The Role And Mechanism Of SDF-1/CXCR4 Axis In Mediating Human Endometrial Regenerative Cells Induced Inhibition In Experimental Colitis Model

Background: Inflammatory bowel disease(IBD)includes ulcerative colitis(UC)and Crohn's disease(CD).UC is a chronic disabling inflammatory disease which mainly damages the ileum,colon and rectum.The mortality and prevalence of UC have rapidly increased in recent years.However,the etiopathogenesis of UC remains unclear.Although some treatments of UC could come into effects to some degree,the symptoms of UC or the side effects caused by the treatments bring great pain to the patients.Mesenchymal stromal cells(MSCs)are considered as a promising treatment therapy for UC.However,the disadvantages of MSCs like the invasive obtainment and limited proliferative capacity limit their clinical use.Therefore,it is necessary to find a new source of regenerative cells,which both have the therapeutic effects in the treatment of UC and overcome shortcomings of MSCs.Endometrial regenerative cells(ERCs)as a new type of mesenchymal-like stromal cells,present many advantages including noninvasively obtained method,plentiful proliferation capacity,lack of immunogenicity,expandability to great quantities,and their therapeutic potential.In our previous studies,the therapeutic effects of ERCs have been tested in a variety of disease models,especially the experimental colitis model in mice.However,the indepth mechanisms of ERCs need to be further investigated,and it is necessary to explore an effective way to enhance the therapeutic effects of ERCs in alleviating colitis.Evidences have demonstrated that ERCs could secret stromal cell derived factor-1(SDF-1)and express C-X-C chemokine receptor type 4(CXCR4).SDF-1 interacts with its ligand CXCR4 to form the SDF-1/CXCR4 axis and this axis plays a chemotaxis role in stem/stromal cells migration.A strong amino acid sequences and coding region nucleotide sequences conservation exists between mouse and human SDF-1,that makes the rodent model feasible for exploring SDF-1 function in various diseases.This study focused on the mechanism of the SDF-1/CXCR4 axis in ERCs treatment of experimental colitis.Objective: The aim of this study was to explore whether the SDF-1/CXCR4 axis could promote ERCs homing and increase the CXCR4 expression on ERCs and the effect ofSDF-1 pretreated ERCs with high level CXCR4 expression on the differentiation of mouse immune cells,as well as to examine whether the therapeutic effect of ERCs on the experimental colitis could be enhanced via SDF-1/CXCR4 axis.Methods: This study included three parts.Part 1 was to observe ERCs' growth morphology and identify their phenotype.Scratch test was performed to investigate the role of SDF-1/CXCR4 axis in ERC wound healing effect.Part 2 was to detect the influence of different concentrations of SDF-1 on the surface CXCR4 expression of ERCs.Flow cytometry was performed and the optimal concentration of SDF-1 was determined.ERCs pretreated with whether SDF-1,AMD3100 or nothing were cocultured with splenocytes in vitro,and the proliferation of tolerant dendritic cells(TolDCs),macrophage type 2 cells(M2)and the regulatory T cells(Tregs)were examined.Part 3 was an in vivo study.Experimental mice colitis model was induced by dextran sulphate sodium(DSS).The therapeutic effect of ERCs pretreated with SDF-1,which highly expresse CXCR4,was investigated in colitis mice.Results: Part 1,ERCs were isolated from menstrual blood and cultured successfully.The phenotypic identification test showed that the surface CD90 and CD105 were positive.The concentration of 50 ng/ml SDF-1 significantly improved the wound healing ability of ERCs compared with other concentrations.Part 2,50 ng/ml SDF-1 effectively increased the CXCR4 expression on ERCs.In vitro study also showed that SDF-1 pretreated ERCs with high level of CXCR4 expression could enhance the generation of Tol-DCs,M2 and Tregs better than untreated ERCs.Part 3,the DSSinduced colitis mice model was established successfully.CXCR4 highly expressed ERCs could remarkably attenuate the severity of experimental colitis and bloody stool,as well as preserve the length of the colon.The inflammatory cell infiltration was significantly decreased in SDF-1 pretreated ERCs group.SDF-1 pretreated ERCs had much better ability in enhancing the generation of Tol-DCs,M2,Th2 and Tregs.CXCR4 highly expressed ERCs could significantly increase the generation of antiinflammation cytokines(IL-4 and IL-10)but decrease the generation of proinflammation cytokines(IL-6 and TNF-?)in the colon.In addition,SDF-1pretreated ERCs with CM-Dil labeling was found in the injured colon.These therapeutic effects of ERCs were suppressed by AMD3100 which blocks the function of SDF-1/CXCR4 axis.Conclusion: In this study,we demonstrated that SDF-1 effectively enhanced the expression of CXCR4 on the surface of ERCs.The chemotactic action of SDF-1/CXCR4 axis could promote the homing of ERCs to the impaired colon tissue in the DSS-induced colitis model in mice.These therapeutic effects of ERCs were suppressed by AMD3100 which block the function of SDF-1/CXCR4 axis.In conclusion,our study aimed at exploring the treatment effect of SDF-1 pretreated ERCs on the colitis model,and investigating their potential in future clinical use.