| Objective:While evaluating the curative effect of Sishen pill and their decomposed recipes essential oil on dextran sodium sulfate(DSS)-induced recurrent ulcerative colitis,the changes in the structure of the intestinal flora and the proportion of follicular helper T cell subsets in mice were observed.From the perspective of TLR/MyD88 signaling pathway,explore the possible mechanism of Sishen pill and their decomposed recipes essential oil regulating follicular helper T cells and intestinal flora balance in the treatment of mouse colitis.Methods:1.Animal model replication and administration method:60 SPF BALB/c male mice were selected and randomly divided into 6 groups:the normal group(Normal),the model group of UC(DSS),and the treatment group of Sishen pill and their decomposed recipes essential oil(DSS+SSP),the treatment group of Ershen Pill essential oil(DSS+ESP),the treatment group of Wuweizisan essential oil(DSS+WWZS),the positive group of Mesalazine group(DSS+5-ASA),each with 10 animals.Except that the mice in the normal group were given normal drinking water,the mice in the other groups were given 3%DSS solution to drink freely for 5 consecutive days;after that,they would drink 2%DSS solution freely for 5 days to replicate the experimental recurrent ulcers.the model group of UC takes the first 3%DSS to the mice as the first day,and start gavage according to the body weight of the mice on the 11th day:the group of Sishen pill and their decomposed recipes essential oil was given 110.84 μL/kg Sishen pill essential oil suspension,the group of Ershen pill essential oil group was given 77.8 μL/kg Ershen pill essential oil suspension by gavage,the group of Wuweizisan essential oil was given 33.04 μL/kg Wuweizisan essential oil suspension by gavage,and the mesalazine group was given 300 mg/kg mesalazine enteric-coated The tablet suspension was given intragastrically,the normal group and the model group were given the same volume of ultrapure water intragastrically.The drug was gavaged once a day at a fixed time for 10 consecutive days.2.Evaluation of the efficacy of Sishen pill and their decomposed recipes essential oil in the treatment of DSS-induced recurrent ulcerative colitis:Closely observe the changes in weight,diet,hair status,stool and rectal bleeding of mice every day.The mice were anesthetized and the peripheral blood was collected and then sacrificed,the colon was separated,the length and weight of the colon were measured,and the colon weight index and the weight of the colon per unit length were calculated.Pathological sections of colon tissue were prepared,and the pathological damage of colon tissue was scored by double-blind method.The inflammatory factors such as IL-4,IL-10,IL-17A,IL-21,IFN-γ and other inflammatory factors in mouse colon were tested by enzyme-linked immunosorbent assay(ELISA)to further verify the experimental treatment of Sishen pill and their decomposed recipess essential oil Effectiveness of recurrent ulcerative colitis.3.The regulatory effect of Sishen pill and their decomposed recipes essential oil on follicular helper T cells:Flow cytometry(FCM)was used to detect the follicular helper T cell subsets Tfhl,Tfh9,Tfh10,Tfh17,Tfh21,Tfr in the peripheral blood of mice Cell changes,to explore whether Sishen pill and their decomposed recipes essential oil can treat DSS-induced colitis in mice by regulating changes in follicular helper T cells.4.Sequencing of the intestinal flora:Analyze the species specificity of the intestinal microflora through Meta 16S rRNA sequencing technology,explore the correlation between the intestinal flora and ulcerative colitis,and the regulatory effect of the essential oil of Sishen pill and their decomposed recipes on the flora.5.TLR/MyD88 signaling pathway related protein detection:Western blotting was used to detect TLR/MyD88 signaling pathway TLR2,MyD88,Rac1,IRAK4,IRAK1,TRAF6,TAB1,TAB2,MKK6,P38 MAPK,CREB protein.The regulation of Shenwan essential oil on TLR/MyD88 signaling pathway.6.Statistical analysis:The SPSS 22.0 software was used for data statistics.The comparison between groups was performed by control t-test and one-way analysis of variance,expressed as x±s,and P<0.05 as the difference was statistically significant,and P<0.01 The statistical results have extremely significant differences.Results:1.Evaluation of the curative effect of the essential oil of Sishen pill and their decomposed recipes in the treatment of recurrent ulcerative colitis.After treatment with essential oil from Sishen pill and their decomposed recipess,the general state of mice with recurrent ulcerative colitis improved significantly.The weight gradually increased,the length of the colon was obviously restored,the weight of the colon,the weight per unit length of the colon,and the weight index of the colon were significantly decreased,and the pathological damage of the colon tissue was obviously repaired under the light microscope.ELISA test results showed that the expression levels of IL-4,IL-17A,IL-21 and other inflammatory cytokines decreased to varying degrees,and the levels of IL-10 and IFN-γ increased(P<0.05 or P<0.01).This indicates that the intestinal inflammation in mice with recurrent ulcerative colitis is significantly relieved,suggesting that the essential oil of Sishen pill and their decomposed recipes may treat ulcerative colitis by regulating the expression of inflammatory cytokines.2.The effect of the essential oil of Sishen pill and their decomposed recipess on the level of helper T cells in recurrent ulcerative colitis.The results of flow cytometry showed that the expression levels of follicular helper T cell subsets Tfhl,Tfh9,Tfh17,Tfh21 cells increased,and the expression levels of Tfr and Tfh10 cells decreased,indicating that Sishen pill and their decomposed recipes essential oil regulates mouse Tfh cells effect.3.The effect of the essential oil of Sishen pill and their decomposed recipess on the intestinal flora of mice with recurrent ulcerative colitis.Through the 16s rRNA sequencing analysis of the intestinal flora,it was found that the fecal colony composition of the mice in each group had a higher similarity.The essential oil of Sishen pill and their decomposed recipess could significantly improve the diversity and richness of the intestinal flora of mice with recurrent ulcerative colitis.Affect the difference in the composition of the mouse intestinal flora,and the results show that the mouse intestinal flora has a significant correlation with follicular helper T cells.4.The regulatory of Sishen pill essential oil on the expression of TLR/MyD88 signaling pathway-related proteins in mice with recurrent ulcerative colitis.Through the detection of TLR/MyD88 signaling pathway related proteins,the results show that Sishen pill and their decomposed recipes and it’s decomposed recipes essential oil can significantly reduce the colon tissue TLR2,MyD88,Rac1,IRAK4,IRAK1,TRAF6,TAB1,TAB2,MKK6,P38 MAPK,P38 MAPK,The expression level of CREB protein suggests that it inhibits the activation of TLR/MyD88 signaling pathway.Conclusion:Sishen pill and their decomposed recipess essential oil can effectively treat DSS-induced recurrent ulcerative colitis in mice,and its mechanism of action may be achieved by intervening in the TLR/MyD88 signaling pathway to regulate the differentiation of Tfh cells and the balance of intestinal flora. |
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