S100A8/A9 Promotes Endometrial Fibrosis In Intrauterine Adhesion Via Regulating RAGE/JAK2/STAT3 Signaling Pathway

Objectives: Intrauterine adhesions(IUA)is mainly caused by damage to the basal layer of endometrium and may lead to the formation of adhesions(scar tissue)between the anterior and posterior walls of the uterus,where the walls partially or completely adhere or stick to each other.Endometrial fibrosis is the main pathological feature of IUA.When the basal layer of the endometrium is damaged by instrumentation or infection,the stromal fibroblasts proliferate and phenotypically differentiate into myofibroblasts.The characteristic features of IUA include excessive deposition and reorganization of the extracellular matrix,replacing the normal endometrium.Increasing evidence suggests that fibrosis is the final stage of a chronic inflammatory response,which are induced by multiple stimuli.As a member of the S100/calgranulin family of proteins,S100A8/A9 is composed of two subunits,S100A8 and S100A9,both forming heterogenous multimers.S100A8/A9 is expressed in myeloid cells,such as neutrophils,and is released in response to cellular injury,where it can stimulate leukocyte recruitment and induce cytokine secretion.S100A8/A9 has been documented to promote the proliferation and differentiation of fibroblasts into myofibroblasts,and accelerate the secretion and deposition of extracellular matrix.However,the role and mechanism of S100A8/A9 in IUA have not been reported yet.Under stress conditions,S100A8/A9 binding to RAGE activates inflammatory pathways that lead to secretion of multiple cytokines that sustain or exacerbate inflammation.RAGE is a multi-ligand pattern recognition receptor that interacts with diverse endogenous ligands and activates multiple signaling cascades,including JAK/STATs,which is implicated in the pathogenesis of inflammatory response.Transcriptome sequencing was conducted to compare the endometrial tissue of IUA patients with that of normal individuals.S100A8/A9 was identified significantly upregulated in the endometria of IUA patients.Accordingly,we hypothesized that S100A8/A9 activates the JAK2/STAT3 signaling pathway by binding to the RAGE receptor to release pro-inflammatory factors and cytokines,promote the proliferation and activation of fibroblasts,increase the expression and deposition of extracellular matrix,and ultimately lead to the formation of fibrosis.At present,the standard method of treatment of IUAs is transcervical resection of adhesion(TCRA).Although TCRA could provide anatomical structure recovery,it lacks functional recovery,with a high recurrence rate of postoperative adhesion.Therefore,there is a need for an effective and safe alternative in IUA treatment.Menstrual blood-derived stromal cells(Men SCs)are shedding endometrial stem cells that are derived from menstrual blood.Men SCs exhibit a multi-lineage potential and have attracted extensive attention in regenerative medicine.We have previously demonstrated that Men SCs could treat IUA by promoting endometrial regeneration and functional recovery.Bama miniature pig is an optimal model to explore the mechanisms underlying IUA due to its anatomical and physiological similarities with humans.Therefore,in this study,female Bama miniature pigs were selected to establish an effective IUA animal model to investigate the therapeutic effect of Men SCs in IUA.In summary,we firstly observed the expression level of S100A8/A9 in the endometrial tissue of IUA patients;secondly,we explored whether S100A8/A9 promoted endometrial fibrosis by regulating the RAGR/JAK2/STAT3 signaling pathway;thirdly,we established a Bama miniature pig IUA model and explored whether S100A8/A9 is involved in the therapeutic effect of Men SCs in IUA.Materials and methods: 1.Patients who underwent surgical intervention for IUA in our hospital were selected as the research object,and the uterine adhesion tissue and adjacent endometrium were selected as the study specimen.Patients who were diagnosed with non-uterine adhesions by hysteroscopy with endometrial biopsy were recruited into the control group.RT-PCR was performed to measure S100A8,S100A9,COLI,αSMA m RNA levels.Immunohistochemistry was used to detect the expression of S100A8/A9 and COLI in endometrial tissue.Immunofluorescence staining was used to examine the co-expression of S100A8/A9 and granulocytes.2.Primary cultured human endometrial stromal cells(h ESCs)were treated with different concentrations of S100A8/A9.CCK8 assay was used to detect cell proliferation,and RT-PCR was used to detect the expression of inflammatory-related factors(IL-1,IL-6,and TNF).Expression levels of COLI and α-SMA were examined by RT-PCR,Western blot,and IF.Western blot was performed to detect the expression of RAGE,JAK2,STAT3,p-JAK2,and p-STAT3.Cells were stimulated with S100A8/A9 following pretreatment with RAGE antagonist(FPS-ZM1)or JAK2/STAT3 pathway inhibitor(AG490).Experiments mentioned above were performed and immunofluorescence assay was used to detect nuclear translocation of STAT3.3.A pig IUA model was established by electrocautery injury to the uterus.Endometrial fibrosis was measured in the tissues with HE staining,Masson staining,immunohistochemistry staining,and hysteroscopy,counted by endometrial thickness,the ratio of the area with endometrial fibrosis to total endometrial area,number of endometrial glands,and microvascular density.Hydroxyproline content was chemically determined.After local multi-point injection of Men SCs,endometrial fibrosis and changes of S100A8/A9 expression were measured.Results: 1.Compared with the control group,the m RNA expressions of S100A8,S100A9,COLI and α-SMA in endometrial tissues of IUA patients were significantly increased.Immunohistochemical results showed that in endometrium tissues of IUA patients S100A8/A9 and COLI expression levels increased.Significantly increased collagen fibers were observed by Masson staining.Immunofluorescence results showed that S100A8/A9 was co-localized to CD15 positive granulocytes,suggesting that S100A8/A9 was mainly secreted by granulocytes.2.In primary cultured h ESCs,100ng/m L S100A8/A9 significantly promoted the proliferation of endometrial stromal cells,the expression of inflammatory factors(IL-1 and IL-6),and the expression of fibrosis related proteins(COLI and α-SMA).S100A8/A9 enhanced RAGE receptor protein expression,activated the downstream JAK2/STAT3 signaling pathway,and promoted the nuclear translocation of STAT3.S100A8/A9-induced proliferation of endometrial stromal cells,expressions of COLI and α-SMA,and activation of JAK2/STAT3 signaling pathway were inhibited by FPS-ZM1(RAGE blocker).Also,FPS-ZM1 inhibited STAT3 nuclear translocation.AG490(JAK2/STAT3 pathway inhibitor)blocked the promoting effect of S100A8/A9 on COLI and α-SMA expressions in endometrial stromal cells.3.After electrocautery injury in the endometrium,we observed a number of morphological changes in the endometrium,including bleeding,tissue edema,and even carbonized in a short period of time.On the 35 th day after the injury,the endometrium displayed a decreased the thickness of the endometrium,an increased the collagen volume fraction,and decreased number of glands and microvessels.A narrow uterine cavity was observed by hysteroscopy,accompanied by a large number of scar tissue and fibrous adhesion strips.Endometrial injury caused electrocautery injury is intensity-dependent and the pig IUA model could be well-established when intensity of electric field reached 70 W.After electrocauterizing,the expression levels of S100A8/A9 and COLI increased significantly compared with the control group.Men SCs treatment could reduce the expression levels of S100A8/A9 and COLI in the endometrial tissue,increase endometrial thickness,reduce collagen volume fraction,increase the number of glands and microvessels,and promote endometrial repair.Conclusions: 1.Transcriptome sequencing was conducted to compare the endometrial tissue of IUA patients with that of normal individuals.Gene ontology(GO)and kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analyses revealed that differentially expressed genes(DEGs)were mainly enriched in inflammatory response,of which S100A8/A9 expressed most differentially.The histopathological results of the samples showed endometrial fibrosis in IUA patients.In addition,there is a significant increase in the expression of the genes of S100A8 and S100A9.Moreover,S100A8/A9 was mainly derived from granulocytes.2.Coupling of RAGE by its ligand S100A8/A9 activates downstream JAK2/STAT3 signaling pathway that ultimately leads to the upregulation of cytokines(IL-1 and IL-6),COLI,α-SMA,and the differentiation of fibroblasts into myofibroblasts.S100A8/A9 may exert pro-inflammatory and pro-fibrotic effects on endometrial fibroblasts.3.Electrocautery injury could successfully establish a stable and effective animal model of IUA in Bama miniature pigs and S100A8/A9 expression was significantly increased in endometrial tissue.Men SCs reduced endometrial fibrosis by decreasing S100A8/A9 concentration at the site of injury.The endometrial thickness,the number of glands,and number of microvessels in pigs with IUA were increased significantly after Men SCs treatment.