Research On Mechanism Of Sishen Pill Regulated Memory T Cells To Relieve Chronic Colitis By Controlling MiRNA-200/ZEB Signal Pathway

Objective:To explore the mechanism of Sishen Pill（SSP）in treatment of chronic ulcerative colitis induced by DSS effectively,and to find out if miRNA-200/ZEB signal was link to interfere differentiation of immune memory T cells.Methods:40 SPF male BALB/c mice were randomly divided into 4 groups,normal group（Control）,control group（Control+SSP,Ctrl+SSP）,model group（DSS）and Sishen Pill group（DSS+SSP）,each group with 10 animals.The model of chronic ulcerative colitis mouse was set up by dextran sodium sulfate（DSS）.Mice in DSS group and Sishen Pill group were given 3%DSS（w/v）solution for 5 days,which dissolved in ultrapure water.Normal water a week from the sixth day.Then 2%DSS（w/v）solution were given for 5 days from the 13th day.After the model was replicated successfully,on the 11th day,Control+SSP group and DSS+SSP group were given SSP(2.5g·kg^-1)for7 days.Monitor the symptoms of the mice’s weight,fecal properties,activity status and other symptoms every day during the experiment.Occult blood（OB）score and disease activity index（DAI）were also calculated.On the 18th day,the mice were weighed and killed after anesthetized.Spleen and colon tissues were collected to be tested.The colonic length and colonic weight were measured,then the index of colonic weight and index of weight/length were calculated.Colon tissue were prepare for HE staining,and the pathological injury point was scored under the microscope;Enzyme-linked immunosorbent assay（ELISA）tests the levels of interleukin 6（IL-6）,interleukin 9（IL-9）,interleukin 12（IL-12）,interleukin 15（IL-15）and Interferon-γ（IFN-γ）inflammatory factors in colon tissue;Flow cytometry（FCM）detects the levels of various subsets of memory T cells in spleen;Western blotting（WB）was used to test the expression of miRNA-200/ZEB signaling pathway related proteins and the related proteins affecting the survival of immune memory T cells in colon tissues.Real-time fluorescent quantitative PCR（RT-PCR）detects the expression of miRNA-200 family members and mRNA of ZEB family on miRNA-200/ZEB signal pathway.From the perspective of miRNA-200/ZEB signal regulating the level and survival of immune memory T cells,we explored the possible mechanism of SSP in the prevention and treatment of chronic ulcerative colitis.Results:1.General clinical efficacy evaluation of Sishen Pill on mice with chronic colitis induced by DSSThe weight of the mice in the control group was relatively stable,and the condition are good.The mice in the DSS group were slightly depressed,tended to flock together,hair color are messy and dark,weight loss,decreased activity,less appetite,perianal filth,loose feces and blood in stool.Given continuous intragastric treatment with SSP for 7 days,the colonic length recovered,the body weight gradually gained back,The colonic weight,index of colonic weight/body weight and weight/length all decreased.In the Control group,the colonic mucosa was intact,the crypts and glands were arranged neatly.However,in the DSS group,the mucosal surface congestion,edema,erosion,partial superficial ulcer formed,the crypt structure changed,goblet cells decreased,inflammatory cell infiltration in the lamina propria and etc.The colonic mucosal surface hyperemia and edema in the DSS+SSP group were significantly reduced,erosion repaired and inflammatory cell infiltration were significantly reduced.The pathological injury score by histopathology was significantly repaired（P<0.01）.The expression of inflammatory factors was tested by ELISA.Compared with Control group,the expression level of IL-9 and IL-12pro-inflammatory factor in the colon tissue of the DSS group were significantly increased（P<0.01）,but IL-2 and other anti-inflammatory factors were decreased significantly（P<0.01）.After the treatment of SSP,the trend of the above inflammatory factors was reversed,and the difference was statistically significant（P<0.05 or P<0.01）,suggesting that SSP can treat chronic colitis mice effectively.2.Effect of Sishen Pill on memory T cell level in mice with ulcerative colitisFlow cytometry revealed that compared with Control group,the levels of Tcm（CD45RA^-CD62L⁺CCR7⁺）cells in DSS group were significantly increased（P<0.01）,the levels of CD4⁺and CD8⁺Tcm cells were both significantly increased（P<0.01）.The levels of Tem（CD45RA^-CD62L^-CCR7^-）also elevated（P<0.05）,the levels of CD4⁺and CD8⁺Tem cells were both significantly increased（P<0.01）.The levels of m Treg（CD45RA^-CCR7⁺Foxp3⁺and CD45RA^-CCR7^-Foxp3⁺）were decreased（P<0.01）,while the levels of m Th1（CD45RA^-CCR7⁺CXCR3⁺and CD45RA^-CCR7^-CXCR3⁺）,m Th2（CD45RA^-CCR7⁺CCR4⁺and CD45RA^-CCR7^-CCR4⁺）,m Th7（CD45RA^-CCR7⁺IL-7R⁺and CD45RA^-CCR7^-IL-7R⁺）and m Th17（CD45RA^-CCR7⁺IL-17⁺and CD45RA^-CCR7^-IL-17⁺）were all significantly increased（P<0.01）.The levels of Blimp-1⁺and TIGIT⁺marked memory T cells were both significantly decreased（P<0.01）,however,the levels of KLRG1⁺and T-BET⁺labeled memory T cells were both significantly increased（P<0.05 or P<0.01）.After treatment with Sishen Pill,the levels of Tcm,CD4⁺and CD8⁺Tcm in spleen of mice were significantly decreased（P<0.01）.The levels of Tem,CD4⁺and CD8⁺Tem in spleen of mice were also significantly decreased（P<0.01）.The levels of m Treg cells were significantly upregulated（P<0.01）.While The levels of m Th1、m Th2、m Th7 and m Th17 cells subsets all decreased significantly（P<0.01）.The levels of memory T cells of Blimp-1⁺、TIGIT⁺、KLRG1⁺and T-Bet⁺were all reversed,and the difference was statistically significant（P<0.05 or P<0.01）.These results suggest that Sishen Pill can regulate the memory T cell subsets in mice with chronic colitis.3.Effects of Sishen Pill on expression levels of miRNA-200 family,mRNA of ZEB1 and ZEB2,in colon tissues of mice with ulcerative colitisRT-PCR results indicated that compared with Control group,the expression levels of miRNA-200 family in colon tissue of DSS group mice was decreased（P<0.05 or P<0.01）,especially miRNA-200a and miRNA-200c were both decreased markedly（P<0.01）;the expression of ZEB1 mRNA was increased（P<0.05）,while ZEB2 mRNA was decreased（P<0.05）.After treatment with Sishen Pill,the expression levels of miRNA-200 family was increased,which did not reach statistical significance,except miRNA-200a and miRNA-200c（P<0.01）.The expression of ZEB1mRNA was significantly decreased（P<0.01）,and ZEB2 mRNA was significantly increased（P<0.01）.These results suggest that Sishen Pill can regulate miRNA-200family and mRNA expression on miRNA-200/ZEB pathway,such as mRNA of ZEB1and ZEB2.4.The expression of miRNA-200/ZEB signal pathway related proteins in mice with ulcerative colitis by Sishen PillWB results showed that compared with Control group,the protein expression levels of ZEB1 and ZEB2 were significantly increased in colon tissue of the DSS group（P<0.01）.Snail,transformation suppressor of miRNA-200 family,was significantly increased（P<0.01）.The target protein of epithelial-to-mesenchymal transition——E-cadherin（epithelial cell markers）and Vimentin（interstitial cell markers）were both significantly increased（P<0.01）.TGF-β1 protein expression levels were also significantly increased（P<0.01）.The protein expression levels of pro-apoptotic proteins such as Fox O3a、Fas、Fas L、ID2、Caspase-3、BAX and NOXa were increased（P<0.05 or P<0.01）,Accompanied by the decline of apoptosis inhibitor protein Bcl-2（P<0.01）.In DSS+SSP Group,the protein expression levels of ZEB1 and ZEB2 were both significantly down-regulated（P<0.01）,Snail、E-cadherin、Vimentin and TGF-β1 were all significantly decreased（P<0.05 or P<0.01）.The expression levels of pro-apoptotic and anti-apoptotic proteins were reversed.These results above suggest that Sishen Pill can regulate the expression of miRNA-200/ZEB signal pathway related proteins and apoptosis related proteins.Conclusion:Sishen Pill has a good therapeutic effect on chronic ulcerative colitis induced by DSS,The internal mechanism mainly through the regulation of memory T cell differentiation by controlling miRNA-200/ZEB signal pathway.