The Molecular Mechanism Of Sishen Pill Intervention On The Activation Of Dendritic Cells Regulated By ATP-P2X7-Panx1 Signal In The Treatment Of Ulcerative Colitis

Objective:In this study,sodium glucan sulfate（DSS）method was used to replicate the chronic colitis model of BALB/c male mice.In addition to evaluating the curative effect of Sishen Pill on colitis mice,the level and differentiation of dendritic cells were observed,and from the perspective of interfering with the ATP-P2X7-Panx1signal pathway,To explore the possible mechanism of Sishen Pill in treating chronic ulcerative colitis mice.Methods:1.Model replication and administration methods:40 SPF BALB/c healthy male mice,22～24 g,were fed adaptive for 3 days and randomly divided into 4 groups:Control group（Ctrl）,Control+Sishen pill group（Ctrl+SSP）,model group（DSS）and model+Sishen pill group（DSS+SSP）,with 10 mice in each group.DSS was used to replicate the chronic ulcerative colitis model of mice.Except mice in the normal group and the normal+Sishen pill group who drank ultra-pure water normally,mice in the model group and the model+Sishen pill group drank ultra-pure water freely for a week to prepare 3%DSS（w/v）solution.After drinking ultra-pure water continuously for 7 days from the 8th day,Then 2%DSS（w/v）solution was used to drink water freely for 7 days.After successful model replication,administration started from the 8th day,and the normal+Sishen pill group and the model+Sishen pill group were given 2.5gkg^-1Sishen Pill suspension by gavage,and the normal group and the model group were given the same volume of normal saline by gavage for continuous 14 days.2.Efficacy evaluation of Sishen Pill in the treatment of DSS induced chronic colitis:The changes of body weight,fecal consistency,hair status,colorectal bleeding and mental activity status of mice in each group were monitored every day,and the changes of body weight were recorded.On the day of death,the mice were anesthetized,and the colon part was taken out,and the colon length was photographed,measured and weighed to complete the calculation of colon mass index（unit length colon mass）.Colon tissue was taken for HE staining,and the transverse section of colon tissue of mice was observed by microscope,and the microscopic tissue was scored according to the pathological damage scoring standard.3.ATP level detection:Enzyme-linked immunosorbent assay（ELISA）was used to detect the levels of ATP,ADP,AMP and Ado.4.Analysis of the level of DC cells and their subpopulations:Flow cytometry（FCM）was used to analyze the level of DC cells and their subpopulations:CD11c⁺CD103⁺E-cadherin⁺,CD11c⁺CD103⁺E-cadherin⁺CD8a⁺,CD11b⁺CD103⁺CD8a⁺,CD11b⁺CD103⁺CD8a^-,CD11b^-CD103⁺CD8a⁺,CD11b^-CD103⁺CD8a^-cell number detection,to explore whether Sishen Pill can regulate DC cell differentiation and its subgroup level.5.ATP-P2X7-panx1 signaling pathway and DC cell differentiation related protein detection:The protein expression levels of P2X7,Panx1,Connexin43,CCR6,CCR7,CCL19,CCL20,CD39,CD73,A2a and A2b in colon tissues were detected by Western bloting method.6.Detection of P2X7 and Panx1 mRNA level:RT-qPCR method was used to detect the expression level of P2X7 and Panx1 mRNA in mouse colon tissue.7.Statistical data analysis:In this experiment,SPSS 22.0 statistical software was used to analyze the statistical data,and unpaired t test,multiple t test,and one-way ANOVA were used to evaluate the statistical significance between different groups.P<0.05 indicates statistical significance,and P<0.01 indicates extremely significant statistical significance.Results:1.Effect evaluation of Sishen Pill on chronic colitis induced by DSS in mice:The weight of mice in Ctrl and Ctrl+SSP groups increased gradually,and the condition was good.The colitis weight of mice in the model group decreased,the diet was less,the hair was yellowish,the feces was thin and thick,and the anus was bleeding.The body weight of mice in the model group decreased sharply after drinking 3%DSS on the fourth day（P<0.05 or P<0.01）.On the 15th day,the mice began to drink 2%DSS.It could be found that the body mass of mice in DSS group increased slowly,and on the 18th day,the body mass of mice in DSS+SSP group decreased slowly and increased rapidly,and was always higher than that in DSS group（P<0.05）.Dietary volume was increased,hair was shiny,in addition,the daily stool viscosity,OB score and DAI score were significantly improved.Compared with normal group,colon length in DSS group was significantly shortened（P<0.01）,but colon mass,colon mass index,colon mass/colon length,body mass/colon length,spleen mass,spleen mass index and colon tissue injury score were significantly increased（P<0.05 or P<0.01）.After treatment,it was significantly improved（P<0.05or P<0.01）.Observation of the pathological sections of mice under the microscope showed that the ulcer was significantly reduced or repaired after the treatment of Sishen Pill,the mucosal epithelium recovered somewhat and tended to be complete,the inflammatory cell infiltration decreased and the glands increased,suggesting that Sishen pill has a curative effect on the treatment of experimental mice chronic colitis.2.ATP level detection:Compared with normal group,the contents of ATP and AMP in colon tissue of DSS group were significantly increased（P<0.01）,while the contents of ADP and Ado were decreased（P<0.05 or P<0.01）.At the same time,the values of ADP/ATP and AMP/ATP dropped sharply（P<0.01）.After treatment with Sishen Pill,the expression levels of ATP and AMP were significantly decreased（P<0.05）,while the contents of ADP and Ado were significantly increased（P<0.01）.The values of ADP/ATP and AMP/ATP were significantly increased（P<0.05）.3.The regulatory effect of Sishen Pill on DSS induced chronic mouse colitis DC cells:DC cells were detected by flow cytometry,and compared with the normal group,CD11c⁺CD103⁺E-cadherin⁺,CD11c⁺CD103⁺E-cadherin⁺CD8a⁺,CD11b⁺CD103⁺CD8a⁺,CD11b^-CD103⁺CD8a⁺increased significantly in DSS group（P<0.05 or P<0.01）.The levels of CD11b⁺CD103⁺CD8a^-and CD11b^-CD103⁺CD8a^-cells were significantly decreased（P<0.05 or P<0.01）,and compared with DSS group,CD11c⁺CD103⁺E-cadherin⁺,CD11c⁺CD103⁺E-cadherin⁺CD8a⁺,CD11b⁺CD103⁺CD8a⁺,CD11b^-CD103⁺CD8a⁺and CD11b^-CD103⁺CD8a⁺in DSS+SSP group decreased sharply（P<0.05 or P<0.01）.The levels of CD11b⁺CD103⁺CD8a^-and CD11b^-CD103⁺CD8a^-cells were increased（P<0.05）,indicating that Sishen Pill could effectively regulate the expression of DC cell subtypes.4.ATP-P2X7-Panx1 signaling pathway and DC cell differentiation related protein detection:ATP-P2X7-Panx1 signaling pathway was detected,and compared with normal group,the expressions of P2X7,Panx1,Connexin43,CCR6,CCR7,CCL19,CCL20 and A2b in DSS group were significantly increased（P<0.01）.The expressions of CD39,CD73 and A2a were highly decreased（P<0.01）.Compared with DSS group,the expressions of P2X7,Panx1,Connexin43,CCR6,CCR7,CCL19,CCL20 and A2b decreased sharply after treatment with Sishen Pill（P<0.01）,while the expressions of CD39,CD73 and A2a increased significantly（P<0.01）.5.Detection of P2X7 and Panx1 mRNA level:The mRNA levels of P2X7 and Panx1 in colon tissues of mice were detected by RT-qPCR.The mRNA levels of P2X7 and Panx1 in DSS group were significantly higher than those in Ctrl group（P<0.01）.The mRNA levels of P2X7 and Panx1 in DSS+SSP group were significantly lower than those in DSS group（P<0.01）.Conclusion:The effective treatment of Sishen pill for DSS induced chronic mouse colitis may be achieved by regulating DC cell differentiation by inhibiting ATP-P2X7-Panx1signaling pathway.