Study On Umbilical Cord Mesenchymal Stem Cells Reverse Fibrosis Of Uterine Adhesions Through Activation Of NR4A1

Background:Intrauterine adhesions(IUAs)is the main cause of secondary uterine infertility,which seriously affects women’s physical and mental health,family harmony and social stability.The reproductive barrier caused by IUAs has also been paid more and more attention,but the low pregnancy rate and high recurrence rate are still the main problems.It is an urgent medical problem to find an effective way to prevent IUAs,promote endometrial proliferation,and restore the reproductive function of women with IUAs.The therapeutic effect of stem cells on many difficult diseases has attracted much attention,and has also brought hope for patients with refractory IUAs.Due to the lack of research on the mechanism of the development of IUAs,the target of stem cell therapy is unclear,and the mechanistic research remains on the surface.The pregnancy rate of previous clinical studies with stem cell is about 38%,which is better than the simple prognosis of traditional intrauterine adhesion separation,which shows the prospect of stem cell therapy.However,nearly two-thirds of the fertility problems of patients with severe IUAs need to be solved.The reason may be that the fibrous scarring of the inner wall of IUAs makes it difficult for stem cells to enter the lower part of the scar of the uterine wall,thus affecting its corresponding role.It is a common phenomenon that fibrotic scar tissue appears in the uterine cavity of patients with moderate and severe IUAs,which is also an important factor leading to poor prognosis.In the past,it was believed that scar tissue was caused by endometrial fibrosis,but the repair of the intrauterine wall,especially the local environment caused by scarring is not conducive to endometrial growth,has not been a breAKThrough for many years.We found that the pathological results of the removed scar tissue often reported as the smooth muscle tissue.So does the source of scar tissue in the uterine cavity also include the smooth muscle tissue of the uterus?What mechanisms may be involved in the occurrence and development of scar tissue?And what treatment methods can further improve the final treatment effect based on the findings?These are issues that need to be discussed urgently.Therefore,it is necessary to improve the resident and cell homing rate of stem cells in clinical application,and to deeply study the development mechanism of IUAs scar tissue and the mechanism of stem cell therapy for IUAs.Previous study of our research group found that cold knife ploughing technique can improve the prognosis of patients with IUAs.And animal experiments found that scratch before transplantation into the mouse uterus can increase the retention rate and retention time of stem cells.However,studies on the disease direction of IUAs have not been conducted.However,there is currently no study to explore whether scratches can promote the effect of stem cell therapy on IUAs.Objective:The purpose of this study is to explore the effect of intrauterine injection of human umbilical cord derived mesenchymal stem cells(hUCMSC)on IUAs,evaluate the effect of scratch operation on hUCMSC;explore the mechanisms of fibrotic scar tissue in the intrauterine wall of patients with IUAs and the molecular mechanism of hUCMSC in the treatment of IUAs,so as to provide a new idea for the study and treatment of intrauterine scarring of IUAs.Methods:(1)The efficacy of hUCMSC treatment on IUAs was evaluated by in vitro cell models and in vivo animal models.In vitro isolated and cultured hUCMSC and human endometrial stromal cells(HESC).TGF-β induced HESC to establish a fibrotic cell model,and explored the effect of hUCMSC on proliferation,apoptosis,migration,and expression of fibrotic marker molecules(TGF-β,Collenge-Ⅰ,and α-SMA)by establishing a coculture cell hUCMSC-HESC.On the basis of the animal model of IUAs established with pregnant mice,the in vivo animal experiments were divided into four groups according to the intervention method,including the control group,the model group,stem cell group and stem cell+scratch group.Uterine tissues were collected at 7,14,and 21 days after intervention,and the number of endometrial glands,uterine fibrosis area ratio(endometrium+myometrium),ER expression,fibrosis factor expression(TGF-β and Collenge-I),and pregnancy outcome were observed and compared by HE staining,Massion staining,immunohistochemistry,WB,and PCR.(2)By comparing the pathological characteristics of fibrous scar tissue in the inner wall of the uterine cavity of the patients with IUAs(experimental group,n=16)and the myometrial tissue of the patients with normal uterus(control group,n=6);by using the second-generation sequencing technology to determine the transcriptome characteristics of fibrous scar tissue,explore the source of scar tissue,find out the mRNA differentially expressed between groups,and analyze the functional pathway that may participate in the formation of fibrous scar in the inner wall of the uterine cavity.(3)Through the sequencing analysis of the uterine tissue of the rat model group and the stem cell group,the intersection of differential genes taken with Part Ⅱ sequencing,and the target genes and pathways for further study were determined.Results:(1)hUCMSC can reduce the expression of fibrosis molecules and the apoptosis of HESC fibrosis cell model,promote the proliferation and migration ability(P<0.05).In animal experiments,compared with the control group,the fibrotic area of endometrium and myometrium and the expression of fibrotic molecules in the uterus of the model group were significantly increased(P<0.05).After the intervention of hUCMSC,the fibrotic area of endometrium and myometrium and the expression of fibrotic molecules decreased,the number of endometrial glands and the expression of ER receptor increased,and the implantation number of embryos was higher in rats with IUAs(P<0.05).Scratch operation before the intervention of hUCMSC can further reduce the area of fibrosis and the expression of fibrosis molecules(P<0.05),increase the number of endometrial glands(P>0.05),increase the expression of ER receptor(P<0.05)and the implantation number of embryos(P>0.05).(2)There were no difference in the histological morphology and theα-SMA expression between the experimental and control groups(P>0.05);but there were statistic difference in the area of fibrosis between groups(P<0.05).There was no significant difference in gene expression between groups(P>0.05).Total 698 differentially expressed genes in the two groups(P<0.05).Compared with the control group,there were 526 down-regulated genes and 172 up-regulated genes.The top 10 most differentially expressed genes were ATF3,NR4A1,CCN1,DUSP1,zfp36,rflna,Gadd45b,atxn712,cxcl2 and bhlhe40.Gene Ontology(GO)analysis showed that these genes were mainly related to cell proliferation,AP-1 complex generation,angiogenesis and other functions.Kyoto Encyclopedia of Genes and Genomes(KEGG)showed that signal pathways such as AGE-RAGE,FOXO and TNF were mainly involved in the development of fibrotic scar tissue.(3)There were intersected 111 different genes between the tissue of human and rat;the most DEG was NR4A1,and the enrichment pathway was MAPK;hUCSMC may treat IUAs by activation NR4A1 through"AKT/JNK/AP-1" pathway.(4)In vitro cell experiment,PCR results showed that hUCMSC could down-regulate the expression of AKT,and up-regulate the expression of JNK,AP-1 and NR4A1(P<0.05).In vivo animal experiments,WB and PCR results showed that hUCMSC could also down-regulate the expression of AKT and p-AKT,and up-regulate the expression of JNK,AP-1 and NR4A1 in the uterine tissue of the IUAs rat;compared with the stem cell group,scratch could down-regulate the expression of AKT and p-AKT,and up-regulate the expression of JNK,AP-1 and NR4A1(P<0.05).In the inhibitor group,Compared with the stem cell group,the fibrotic area and the expression offibrotic molecules were increased,and the number of endometrial glands and the expression of ER receptor were decreased,the difference was statistically significant(P<0.05);at the same time,AKT and p-AKT were up-regulated,and JNK,AP-1 and NR4A1 were down-regulate.Conclusion:(1)hUCMSC has the effect of reversing cell and uterine fibrosis,can effectively reduce the degree of uterine fibrosis,increase the number of uterine glands,ER receptor expression and embryo implantation number of rats with IUAs.Moreover,the scratch operation before hUCMSC transplant may further promote the role of hUCMSC.(2)The histological origin of scar tissue formation in the intrauterine wall of patients with IUAs may be related to myometrial fibrosis.The expression of ATF3,NR4A1,CCN1,DUSP1,zfp36,rflna,Gadd45b,atxn712,cxcl2 and bhlhe40 genes and signal pathways such as AGE-RAGE,FOXO and TNF may be involved in the occurrence and development of fibrous scar.(3)hUCMSC may reverse fibrosis of IUAs by activation NR4A1 through "AKT/JNK/AP-1" pathway,and NR4A1 antagonist can block the effect of hUCMSC on reversing uterine fibrosis;Scratch operation can promote the regulation of "AKT/JNK/AP-1" pathway and NR4A1 by hUCMSC.