| ObjectiveUlcerative colitis(UC)is a chronic non-specific inflammatory disease of the colon and rectum whose etiology is not very clear.The clinical manifestations are characterized by recurrent abdominal pain,diarrhea,mucus pus and blood,which can be accompanied by fever,appetite decline and so on.Recent studies have found that Th17/Treg imbalance is an important factor in the onset and progression of ulcerative colitis.miR-155 is a typical multifunctional gene.It is mediated by its downstream genes and is involved in many physiological and pathological processes,such as inflammation,immunity,and tumorigenesis.Studies have shown that miR-155 can regulate T cell differentiation and affect Th17/Treg balance,but whether miR-155regulates Th17/Treg balance in UC patients through the Jarid2/Wnt/β-catenin signaling pathway is not well understood.Therefore,the purpose of this experimental study is to investigate the role of miR-155 in regulating Th17/Treg balance in DSS-induced colitis in mice,and to study the intervention effect of Compound Sophorae Decoction in this process.This experiment provides a basis for discovering new treatments for ulcerative colitis.Methods1.Naive CD4~+T cells were obtained by magnetic bead sorting.The induction factors were added to induce the differentiation of Th17 and Treg cells.On this basis,naive CD4~+T cells were divided into three groups,and miR-155 inhibitor,negative control inhibitor and Compound Sophorae Decoction containing serum were intervened.The differentiation results were detected by flow cytometry after 72 hours,and miR-155 and Jarid2 expression were detected by qRT-PCR.2.In animal experiments,48 C57 mice were randomly divided into 6 groups after one week of adaptive feeding,8 in each group:normal control group,model group,miR-155 antagomir group,negative control group,compound sophorae decoction group,and mesalazine group.On the first day,except the normal control group,the other groups were given free drinking of 3%DSS for 7 days,the compound sophorae decoction group was administered with compound sophorae decoction for 7 days,and the mesalazine group was administered with mesalazine for7 days.Starting on day 5,the miR-155 antagomir group was injected intraperitoneally with miR-155 antagomir for 3 days,and the negative control group was injected intraperitoneally with the same amount of negative control agent for 3 days.During the modeling period,the mice disease activity index score(DAI)were observed and recorded daily.All mice were sacrificed on the 8th day,colon tissue in each group were taken for length measurement and fixed to HE staining.The spleen and mesenteric lymph nodes were measured by flow cytometry for Th17 and Treg cells.The expression of miR-155 was detected by hybridization;Jarid2,Wnt1,β-catenin,sfrp1,IL-6,IL-17A,RORγt,TGF-βmRNA and miR-155 content were was detected by RT-PCR;protein expression of IL-6,IL-10,IL-17A,TGF-βwere was detected by Elisa;western blot was used to measure the expression of Jarid2,Wnt1,β-catenin protein;immunofluorescence was used to detect the expression of Jarid2 in colon tissue,and immunohistochemical was used to detect the expression of Wnt1,β-catenin,sfrp1,TCF-4 and Cyclin D1;Results1.The results of in vitro experiments showed that compared with the negative control group,the proportion of Th17 cells in the miR-155 inhibitor group and the compound sophorae decoction containing serum group was significantly reduced,the proportion of Treg cells was significantly increased,and the expression level of miR-155 was reduced to varying degrees and the Jarid2 was increased.2.The results of animal experiments showed that compared with the normal group,the DAI of the mice in each group increased,which indicates that the modeling was successful;compared with the model group,the DAI of the other groups of mice decreased to varying degrees.Compared with the normal group,the proportion of Th17 cells in the spleen and mesenteric lymph nodes of the model group was significantly increased(P<0.01),and the proportion of Treg cells was slightly increased;the miR-155 content of the colon tissue was significantly increased(P<0.01);sfrp1,IL-6,IL-17A,RORγt mRNA and sfrp1,IL-6,IL-17A protein expression levels were significantly increased(P<0.01),while Jarid2,Wnt1,β-catenin,TGF-βmRNA and protein expression levels were significantly decreased(P<0.01).The protein expressions of TCF-4 and Cyclin D1 were significantly decreased(P<0.01),and the expression of Treg-related anti-inflammatory factor IL-10 was also significantly decreased(P<0.01).Compared with the model group,the proportion of Th17 cells in the spleen and mesenteric lymph nodes of the miR-155 inhibitor group,the compound sophorae decoction group,and the mesalazine group were significantly reduced(P<0.01),and the Treg cells were significantly increased(P<0.01);miR-155content was significantly reduced(P<0.01);the mRNA levels of sfrp1,IL-6,IL-17A,RORγt and protein expression of sfrp1,IL-6,IL-17A were significantly reduced(P<0.01),while Jarid2,Wnt1,β-catenin,TGF-βmRNA and protein expression levels were significantly increased(P<0.01),and the protein expressions of TCF-4 and Cyclin D1 were significantly increased(P<0.01);The protein expression of 10 was significantly increased(P<0.01).Conclusions1.miR-155 antagomir can relieve DSS-induced colon damage in mice2.miR-155 antagomir affects the differentiation and function of Th17 and Treg cells in colonic mucosa of mice induced by DSS3.miR-155 antagomir may regulate Th17/Treg balance through Jarid2/Wnt/β-catenin signaling pathway4.Compound Sophorae Decoction can affect the expression of miR-155 and relieve DSS-induced ulcerative colitis... |
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