| Objective:The mouse model of spleen-kidney yang-deficiency colitis was established by dextran sodium sulfate(DSS)+subcutaneous injection of hydrocortisone+rhubarb gavage method,and the effect of Sishen Pill(SSP)on UC and dendrites were observed,and the regulatory effect on the dendritic cells was observed.The mechanism of SSP regulating the differentiation of dendritic cells and the treatment of UC were discussed.Methods:1.Reproduction of UC model of spleen and kidney yang deficiency in mice and drug treatment methods:40 mice were adaptively fed for 1 week,and they were randomly divided into 4 groups,namely the normal group(Normal),and normal mice with the addition of Si Shen The pill group(Normal+SSP),the model group(DSS)and the Sishen pill group(DSS+SSP),each group has 10 pills.The first stage is to replicate the“spleen deficiency”model:the normal group and the normal mice plus the Sishen pill group were irrigated with distilled water,1mL/d each,and the mice in each group in the model group were irrigated with rhubarb solution,1m L each/d,continuous gavage for 7 days;The second stage is to replicate the“spleen and kidney yang deficiency”ulcerative colitis model:on the 8th day,25mg/(kg·d)hydrocortisone was injected alternately on the left and right hips of the model group,and 2.5%(w/v)was given at the same time The DSS solution was drunk freely for 7 consecutive days,and the other groups were drunk with distilled water.On the day after the last injection of hydrocortisone and DSS,the normal mice were treated with Sishen pill group and Sishen pill group by gavage with 5g/kg Sishen pill suspension for 7 days,normal Group and model group were given an equal volume of distilled water by gavage.The mice were sacrificed 7 days after the administration,and the peripheral blood,colon tissue and spleen of the mice were collected.2.Evaluation of the efficacy of Sishen Pills in treating UC of spleen and kidney yang deficiency:mice in each group were weighed at 15 o’clock every afternoon and their body weight was recorded,and the general changes of mice were observed.On the8th day,collect fresh feces from each mouse and operate according to the instructions of the occult blood test kit.The mice were deeply anesthetized with 2%sodium pentobarbital,fresh blood was collected,and T3,T4,T indicators were detected;Separate the mouse from the anus to the ileocecal colon and the mouse spleen,measure the length of the colon,weigh the mass of the colon and the spleen,calculate the weight per unit length of the colon and the mass index of the spleen,and score the disease activity index(DAI);Make paraffin sections of colon tissue and H&E staining,observe the pathological conditions of the colon under a microscope and collect pictures,use double-blind method for pathological scoring,and use ELISA to detect IL-10,TNF-α,IFN-γ,IL-12 of colonic mucosa,IL-6,IL-1βand other factors.3.Analysis of dendritic cells:use flow cytometry to detect the number of dendritic cell subsets CD103~+CD11c~+CD324~+,CD103~+CD11c~+TNF-α~+,CD103~+CD11c~+INOS~+cells,and explore the treatment of spleen by Sishen pills The immune mechanism of kidney-yang deficiency type UC.4.TLR4/NF-κB pathway protein analysis:Western Bloting method was used to detect and analyze the core protein and related proteins of the TLR4/NF-κB signaling pathway,and explore the regulatory effect of Sishen pills on the TLR4/NF-κB signaling pathway.5.Experimental data analysis:using graphpad prism software 8.0 software for statistics,comparison between groups using one-way ANOVA,expressed by the mean standard deviation((?)±s),with P<0.05 as the statistical result,there was significant difference.Results:1.Evaluation of the curative effect of Sishen Pill in treating UC of spleen and kidney yang deficiencyCompared with the normal group,the mice in the model group showed bleeding with the naked eye,dirty anus,slower or even negative weight gain,and a significant increase in the DAI score.The mice showed a significant decrease in body weight,a significant decrease in colon length,weight per unit length of the colon and mouse spleen weight gain and spleen weight index were increased,the difference was statistically significant(P<0.05 or P<0.01);Compared with the model group,the mice in the treatment group were treated with Sishen pills for 3-5 days,and the symptoms reversed,and the mice recovered significantly after 6-7 days.The weight of the mice gradually stabilized,the DAI score decreased,the length of the mouse colon was extended,the mouse spleen and the spleen weight index were decreased,the difference was statistically significant(P<0.05 or P<0.01).It suggests that Sishen Pill can effectively improve the symptoms of UC mice with spleen and kidney yang deficiency.Observation of the colon pathological section under the microscope revealed that the colon mucosa of the model group showed obvious epithelial shedding,reduced glands,ulcer formation,and inflammatory cell infiltration.The colonic epithelium of the mice treated with Sishen Pills recovered and tended to be intact,the abundance of goblet cells increased,the infiltration of inflammatory cells was reduced,and no obvious ulcers were formed.It suggests that Sishen Pill can effectively alleviate the pathological damage of UC mice with spleen and kidney yang deficiency.Compared with the normal group,the serum levels of T3,T4,and T in the model group decreased significantly(P<0.05 or P<0.01),and the difference was statistically significant;after treatment with Sishen pills,the T level increased significantly(P<0.01)),suggesting that Sishen Pill can increase the T level of UC mice with spleen and kidney yang deficiency.ELISA detection of colonic cytokines found that TNF-α,IL-12,IL-6,and IL-1βfactors were highly expressed in the colon mucosal tissues of mice in the model group,and the expression levels of IL-10 and IFN-γfactors were reduced,and the difference was statistically significant.Significance(P<0.05 or P<0.01);after 7 days of intragastric treatment,the expression of TNF-α,IL-12,IL-6,and IL-1βin the Sishen Pill group was significantly inhibited,and IL-10.The expression of IFN-γfactor increased,and the difference was statistically significant(P<0.05 or P<0.01).It is suggested that Sishen Pill can regulate the level of cytokines in the colon of UC mice with spleen and kidney yang deficiency.2.The effect of Sishen Wan on the level of dendritic cells in UC mice with spleen and kidney yang deficiencyCompared with the normal group,the model group CD103~+CD11c~+CD324~+,CD103~+CD11c~+TNF-α~+,CD103~+CD11c~+INOS~+increased,the difference was statistically significant(P<0.05 or P<0.01);compared with the model group In the treatment group,the expression levels of CD103~+CD11c~+CD324~+,CD103~+CD11c~+TNF-α~+,CD103~+CD11c~+INOS~+decreased(P<0.05 or P<0.01),and the difference was statistically significant.It is suggested that Sishen Pill can reduce the expression of inflammatory dendritic cells in UC mice with spleen and kidney yang deficiency.3.Sishenwan on the expression of TLR4/NF-κB pathway signaling protein in UC mice with spleen and kidney yang deficiencyCompared with the normal group,the expression of TLR4,NF-κB,TRAF6,Myd88,TAB2 protein in the colon tissue of the model group was significantly up-regulated,and the level of IκB protein was significantly suppressed,and the difference was statistically significant(P<0.01);compared with the model group The expression of TLR4,NF-κB,TRAF6,Myd88,TAB2 protein in the colon tissue of the Sishenwan group mice all decreased,and the expression level of IκB protein increased,and the difference was statistically significant(P<0.01).It is suggested that Sishen pills can effectively regulate the expression of the core protein and downstream proteins of the TLR4/NF-κB pathway signal.Conclusion:Sishen pill can effectively treat UC of spleen kidney yang deficiency type,which may be achieved by intervening TLR4/NF-κB signaling pathway,reducing the expression of inflammatory genes and regulating the expression of inflammatory DC. |
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