| Aim:In this experiment,the method of DSS was used to replicate the ulcerative colitis model in mice.First of all,the effect of Astragalus Polysaccharide on colitis was evaluated.The changes of JAK/STAT signaling pathway related proteins in colon and Treg/Th17 cell balance in peripheral blood of mice were observed at the same time.And further clarified whether Astragalus Polysaccharide can regulate Treg/Th17 balance to restore intestinal immune homeostasis via JAK/STAT signaling pathway,so as to treat ulcerative colitis.Methods:1.DSS-induced ulcerative colitis model replication and drug treatment:Sixty healthy male Balb/c mice were randomly divided into the normal control group(Control),the DSS group(DSS),the low dose group of Astragalus Polysaccharide group(APS-L),the medium dose group of Astragalus Polysaccharide group(APS-M),the high dose group of Astragalus Polysaccharide group(APS-H)and the positive control group of Mesalazine(5-ASA),with10 mice in each group.3%(w/v)DSS(36 000-50 000 k D)solution was prepared with ultrapure water.Except for the normal control group,the other five groups were given 3%DSS solution freely for 7 consecutive days,and the normal control group drank distilled water freely during this period.The mice were given 3%DSS for the first time on the first day,from the 5th day,mice in the APS-L group were given 50 mg/kg Astragalus Polysaccharide by gavage,mice in the APS-M group were given 100 mg/kg Astragalus Polysaccharide by gavage,mice in the APS-H group were given 200 mg/kg Astragalus Polysaccharide by gavage,mice in the 5-ASA group were given 300 mg/kg Mesalamine by gavage,mice in the normal control group and the DSS group were given the same volume of saline by gavage once a day for 5 days.Another forty healthy male Balb/c mice were randomly divided into the normal control group(Control),the model group(DSS),the Astragalus Polysaccharide group(DSS+APS)and the positive control group of Mesalazine(DSS+5-ASA).Except the the normal control group,other three groups were given 3%DSS solution for 7 consecutive days to establish the mouse model of acute ulcerative colitis.On the first day after modeling,the DSS+APS group was given 200 mg/kg Astragalus Polysaccharide suspension by gavage,and the DSS+5-ASA group was given 300 mg/kg 5-ASA suspension by gavage,both the normal control group and the DSS group were given the same volume of normal saline by gavage once a day for 5 consecutive days.2.Evaluation of the efficacy of Astragalus Polysaccharide in the treatment of DSS-induced ulcerative colitis model:The body weight of the mice was recorded at a fixed time every day,and the mental state,food intake,coat smoothness,activity,blood in stool and sticky degree of feces also needed to be observed daily.After the mice stopped taking the drug,the body weight,colon weight and colon length of mice in each group were measured,the intestinal weight index and unit colon weight were calculated,and the pathological injury score of colon tissue was evaluated under colonoscopy on the day of death.3.Analysis of Treg cells,Th17 cells and related cells:Using flow cytometry to measure the percentage of CD4~+CD25~+,CD4~+CD25~+Foxp3~+,PD-1~+CD4~+CD25~+,PD-L1~+CD4~+CD25~+,PD-1~+CD4~+CD25~+Foxp3~+,PD-L1~+CD4~+CD25~+Foxp3~+and CD4~+CD44~+IL-17A~+cells to clarify whether Astragalus Polysaccharide can regulate Treg/Th17 cell balance in colitis mice.4.Detection of TLR/My D88 signaling pathway related protein:Janus kinase 1,3(JAK1,JAK3),signal transducers and activators of transcription 5(STAT5),phosphorylated signal transducer and activator of transcription protein 5(p-STAT5),protein inhibitors of activated stats 3(PIAS3),suppressors of cytokine signaling 1(SOCS1),caveo Lin-1(Cav-1)and protein phosphatase 2(PP2A)in mice colon were detected by western blotting to explore whether Astragalus Polysaccharide can regulate JAK/STAT signaling pathway in colonic mucosa of mice with colitis.5.Statistical analysis:Graph Pad Prism 9.0 software was used for statistical analysis,measurement data were expressed as Mean±standard deviation((?)±s).One-way ANOVA was used for comparison between groups,with P<0.05 means statistically significant difference,and P<0.01 means statistically highly significant difference.Results:1.Evaluation of effective dose of Astragalus polysaccharide in the treatment of UC:Compared with the normal control group,the body weight and colon length of the DSS group were significantly decreased(P<0.01),and the colon weight,unit colon weight and intestinal weight index were significantly increased(P<0.01),indicating that the model was successfully established.Compared with the DSS group,after Astragalus Polysaccharide intervention,Only the body weight of mice in the APS-H group was significantly increased,and intestinal weight index was significantly decreased(P<0.01),colon weight and unit colon weight of mice in APS-M and APS-H groups were significantly decreased(P<0.05,P<0.01),and colon length of mice in APS-M and APS-H groups was significantly increased(P<0.05).Although there was no statistically significant difference in the indicators between the medium and high dose groups of Astragalus Polysaccharide,the decline trend of colonic mass in the APS-H group was significantly lower than other groups,and only the APS-H group could significantly improve the body weight and intestinal weight index of mice,with highly significant differences.The results suggested that Astragalus Polysaccharide can effectively treat ulcerative colitis in mice and high dose is the best.2.Evaluation of the efficacy of high-dose Astragalus Polysaccharide retreatment in UC mice:Compared with the normal control group,the body weight and colon length of mice in the DSS group were decreased significantly,and the intestinal weight index,disease activity index(DAI)score and tissue damage score were significantly increased(P<0.01,P<0.05),indicating that the model was successfully established.Compared with the DSS group,after Astragalus Polysaccharide and Mesalamine intervention,the body weight of mice in the treatment group was recovered,the intestinal weight index and DAI score were significantly decreased(P<0.01,P<0.05),The mucosa of colitis mice was more intact,most of the golet cells and crypts began to recover,the glands were more orderly,the degree of vascular hyperplasia was smaller,and the ulcer area was reduced.The infiltration of inflammatory cells was improved,and the pathological injury score was significantly reduced at the same time(P<0.01).Compared with the DSS group,the colonic length was significantly increased in the DSS+APS group.These results indicated that Astragalus Polysaccharide could effectively alleviate ulcerative colitis.3.Changes of Treg cells,Th17 cells and related cells:Compared with the normal control group,the percentage of PD-1~+CD4~+CD25~+,PD-L1~+CD4~+CD25~+and CD4~+CD44~+IL-17A~+cells in the peripheral blood of mice in the DSS group was significantly increased(P<0.01,P<0.05),CD4+CD25~+,CD4~+CD25~+Foxp3~+,PD-1~+CD4~+CD25~+Foxp3~+and PD-L1~+CD4~+CD25~+Foxp3~+cells was significantly decreased(P<0.01,P<0.05);compared with the DSS group,the percentage of PD-1~+CD4~+CD25~+,PD-L1~+CD4~+CD25~+and CD4~+CD44~+IL-17A~+cells in the peripheral blood of mice in the treatment group was significantly decreased(P<0.01,P<0.05),the percentage of CD4~+CD25~+,CD4~+CD25~+Foxp3~+,PD-1~+CD4~+CD25~+Foxp3~+and PD-L1~+CD4~+CD25~+Foxp3~+cells in the peripheral blood of mice in the DSS+APS group was significantly increased(P<0.01,P<0.05),the percentage of CD4~+CD25~+、CD4~+CD25~+Foxp3~+and PD-L1~+CD4~+CD25~+Foxp3~+in peripheral blood of mice in the DSS+5-ASA group was reduced obviously(P<0.01).The experiment results above demonstrated that Astragalus Polysaccharide could effectively regulate the balance of Treg/Th17 cells.4.Changes of JAK/STAT signaling pathway related proteins:Compared with the normal control group,the protein expressions of JAK1,JAK3,STAT5,p-STAT5 and PP2A in the colon tissue of the DSS group mice were significantly increased(P<0.01),the protein expressions of PIAS3,SCOS-1and Caveo Lin-1 were significantly decreased(P<0.01);compared with the DSS group,the protein expressions of JAK1,STAT5,p-STAT5 and PP2A in the colon tissue of the mice in the DSS+APS group were significantly decreased(P<0.05,P<0.01),the protein expressions of JAK3,PIAS3,SCOS-1 and Cav-1 were significantly increased(P<0.01);compared with the DSS group,the protein expressions of STAT5 and p-STAT5 in colon tissue of mice in the ASA+DSS group were significantly decreased(P<0.05,P<0.01),while the protein expressions of PIAS3 and SCOS-1 were significantly increased(P<0.05).The experiment results above indicated that Astragalus Polysaccharides could effectively regulate the JAK/STAT signaling pathway.Conclusion:The effective treatment of DSS-induced ulcerative colitis with Astragalus polysaccharide may be achieved by inhibiting the JAK/STAT signaling pathway to regulate Treg/Th17 cells. |
