Mechanism Of Sishen Pill Treated Chronic Relapsed Ulcerative Colitis By Regulating Differentiation Of Memory T Cell Via AMPK-TSC Signaling Pathway

Objective:Observe the effect of Sishenwan?SSP?on UC and the regulation of immune memory T cells through a mouse model of colitis induced by dextran sodium sulfate?DSS?;at the same time,the regulation of SSP is discussed that the target and mechanism of memory T cell differentiation to treat UC from the AMPK-TSC signaling pathway.Methods:The animal model of colitis was induced by sodium dextran sulfate?DSS?.After being treated with Sishen Pill for 7 days,the efficacy of Sishen Pill in the treatment of ulcerative colitis was verified by pathomorphological analysis,and the classification and subsets of memory T cells were detected by flow cytometry.At the same time,enzyme labeling,colorimetry,PCR array analysis and RT-PCR were used to investigate the level of energy metabolism,the activity of related ATP enzymes and the expression of genes related to mitochondrial energy metabolism pathway in colitis mice.At the same time,the expression of upstream and downstream proteins of AMPK-TSC signal was detected by Western blotting.To detect the activities of ATP-related enzymes,and the expression of upstream and downstream proteins of AMPK-TSC signal,in order to clarify the effect of Sishen pill on energy metabolism regulating memory T cells in the treatment of ulcerative colitis,and to explore the mechanism of Sishen pill intervention on energy metabolism regulating memory T cell differentiation in ulcerative colitis from AMPK-TSC signal.Results:1. Preparation of mouse model of ulcerative colitis and evaluation of therapeutic effect of Sishen Pill.Compared with the normal group,the mice in the model group were slightly dispirited,lost weight,reduced activity,dishevelled and dull color,yellowish urine,sparse stool,yellow color,perianal filth,Sishen pill and mesalazine group mice improved,their body weight was gradually stable,and the movement was flexible.The hair color is messy and supple,the urine is yellowish,the stool is fusiform,the dry and wet is moderate,the color is black,and the perianal area is clean.Compared with the normal group,the DSS-induced model mice showed a significant decrease in body weight,an increase in colon weight,a significant decrease in colon length and a significant increase in intestinal weight index?P<0.05or P<0.01?,and the above-mentioned indexes in Sishen pill group and mesalazine group were significantly reversed compared with the model group?P<0.05or P<0.01?.In the normal group,the colonic mucosa was intact,the crypts and glands were arranged neatly;in the model group,the mucosal surface congestion,edema,erosion and partial superficial ulcer formed,the crypt structure changed,goblet cells decreased,inflammatory cell infiltration in the lamina propria,etc.;the colonic mucosal surface hyperemia and edema in the Sishen pill group and mesalazine group were significantly reduced,erosion repair and inflammatory cell infiltration were significantly reduced.Compared with the normal group,the colonic mucosa of the model group showed obvious inflammatory cell infiltration and tissue injury,and the pathological score increased significantly?P<0.01?.The pathological score of colonic mucosa decreased significantly after treatment with Sishen Pill and Mesalazine?P<0.05?.2. Effect of Sishen Pill on the level of memory T cells in mice with ulcerative colitis.Compared with the normal group,the levels of CD45RA^-CD62L⁺CCR7⁺Tcm cells and CD45RA⁺CD62L⁺CCR7⁺Tcm cells in the model group decreased significantly,while the level of CD45RA^-CD62L^-CCR7^-Tem cells increased significantly?P<0.01?.Compared with the model group,after treatment with Sishen pill or mesalazine,the level of CD45RA^-CD62L⁺CCR7⁺Tcm cells in colitis mice in Sishen pill group and mesalazine group increased significantly,the level of CD45RA⁺CD62L⁺CCR7⁺Tcm cells increased significantly,and the level of CD45RA^-CD62L^-CCR7^-Tem cells decreased significantly?P<0.01?.In the CD45RA^-CD62L⁺CCR7⁺Tcm cells,compared with the normal group,the levels of CD4⁺CD45RA^-CD62L⁺CCR7⁺Tcm and CD8⁺CD45RA^-CD62L⁺CCR7⁺Tcm cells in the colon tissue of the model group were significantly increased?P<0.05 or P<0.01?.In the CD45RA⁺CD62L⁺CCR7⁺Tcm cells,compared with the normal group,the levels of CD4⁺CD45RA⁺CD62L⁺CCR7⁺Tcm andCD8⁺CD45RA⁺CD62L⁺CCR7⁺Tcm cells in the colon tissue of the model group were significantly lower than those of the normal group?P<0.05or P<0.01?.Compared with the model group,after treatment with Sishen pill or mesalazine,the levels of CD4⁺CD45RA^-CD62L⁺CCR7⁺Tcm and CD8⁺CD45RA^-CD62L⁺CCR7⁺Tcm cells in colon tissue of mice in both groups decreased significantly,while the levels of CD4⁺CD45RA⁺CD62L⁺CCR7⁺Tcm and CD8⁺CD45RA⁺CD62L⁺CCR7⁺Tcm cells in colon tissue increased significantly?P<0.05or P<0.01?.Compared with the normal group,the level of CD4⁺CD45RA^-CD62L^-CCR7^-Tem cells in the colonic tissue of the model group increased significantly,while the level of CD8⁺CD45RA^-CD62L^-CCR7^-Tem cells decreased significantly?P<0.01?.Compared with the model group,after treatment with Sishen pill or mesalazine,the level of CD4⁺CD45RA^-CD62L^-CCR7^-Tem cells in colon tissue of the two groups decreased significantly,while the level of CD8⁺CD45RA^-CD62L^-CCR7^-Tem cells increased significantly?P<0.05?.3. Effect of Sishen Pill on Energy Metabolism in mice with Ulcerative Colitis.Compared with the normal group,LDH activity value,SDH activity value,Na⁺-K⁺-ATPase,Ca²⁺-Mg²⁺-ATPase activity value and ATP content in the model group were significantly lower than those in the normal group?P<0.05,P<0.01?,and the activity values of Na⁺-K⁺-ATPase,Ca²⁺-Mg²⁺-ATPase and ATP were significantly lower than those in the normal group?P<0.01?.Compared with the model group,the content of ATP in the Sishen pill group increased,and the activity values of Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase increased?P<0.05?.The activity values of LDH and ALDH decreased,which were close to those of the normal group.Both treatment groups could significantly reduce the activity values of LDH,ALDH,SDH and increase the activity values of Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase.4. Regulatory effect of Sishen Pill on the expression and activity of AMPK-TSC and other signal pathway proteins in mice with ulcerative colitis.Compared with the normal group,the protein expression levels of p-AMPK?,TSC1,TSC2,Raptor and p70S6K in the colonic tissue of the model group were significantly inhibited,while the protein expression levels of PI3K,AKT,p-Akt,Rheb,Kif2a,T-bet,4E-BP2,c-Myc and HIF-1a were significantly increased in the model group.Compared with the model group,the protein expression levels of AMPK?,p-AMPK?,TSC1,TSC2,Raptor,Kif2a,4E-BP2 and p70S6K in colon tissue of mice in Sishen Pill group were significantly increased,while the protein expression levels of PI3K,AKT,p-Akt,Rheb,T-bet,HIF-1a and c-Myc were significantly decreased in Sishen Pill group.In the colonic tissue of model group,the expression of 66 m RNA genes related to ATP,such as Atp5f1,Atp2b3,Atp8a2 and Atp6v1e1,decreased significantly,while the expression of 20 genes such as Atp5e,Atp6v1c2 and Atp6v0c increased significantly.The expression trend of ATP synthase related m RNA genes in Sishen pill group was similar to that in normal group,such as Atp8a2,Atp6v0c,Atp5e,Atp1a2,Atp1a3 and so on.Sishen Pill can up-regulate the gene expression levels of mi R-455-3p,mi R-551b-3p,mi R-1249-3p,mi R-874-3p and mi R-139-5p,decrease the gene expression levels of mi R-300-5p,mi R-325-5p,mi R-770-5p,mi R-337-3p and mi R-127-5p.ATP-related genes were mainly concentrated in cell differentiation,metabolic process,cell and cellular components,molecular synthesis,etc.,and could also regulate DNA transcription and biological process.Conclusion:1.Sishen Pill has a good therapeutic effect on ulcerative colitis induced by DSS in mice.2.Sishen Pill can treat colitis induced by DSS by regulating the levels of memory T cells and their CD4⁺and CD8⁺subsets.3.The therapeutic effect of Sishen Pill on ulcerative colitis may be closely related to the energy metabolism regulated by AMPK-TSC,PI3K-Akt and their downstream m TOR signals.Its internal mechanism is mainly through the intervention of cell energy metabolism pathway,and then regulate the differentiation of memory T cells to achieve the purpose of treatment.