| Aim:While confirming the efficacy of Xiangshen pill in the treatment of experimental ulcerative colitis,the possible mechanism of Xiangshen pill was explored by interfering with TLR/NF-κB signal pathway to regulate the level of inflammatory dendritic cells.Methods:1.Animal model replication and administration methods:40 SPF male BALB/c mice were randomly divided into 4 groups,namely normal group,model group,Xiangshen Pill group and mesalazine group,with 10 mice in each group;except for normal control group,the rest groups were prepared with 3%(w/v)DSS solution prepared with ultrapure water for free drinking for 5 days,and then changed to normal drinking water for 7 days after stopping drinking DSS,and then changed to 2%DSS solution for free drinking for 5 days.Mice began to drink 3%DSS solution as the first day.From the 11th day,according to the body surface area,Xiangshen Pill group was given 5g/kg Xiangshen Pill suspension by gavage;mesalazine group was given 300mg/kg mesalazine suspension by gavage;normal group and model group were given the same volume of physiological saline by gavage at a fixed time every day for 10 days.On the 21th day,the peripheral blood and colon tissues of mice were collected.2.Pharmacodynamic evaluation of Xiangshen Pill on DSS induced recurrent ulcerative colitis:observe and record the weight change,hair color,fecal characteristics,hematochezia,activity and mental state of mice in each group at the same time every day.After the mice were anesthetized and euthanized,the colon tissue was cut off,the length of the colon tissue was measured and weighed,the intestinal weight index was calculated,and the colon pathological sections were prepared.The pathological status of the colon in each group was observed under the microscope,and the pathological injury score was evaluated by double-blind method.The levels of IL-13,IL-4,IL-33,IFN-γand CCL-2 were detected by ELISA.3.Effect of Xiangshen Pill on intestinal microflora of colitis mice:the feces of ileocecal part of intestinal tract of mice were quickly collected with sterilized scissors and forceps,and then frozen in dry ice in a 1.5 m L cryopreservation tube,and then moved to-80℃for storage.After that,DNA extraction and PCR amplification were used to obtain the base sequence of mice feces,and the database was established to explore the correlation between intestinal flora and recurrent colitis and the effect of Xiangshen Pill on intestinal flora,and the expression of TLR2,TLR4 and TLR5 protein in colon was detected by Western blot.4.Regulatory effect of Xiangshen Pill on inflammatory dendritic cells in mice with colitis:flow cytometry was used to detect the number of inflammatory dendritic cell subsets CD11c~+CD103~+CXCR5~+,CD11c~+CD103~+i NOS~+,CD11c~+CD103~+TNF-α~+,CD11c~+CD103~+E-cadherin~+cells to explore the immune mechanism of Xiangshen Pill in the treatment of experimental colitis.5.Regulation of Xiangshen Pill on TLR/NF-κB signaling pathway in colitis mice:Western blot was used to detect the related protein of TLR/NF-κB signaling pathway,and to explore the regulatory effect of Xiangshen Pill on TLR/NF-κB signaling pathway.6.Statistical analysis:all the experimental data were processed by SPSS 22.0 and graphpad prism software 8.0,independent sample t-test or one-way ANOVA were used,and the experimental results were analyzed by mean±SEM,P<0.05 was statistically significant.Results:1.Pharmacodynamic evaluation of Xiangshen Pill on DSS induced recurrent ulcerative colitis:compared with the normal group,the mice in the model group showed weight loss,loose stool and loose stools.On the 5th day,bloody stools,mental depression,loss of appetite,dull hair color,significantly shortened colon length,significantly increased colon weight and intestinal weight index were observed by naked eyes(P<0.01或P<0.05).Compared with the model group,the mice in the model group began to be gavaged on the 11th day After treatment,the symptoms of the treatment group were improved in varying degrees,mental state was good,appetite increased,stool and blood decreased,colon length recovered,colon weight and intestinal weight index decreased significantly(P<0.01或P<0.05).It is suggested that Xiangshen Pill can effectively improve the symptoms of experimental colitis in mice.Microscopic observation of colonic histopathological sections showed that the colonic mucosa of normal group mice was complete and smooth,without obvious ulcer,inflammatory cell infiltration and hyperemia and edema;while the colonic mucosa epithelium of model group mice fell off,crypt structure changed,arranged disorderly,the lamina propria and submucosa have a large number of inflammatory cell infiltration,ulcer formation,hyperemia and edema in many places.and the score of pathological injury increased significantly(P<0.01);Xiangshen Pill group and control group In mesalazine group,the colonic epithelium recovered and tended to be intact,the goblet cell richness increased and arranged relatively regularly,the inflammatory cell infiltration decreased,and there was no obvious ulcer formation,and the score of pathological injury decreased significantly(P<0.01 or P<0.05).ELISA showed that the levels of IL-13 and IL-4 in colon tissue of model group were significantly decreased,while the levels of IL-33,IFN-γand CCL-2 were significantly increased;after intragastric administration of xiangshen pill and mesalazine,the levels of IL-13 and IL-4 were significantly increased,and the expression levels of IL-33,IFN-γand CCL-2 were significantly inhibited.It is suggested that Xiangshen Pill can regulate the expression of inflammatory factors in mice with recurrent colitis.2.Effect of Xiangshen Pill on intestinal flora of colitis mice:compared with the normal group,the diversity of intestinal flora in the model group decreased,and the abundance of Staphylococcus and bacteroidaceae in the model group increased significantly,while the abundance of muribaculaceae,lachnospiraceae and ruminococcaceae in the model group decreased significantly;Compared with the model group,the abundance of lachnospiraceae and ruminococcaceae in xiangshen pill group and mesalazine group increased,while the abundance of bacteroidaceae decreased significantly.The results showed that the intestinal flora of DSS induced colitis mice was unbalanced,the pathogenic bacteria increased and the beneficial bacteria decreased.The expression of TLR2,TLR4 and TLR5 was significantly increased in the model group(P<0.01),and the correlation analysis with intestinal flora showed that the intestinal flora in the model group was positively correlated with TLR2,TLR4 and TLR5,and more closely with TLR4 and TLR5;while the normal group and Xiangshen Pill group were negatively correlated with TLR2,TLR4 and TLR5.It is suggested that Xiangshen Pill may treat colitis by regulating intestinal flora and reducing the expression of TLR2,TLR4 and TLR5.3.Regulatory effect of Xiangshen Pill on inflammatory dendritic cells in mice with colitis:the inflammatory dendritic cells were detected by flow cytometry.Compared with the normal group,the subtypes of inflammatory dendritic cells in the model group were CD11c~+CD103~+CXCR5~+,CD11c~+CD103~+i NOS~+,CD11c~+CD103~+E-cadherin~+,CD11c~+CD103~+TNF-α~+Compared with the model group(P<0.01),the expressions of CXCR5,i NOS,E-cadherin and TNF-αwere significantly decreased after treatment with Xiangshen Pill and mesalazine(P<0.01).It is suggested that Xiangshen Pill can effectively inhibit the expression of inflammatory dendritic cells in colitis mice.4.Effect of Xiangshen Pill on TLR/NF-κB signaling pathway in mice with colitis:Western blot analysis Compared with the normal group,the expression levels of My D88,IRAK4,TRAF6,TAK1,P38,NF-κB,IRAK4,TAB1,TAB2,MKK3,MKK6and CREB in the colon tissue of the model group were significantly increased(P<0.01or P<0.05);after drug treatment,the expression levels of the above proteins in the colon tissue of the treatment group were different from those in the model group The degree of decline(P<0.01 or P<0.05).It is suggested that Xiangshen Pill can inhibit the activation of TLR/NF-κB signaling pathway.Concussion:1.Xiangshen pill can effectively treat experimental recurrent ulcerative colitis,relieve the symptoms of ulcerative colitis,alleviate the pathological damage of colonic mucosa in mice,effectively increase the expression of anti-inflammatory factors IL-13and IL-4,and inhibit the expression of pro-inflammatory factors such as IL-33,IFN-γand CCL-2.2.Xiangshen pill may regulate the diversity and structure of intestinal flora in DSS induced recurrent ulcerative colitis mice by inhibiting the expression of TLR2,TLR4and TLR5.3.Xiangshen pill can inhibit the expression of inflammatory dendritic cells in peripheral blood of mice with recurrent ulcerative colitis.4 xiangshen pill can effectively inhibit the activation of TLR/NF-κB signaling pathway in colonic tissue of mice with recurrent ulcerative colitis. |
