| Objective:To verify that Sishen pill regulates the balance of memory Treg/Th17 cells in the treatment of dextran sodium sulfate(DSS)-induced chronic recurrent ulcerative colitis(UC),and to observe the effects of Sishen pill on various proteins and genes in Numb/Notch-glucose metabolism pathway.To explore the possible mechanism of Sishen Pill in regulating the balance of memory Treg/Th17 cells in the treatment of ulcerative colitis(UC)by interfering with Numb/Notch-glucose metabolism pathway.Methods:1.Replication and administration of animal models:40 SPF BALB/c male mice aged 6-8 weeks were randomly divided into 4 groups,namely Control group(Control),Control+Sishen Pills group(Ctrl+SSP),model group(DSS)and model+Sishen Pills group(DSS+SSP),with 10 mice in each group.Among them,the Control group and the Control+Sishen Pills group drank distilled water freely throughout the whole process,the model group and the model+Sishen Pill group were given 3%(w/v)DSS solution for continuous free drinking for 7 days,the DSS solution was stopped and replaced with distilled water for 7 days,and then replaced with 2.5%(w/v)DSS solution for 7 days.Finally,the mice were sacrificed 1 day after DSS solution was stopped,namely,the model replication of chronic recurrent ulcerative colitis was completed.The mice were treated with 3%(w/v)DSS solution on the first day as day1,and the 3%(w/v)DSS solution was stopped on day 7,and the mice were treated with Sishen Pills by ingavage according to their body weight.The normal group,the normal+Sishen Wan group and the model group were given distilled water,According to the preliminary experimental results,and the model+Sishen Pills group was given 5g/kg Sishen Pills suspension,once a day.The rats were given intragastric treatment at a fixed time.After two weeks of continuous administration,the rats were anesthetized and sacrificed.Spleen,colon and mesenteric lymph nodes were separated immediately.2.Efficacy evaluation of Sishen Pills in the treatment of chronic relapsed UC mice induced by DSS:Daily record the change of the mice weight,calculating the weight change rate of mice[weight rate=mice daily weight(g)/initial body weight in mice(g)×100%],at the same time during the period of experiment to each mouse droppings state changes(including viscosity),to evaluate fecal occult blood,and to observe the mice’s state of mind every day,diet,etc.,Disease active index(DAI)[DAI=weight change score+fecal viscosity score+stool blood score]was used to evaluate the specific conditions of mice.2 weeks after administration,the mice were anesthetized to death,and spleen,colon and mesenteric lymph nodes were separated.Colon length was measured,spleen and colon weight were weighed,and intestinal weight index,spleen weight index,colon weight index and colon weight per unit length were calculated.Paraffin sections of colon tissues were prepared,and the changes of pathological morphology of colon tissues were evaluated by double-blind microscope observation3.Regulation of Sishen Pills on the balance of memory Treg/Th17 cells:Flow cytometry(FCM)was used to detect the expression levels of memory Treg cells and memory Th17 cells in T cell subsets of mouse mesenteric lymph node cells,specifically effector memory Treg cells(CD4~+CCR7~-Foxp3~+,TEM-m Treg),central memory Treg cells(CD4~+CCR7~+Foxp3~+,TCM-m Treg),effector memory Th17 cells(CD4~+CCR7~-IL-17~+,TEM-m Th17)and central memory Th17 cells(CD4~+CCR7~+IL-17~+,TCM-m Th17),to verify the effect of Sishen Pills on regulating Treg/Th17 cell balance.4.Detection of related indexes of sugar metabolism and energy metabolism pathway:Mouse colon hexokinase 2(HK2),pyruvate kinase(PK),glucose6-phosphatase(G6pase),phosphoenolpyruvate carboxykinase(PEPCK),aldenase A(ALDA),glucokinase(GCK),lactate dehydrogenase A(LDH-A),adenosine triphosphate(ATP)were determined by enzyme-related immunosorbent assay(ELISA).ATP,adenosine diphosphate(ADP),adenosine monophosphate(AMP),adenosine(Ado)were detected to explore the effects of Sishen pills on energy metabolism and sugar metabolism.5.Detection of Numb/Notch-related proteins in Glucose Metabolism Pathway:Using Western Blot method.Numb/Notch-glucose-related proteins such as Numb,Notch3,DLL1,DLL2,DLL4,MSI2,Maml1,Jagged1,GLlut1,Glut4,Gsk3β,STAT4,smad2/3,To explore the regulatory effect of Sishen Pill on Numb/Notch-glucose metabolism pathway.6.Data analysis:SPSS22.0 software was used for statistical analysis.Comparison between groups was performed by control t test and one-way analysis of variance(Mean±SEM).P<0.05 meant that statistical results had significant differences,and P<0.01 meant that statistical results had extremely significant differences.Statistical charts were then made using Graph Pad Prism 8.0 software.Results:1.Effect evaluation of Sishen Pills on DSS induced chronic relapsed UC miceAfter treatment with Sishen Pills,compared with model group,the general state of mice in model+Sishen Pills group was significantly improved,diet and body weight were significantly increased,mental state was significantly improved,stool characteristics,daily OB score and DAI score were significantly decreased(P<0.05or P<0.01),colon length was significantly recovered(P<0.05).Microscopically,the pathological lesion of colon tissue recovered significantly(P<0.05),and the colon weight,colon weight per unit length,colon weight index,spleen weight and spleen weight index all decreased significantly(P<0.05 or P<0.01).These results indicated that Sishen Pills could restore the disease state of ulcerative colitis mice.2.Balance effect of Sishen Pills on memory Treg/Th17 cells in chronic relapsed UC miceCompared with normal group,the number of central memory and effector memory Treg cells in model group decreased(P<0.05 or P<0.01),and the number of central memory and effector memory Th17 cells increased.After drug administration,the number of memory Treg cells increased(P<0.05 or P<0.01),the number of Th17 cells decreased significantly(P<0.05 or P<0.01),and the ratio of memory Treg/Th17 increased(P<0.05 or P<0.01)in model+Sishen Pills group.The level of IL-10 was also significantly increased(P<0.05).These results indicated that Sishen Pills could regulate the balance of memory Treg/Th17 cells.3.Regulation of Numb/Notch pathway and glucose metabolism pathway in chronic relapsed UC mice by Sishen PillsCompared with the control group,the expression levels of Notch3,DLL1,DLL2,and DLL4 in the model group were significantly increased(P<0.05 or P<0.01),while the expression level of Numb was significantly decreased(P<0.05 or P<0.01).Compared with the model group,after drug administration,the expression levels of Numb were significantly increased(P<0.05 or P<0.01).The expression levels of Notch3,DLL1,DLL2 and DLL4 in model+Sishen Pills group were significantly decreased(P<0.05 or P<0.01),while the expression level of Numb was significantly increased(P<0.05 or P<0.01).ATP,ADP,AMP,Ado,HK2,PK,G6pase,PEPCK,ALDA,GCK and LDH-A were detected by enzyme-linked immunosorbent assay.Compared with control group,Ado expression in model group was decreased(P<0.05).The expression levels of other indicators were significantly increased(P<0.05 or P<0.01).After treatment with Sishen pills,the expression levels of ATP,ADP,AMP,Ado,HK2,PK,G6pase,PEPCK,ALDA,GCK and LDH-A were significantly decreased(P<0.05 or P<0.01).Moreover,Ado expression level was increased(P<0.05).These results suggest four gods pill could inhibit Numb/Notch signal activation and correct the sugar metabolism and correct glucose metabolism level.Conclusion:1.Sishen Pills can effectively treat DSS-induced chronic recurrent ulcerative colitis.2.The mechanism of Sishen pills in the treatment of DSS induced chronic recurrent ulcerative colitis may be realized by intervening in the Numb/Notched-glucose metabolic pathway to regulate the balance of memory Treg/Th17cells. |
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