target for the pathogenesis,and it cannot explain that up to 90%of women have menstrual reflux while only 10%to 20%of women suffer from EM.Therefore,further research on the mechanism of EM is urgently needed.The latest research has found that retrograde menstrual blood and endometrial debris enter the abdominal cavity,triggering a series of inflammatory and immune responses.The disruption of the local immune microenvironment in the abdominal cavity plays a crucial role in the process of EM ectopic implantation,adhesion,invasion,etc.However,the specific immune imbalance mechanism is still unclear.Therefore,exploring the molecular mechanisms of EM pathogenesis from an immunological perspective and finding targets to develop personalized EM diagnosis and treatment plans has promising prospects.In the abdominal immune microenvironment,macrophages are the dominant immune cells,involved not only in the response of intrinsic immunity but also in the maintenance of local homeostasis in the complex environment of the organism.The immunomodulatory function of macrophages is turned on by their activation and polarization,and different polarization types dominate different immunomodulatory effects,thus macrophages are a double-edged sword in the progression of EM development.The current findings are contradictory:multi-factor assays of peritoneal macrophages in EM patients revealed higher than normal indices of both M1 and M2 polarization;while intraperitoneal injection of M1 macrophages into EM model mice inhibited the growth of ectopic lesions and injection of M2 macrophages promoted EM progression.Therefore,the mechanism of polarization regulation of EM peritoneal macrophages is not clear.Oxidative stress is an important influence on macrophage polarization,and studies related to inflammatory and neoplastic diseases have shown that reprogramming macrophages with oxidative stress-related genes to regulate their polarization type constitutes reprogrammed macrophages that can be used for disease treatment,but little research has been done in EM.It has been shown that in EM,due to menstrual blood reflux,endometrial fragments enter the peritoneal cavity to undergo ectopic adhesion colonization,and oxidative intermediates such as oxygen radicals are produced due to changes in cellular metabolism caused by ischemia and hypoxia,which disrupt the balance of oxidation and antioxidation,leaving the EM ectopic lesions in a long-term state of oxidative stress with high concentrations of reactive oxygen species and free iron.Therefore,exploring the effect of abnormal EM peritoneal macrophage mechanism is very important.There are many regulatory pathways involved in the impact of oxidative stress on macrophage polarization,among which the Hippo kinase pathway(a conserved signaling pathway,regulating embryonic development and tissue remodeling)is one of the key pathways affecting cell apoptosis and oxidative stress.In recent years,the core molecule in this signaling pathway,Mammalian sterile 20-like kinase 1(MST1),has been found to play a role in regulating innate and adaptive immunity.Humans and mice lacking MST1 exhibit complex immune deficiency syndrome,characterized by early onset of severe infections and other autoimmune diseases.Research has shown that MST1 is involved in the process of macrophage inflammatory factors,as well as regulating the production of reactive oxygen species.The research on the regulatory mechanism of MST1 in EM peritoneal macrophages is still uncertain.The polarization of macrophages plays an important role in the survival of EM ectopic endometrial cells.The powerful phagocytic function and pro-inflammatory factor secretion of M1 type macrophages have a certain anti ectopic endometrial cell proliferation effect,while the weakened phagocytic function and promoting repair of M2 type macrophages promote the colonization and proliferation of ectopic endometrial cells.As a way of self-redemption—autophagy in ectopic endometrial cells’ survival effect cannot be ignored.Autophage is a special programmed death,a highly conservative cell death program that has been preserved in the long-term evolution process.When cells are stimulated by hypoxia,starvation,etc.,the double membrane structure of the rough Endoplasmic reticulum without Ribosome attachment zone wraps abnormal proteins,damaged organelle,and other components to form Autophagosome,and then fuses with Lysosome to form autophagic Lysosome,which degrades and recycles the contents,Realize the "waste utilization" in cells and the renewal of Organelle.From this,autophagy is an energy and material reserve station for the sustained survival of tissues and cells.EM ectopic lesions are in the peritoneal immune microenvironment of ischemia,hypoxia,inflammation,etc.for a long time,and hypoxia and various cytokines will inevitably affect their autophagy.However,the regulation of ectopic endometrial cells by macrophages in the peritoneal immune microenvironment where EM lesions are located is not yet clear.In EM,ovarian endometriosis(OEM)accounts for about 75%,so our study mainly explores the pathogenesis of OEM.In summary,we speculate that in OEM,MST1 is involved in the regulation of oxidative stress and polarization of OEM peritoneal macrophages,which affects autophagy of ectopic endothelial cells and leads to disease progression.We found that MST1 expression was absent in OEM patients’ peritoneal macrophages,accompanied by oxidative stress and M2 polarization;through in vivo and in vitro experiments,we found that the MST1-p38-MAPK signaling pathway was involved in regulating macrophage oxidative stress and polarization,while leading to reduced phagocytosis and secretion of-IL-10 to promote autophagy of co-cultured endothelial cells;and constructed a novel OEM mouse model.Through multidimensional evaluation and validation,we found similarities between the level of oxidative stress and polarization type of peritoneal macrophages in OEM mice and OEM patients,while detecting the regulatory role of MST1-p38-MAPK signaling pathway,and finally we injected MST1 overexpressing adenovirus into the peritoneal cavity of OEM mice and found that the ovarian ectopic lesions in OEM mice were suppressed and disease progression was delayed.Objective1.To evaluate the oxidative stress status and polarization type of peritoneal macrophages in patients with ovarian endometriosis,and explore its mechanism;2.To evaluate the autophagy level of ectopic endometrial tissue and cells in patients with ovarian endometriosis;3.To explore the regulation of macrophage on autophagy of ectopic endometrial cells;4.To construct ovarian endometriosis mice model to verify the relevant phenomena and molecular mechanisms in vivo,and then we conduct rescue treatment.Methods1.This study focuses on the functional status of peritoneal macrophages and the autophagy level of ectopic endometrial cells in patients with ovarian Endometriosis from the clinical perspective.(1)Collect peritoneal fluid and/or peritoneal flushing fluid from OEM patients and control group patients:[1]We use flow cytometry to detect oxidative stress levels and polarization types of peritoneal immune cells;[2]Elisa was used to detect the concentration of IL-10 in the supernatant of abdominal fluid;[3]Separating and cultivating peritoneal macrophages,and we detect the expression levels of MST1,p38-MAPK,and Nrf2 through Western Blot,RT-PCR,and others.(2)And we collect lesion tissue of both two group,including ectopic endometrium and eutopic endometrium:[1]Western Blot,RT-PCR,immunohistochemistry,and multiple immunofluorescences were used to detect their autophagy levels;[2]Isolate and culture endometrial cells(in situ and ectopic),and transfect them with GFP-mCheer-LC3 adenovirus to detect their autophagy flux.2.siRNA-MST1-THP-1(via PMA induced to macrophages,later collectively referred to as THP-1)was constructed by using transfection technology to downregulate MST1 expression and investigate the mechanism of the regulation of MST1 in macrophage immunomodulatory function.[1]Flow cytometry was used to detect the oxidative stress level and polarization type of siRNA-MST1-THP-1;[2]Elisa was used to detect the concentration of IL-10 in the supernatant of siRNA-MST1-THP-1 culture medium;[3]Western Blot,RT-PCR and others were used to detect the expression level of p38-MAPK;[4]After co-cultured with siRNA-MSTl-THP-1,HESCs was collected to detect the expression of autophagy related genes and autophagy flux by using Western Blot,RT-PCR,and GFP-mCheer-LC3 adenovirus transfection;[5]We add IL-10 into HESCs and then use Western Blot and transfection with GFP-mCheer-LC3 adenovirus to detect and evaluate the effect of IL-10 on autophagy of HESCs.3.OEM mice model was constructed to verify the above results,and MST1 overexpression adenovirus was constructed and injected intraperitoneally into OEM mice to assess the volume and weight of ovarian ectopic lesions after treatment.Results1.The expression of MST1 in peritoneal macrophages of ovarian endometriosis(OEM)patients;2.OEM peritoneal macrophages are in oxidative stress and with M2 polarization;3.The concentration of IL-10 in the peritoneal fluid of OEM patients is increased;4.Autophagy activation of ectopic lesion tissue in ovarian EM(OEM)patients;5.Oxidative stress occurs in siRNA-MST1-THP-1 then with M2 polarization;6.The expression of p38-MAPK of siRNA-MSTl-THP-1 is increased;7.After co-cultured with siRNA-MST1-THP-1,the autophagy of HESCs is activated;8.siRNA-MST1-THP-1 secretes IL-10;9.IL-10 promotes autophagy of HESCs;10.The expression of MST1 in peritoneal macrophages of OEM mice is decreased while p38-MAPK is increased;11.Oxidative stress occurs in peritoneal macrophages of OEM mice and with M2 polarization;12.Intraperitoneal injection of MST1 overexpressing adenovirus in OEM mice inhibits peritoneal macrophage oxidative stress and M2 polarization;13.The size and weight of ectopic lesions in OEM mice is decreased after intraperitoneal injection of MST1 overexpressing adenovirus.Conclusion1.Abnormal immune function of peritoneal macrophages in ovarian EM,and increased autophagy of ectopic lesion tissue(cells);2.MST1 regulates macrophage oxidative stress and M2 polarization through p38-MAPK with Nrf2,which in turn induces endothelial cell autophagy through secretion of IL-10;3.Intraperitoneal injection of MST1 overexpressing adenovirus improves the progression of OEM mice. |
PDF Full Text Request