The Role Of Dickkopf-1 In Endometrial Regenerative Cells Mediated Inhibition Of Ulcerative Colitis In Mice

Background:Ulcerative colitis（UC）is a chronic,relapsing and non-specific inflammation disease which usually occurs in the colorects.UC is considered as a main form of inflammatory bowel disease（IBD）,and its clinical course is always prolonged with many complications.Till now,the etiology of UC is still unclear,which is now considered being related with personal genetic,immune and environmental factors.Therefore,its clinical treatment is lack of effective methods.In recent years,on basis of the rapid development and research of mesenchymal stem cells（MSCs）,stem cells have made a new breakthrough in treatment of ulcerative colitis.Endometrial regenerative cells（ERCs）is a new kind of stem cells extracted from human menstrual blood and has been demonstrated possessing the similar phenotypic characteristics and capabilities with MSCs,such as multiple differentiation potential,self-renewal potential and immunomodulatory effects.ERC also has its own unique advantages,including strong proliferation,non-invasive source and low immunogenicity.Many studies have proved that ERCs can induce immune tolerance in the inflammatory response and play a significant role in immunosuppressive effects.Dickkpf-1（DKK1）is a kind of secretory glycoprotein which is widely expressed in various tissues and cells.DKK1 can be secreted by MSCs in a large amount,and it has more than 86%homology between human and mouse in coding gene sequence and amino acid sequence.DKK1 is recognized as a specific inhibitor of Wnt/β-catenin signaling pathway.It can competitively bind the receptor of Wnt/β-catenin pathway and thus inhibit its activation specially.Wnt/β-catenin signaling is a conservative signaling pathway,playing an crucial role in embryonic development,stem cell proliferation and differentiation,and tissue homeostasis.In recent years,Wnt/β-catenin has been found that it also regulates the proliferation and differentiation of various immune cells in vivo,and participates in the induction of immune tolerance.Wnt/β-catenin pathway is closely related to immune-mediated chronic inflammation and tumor-mediated immune response.Therefore,we hypothesize that ERCs can also secrete a large number of DKK1 factors,and DKK1^low-ERC may have a stronger immunomodulation and more effective therapy in delaying the progression of ulcerative colitis in mice.Objective:1)To isolate and culture ERCs,determine whether ERCs can secrete DKK1,and whether glucocorticoid（GC）and IL-1βcan significantly change the secretion of DKK1.2)To establish colitis models in mice by dextran sodium sulfate（DSS）and investigate whether DKK1^low-ERCs（ERCs with low expression of DKK1）exhibits the better immunomodulation and therapeutic effects on mice with colitis.Methods:1)To isolate ERCs,determine whether ERCs secrete DKK1,and explore the effect of glucocorticoid（GC）and IL-1βon DKK1 secretion（1）Menstrual blood was collected with the Diva cup on the 2^nd-3^rd day of menstrual period.ERCs in the blood was separated by density gradient centrifugation and drawn out into culture dishes.Cells were cultured in 5%CO2 incubator at 37℃and passaged down in vitro.The 5^th generation of ERCs were prepared for identifying the phenotype by flow cytometry（FCM）.（2）The 3^rd generation to the 7^th generation cells were respectively collected and divided into three groups:unmodified ERC group,GC-treated ERC group and IL-1β-treated ERC group.Cell culture media were collected for ELISA to determine the DKK1 secretion level.Total m RNA were extracted from the 5^th generation of ERCs for real-time PCR（RT-PCR）to analysis the m RNA expression of DKK1.2)To explore the therapeutic effects of ERCs with different DKK1 expression on DSS-induced colitis.（1）Colitis establishing:colitis was induced by 3%DSS（solved in distilled-water）via free drinking for seven days.（2）Groups and interventions:twenty-four male BALB/c mice aged 6-8 weeks and weighing 22±2g were randomly divided into 4 groups（n=6）:untreated group,unmodified ERC group,DKK1^high-ERC group and DKK1^low-ERC group.All mice were firstly given distilled water containing 3%DSS for 7 days,then changed to non-DSS water to induce colitis.On day 2,5,8 after DSS-induction,untreated group was administrated with 200μl phosphate buffered saline（PBS）by intraperitoneal injection as control;unmodified ERC group,DKK1^high-ERC group and DKK1^low-ERC group were respectively injected with 200μl PBS containing 1×10⁶pretreated ERCs.（3）During the period of experiment,mouse general conditions,body weights,fecal features and bloody stools were observed and recorded every day.Disease activity index（DAI）was calculated to evaluate the activity of UC.（4）On 10^th day after DSS-induction,mice were all sacrificed.Colons were severed and measured.Pathological changes were presented by H&E staining.The inflammatory factor andβ-catenin level in colons and in spleens were detected by ELISA kits.The m RNA changes of inflammatory profiles andβ-catenin was detected by RT-PCR.The proportion of immune cells in spleens were analyzed by FCM.Results:1)（1）ERCs were successfully isolated from menstrual blood by density gradient centrifugation.The cells are spindle-like,and arranged in a nest or braid pattern.After about thrird generations,morphological and biological characteristics of ERCs tend to be consistent.Flow cytometry results showed that the phenotype of ERCs was similar to that of MSCs,assuming high expression of CD24,CD44 and CD90,but low of hematopoietic stem cell specific antigens（CD45）.（2）ELISA data showed that the 3^rd-7^th generation of ERCs secrete a lot of DKK1,and the level of 5^th generation cells went to the least.Compared the DKK1level of each generation cells,the DKK1 secretion in GC-treated ERC group was obviously higher than that in unmodified ERC group,while that in IL-1β-treated group was lower.（3）RT-PCR analysis showed DKK1 m RNA expression of GC-treated ERCs was about three times higher than that of unmodified ERCs.As the meanwhile,the level in IL-1β-treated ERCs was 0.4 times.The difference was significant in statistics.These results demonstrated that both at protein and m RNA levels,GC significantly enhanced DKK1 secretion of ERCs,and IL-1βcould effectively block its secretion.2)To explore the therapeutic effects of ERCs with different DKK1 secretion on colitis,we found:（1）DKK1^low-ERCs significantly ameliorated the general condition,weight loss,stool characteristics and fecal blood of colitis mice.（2）Colonic length in DKK1^low-ERC group was increased when compared with other experimental groups.The situation of intestinal rigidity,intestinal congestion dilatation and stenosis in this group were also significantly relieved.（3）Compared with unmodified ERC group,DKK1^low-ERCs further ameliorated the damage of intestinal structure and gland crypt induced by DSS.DKK1^low-ERCs maintained the integrity of mucosal structure and reduced inflammatory infiltration,hyperemia and edema in intestinal walls.（4）DKK1^low-ERCs increased the proportion of Th2,Treg,and M2 phenotype macrophages in spleens,and inhibited the production of Th1,Th17 and mature DCs.Interestingly,these immunosupressive functions of ERCs were significantly weakened after we raised the DKK1 secretion,which resulted in a poor therapeutic effect when compared with unmodified ERCs.（5）DKK1^low-ERCs further inhibited the expression of TNF-α,IFN-γ,COX-2,i NOs and MPO in colonic tissue,but improved the synthesis of the protective anti-inflammatory factors（IL-4,IL-10 and SOD）.（6）DKK1^low-ERCs also enhanced theβ-catenin expression not only in colons but also in spleens,while in DKK1^high-ERC group,the level ofβ-catenin was reduced when compared with unmodified ERC group.Conclusions:1)ERCs can secrete DKK1 in large quantities,and the secretion of DKK1 can be increased by GC stimulation and inhibited by IL-1βstimulation.2)The low expression of DKK1 in ERC(DKK1^low-ERCs)has a stronger immunoregulation and more effective therapy in delaying the progression of ulcerative colitis in mice.