Study On The Therapeutic Effect And Mechanism Of TAT-N15 In Ulcerative Colitis And Colitis-associated Colorectal Cancer

Object:This part of the study aims to determine the successful construction of model of ulcerative colitis induced by DSS, and the significant role of PIK3R3 in ulcerative colitis.Methods:Through DSS induced ulcerative colitis, we detected the clinical indicators, pathological changes and the infiltration of inflammatory cells, meanwhile evaluated the expression level of PIK3R3 in this colitis model. We also tested the expression level of PIK3R3 in the clinical specimens of ulcerative colitis using immunihistochemical method.Results:The mice induced by DSS had weight loss, the shortening of the colon; colon epithelial structure damaged, and increased infiltration of immune cells; PIK3R3 showed high expression level in DSS induced mice and clinical specimens of ulcerative colitis.Conclusion:Construction of DSS induced ulcerative colitis model is successful. PIK3R3 expression level is high in DSS induced ulcerative colitis and clinical specimens. PIK3R3 plays significant role in the ulcerative colitis.Object:This part aims to evaluate the therapeutic effect of TAT-N15 in ulcerative colitis induced by DSS, and compare with the negative control (control peptide) and positive control (prednisolone).Methods:We detected body weight, length of colon, disease activity index in every group. We used hematoxylin and erosin staining to measure the pathological changes. We assessed epithelial injury by using of Ki-67 of immunohistochemistry staining. At last, we used real-time PCR, ELISA, and CBA methods to evaluate expression of related cytokines.Results:TAT-N15 can inhibit decline in body weight of mice, reduce disease activity, and relieve shortening of colon length. Meanwhile, TAT-N15 can improve the pathological changes and immune cell infiltration. It can reduce the Ki-67 index. TAT-N15 can reduce proinflammatory cytokines production.Conclusion:TAT-N15 can effectively reduce the clinical indicators of ulcerative colitis, improve the pathological changes of the disease, promote the recovery of inflammation as soon as possible, and reduce the expression of proinflammatory cytokines.Object:This study aims to prove that TAT-N15 can effectively inhibit the malignant transformation of colorectal cancer associated with ulcerative colitis.Methods:Through the construction of AOM/DSS induced colitis-associated colorectal cancer, we given TAT-N15 treatment. Then, we observed the precancerous lesions, ultimate tumor (number, size, distribution and tumor load, etc), clinical indicators (the incidence of ulcerative colitis, disease activity index, colon length), pathological changes, tumor proliferation and apoptosis (Ki-67 and TUNEL), and cytokine expression.Results:TAT-N15 could effectively inhibit the precancerous lesions, reduce visible tumors. HE staining showed precancerous lesions reduced. We found that the final formation of the tumor was less number, smaller volume, lower tumor load. We found the morbidity of ulcerative colitis and disease activity index were lower, and colon length was longer. Pathological changes were lighter. Tumor proliferation was lower and apoptosis index was higher. Proinflammatory cytokines had lower expression and anti-inflammatory cytokines were elvated.Conclusion:TAT-N15 can effectively inhibit the precancerous lesion of the tumor, and inhibit the formation of colitis-associated colorectal cancer.Object:This part aims to validate that TAT-N15 can inhibit NF-?B pathway effectively, and determine the effect of TAT-N15 on the expression of ZO-1 and Treg cells.Methods:After construction of AOM/DSS induced animal model, we detected the effect of TAT-N15 on p-NF-KB p65. We determined the effect of TAT-N15 on p-NF-KB p65 in LoVo cell line in vitro. We detected the effect of TAT-N15 on the expression of ZO-1 in animal model, and then the effect of PIK3R3 (low expression and high expression) on ZO-1 in vitro. Then we constructed the LPS induced colon permeability assay to detect the effect of TAT-N15 on permeability ability of the colon. In the animal model, we determined Treg cells using flow cytometry and immunohistichemical staining.Results:In AOM/DSS induced animal model, TAT-N15 could effectively inhibit p-NF-KB p65 and p-STAT3 protein level, increase the expression of ZO-1 protein, and also increase the proportion of Treg cell. In cell experiment, TAT-N15 inhibited NF-?B activation showed a time-dose and concentration-dose dependent relation, but we found that TAT-N15 had no effect on PIK3R3 mRNA and protein level expression. In LPS induced permeability assay, TAT-N15 could reduce colonic permeability.Conclusion:TAT-N15 inhibits the inflammation through NF-?B pathway, increases the expression of intestinal tight junction protein, and increases the proportion of Treg cell to protect the immune function of intestinal tract.