| Objective:The incidence of gastrointestinal diseases is increasing,and colorectal cancer has become the third most common malignant tumor in the world.Intestinal inflammation is one of the most important and critical factors inducing the onset of colon cancer.Studies have shown that the incidence of inflammation-related colon cancer is 5-10 times higher than that of sporadic colon cancer.Nearly 50%of patients with colitis follow the onset over time it will eventually develop into colon cancer.Therefore,exploring safe and reliable colon cancer prevention methods,combining different treatment modes,focusing on the application of preventive strategies in high-risk colon cancer patients(such as patients with ulcerative colitis and colorectal adenoma),will be treatment one of the important strategies for colon cancer.Previous studies have found that evodiamine has a good inhibitory activity on inflammation and tumors.Therefore,we studied the preventive and therapeutic effects of evodiamine on colitis,as the most important risk factor for inflammation-related colorectal cancer,to analyzed the preventive and therapeutic effects of evodiamine on different stages of colitis,and analyzed its effect on inflammatory cells and intestinal epithelium in the intestinal inflammation microenvironment.The influence of cell activity and function,and the clarification of the mechanism of its regulation,have important theoretical significance for the full development and use of evodiamine in the treatment of related diseases.Methods:First,in vivo experiments proved the preventive and therapeutic effects of evodiamine on various stages of inflammation-related colorectal cancer precancerous lesions.Through different concentrations of DSS,the duration and dose of DSS were used to induce mice acute,chronic colitis models.HE staining,enzyme-linked immunosorbent assay,immunohistochemistry,Western blot and other methods to measure the preventive effects of evodiamine on intestinal inflammation,oxidative stress,and inflammatory cancer transformation in mice;then we investigated the two effects of evodiamine on the intestinal inflammation microenvironment large class of cells-the selective effect of intestinal epithelial cells and inflammatory cell activity.LPS stimulated inflammatory cells to mimic the intestinal inflammatory microenvironment,enzyme-linked immunosorbent assay,Western blot,flow cytometry,etc were used to investigate the effect of evodiamine on LPS-induced Inflammatory cells secrete pro-inflammatory factors and affect the phosphorylation level of STAT-3;then,through CCK-8,AnnexinV/PI staining,PI single-label detection,flow cytometry and other detection methods,explore the effect of evodiamine in variety of colon cancer cells and the effects of normal colonic epithelial cell proliferation inhibition,apoptosis,cycle and ROS levels;finally,the regulatory mechanism of evodiamine on the IL-6/JAK/STAT-3 signaling pathway was clarified,and it was obtained from evodiamine by Western blot and computer simulation of drug molecular docking.Whether the base acts directly on IL-6,whether it regulates the upstream related non-receptor tyrosine kinase family JAKs,whether it can directly target the SH2 domain and N-terminal structure of STAT-3,and whether it regulates protein tyrosine Phosphatase level to inhibit the STAT-3 signaling pathway.Results:The results of in vivo experiments show that evodiamine has significant protective and therapeutic effects on all stages of the transformation of colitis to intestinal cancer.It can significantly reduce intestinal inflammation,reduce the level of oxidative stress,protect the intestinal barrier damage,and reduce colon tissue.Inhibit STAT-3 phosphorylation level can prevent the occurrence and progression of cancer in the stage of acute colitis.The results of in vitro experiments show that evodiamine can reduce the expression of inflammatory factors and the level of oxidative stress in Raw 264.7 cells caused by LPS,and significantly reduced the level of STAT-3 phosphorylation expression.Evodiamine had almost no inhibitory effect on human normal colonic epithelial cells NCM-460,but had a significant inhibitory effect on variety of colon cancer cells,blocking colorectal cancer cells in G2 stage,increasing ROS levels,and promoting colorectal cancers apoptosis,inhibition of STAT-3 activation and nucleus,the mechanism of action is closely related to the inhibition of STAT-3 pathway.Evodiamine does not directly act on IL-6 and has no effect on the expression of upstream JAK family proteins.Computer simulation of molecular docking show that the possibility of Evodiamine directly targeting the STAT-3 binding site is not high,mainly by up-regulating the expression of protein tyrosine phosphatase SHP-1 and inhibiting STAT-3 phosphorylation.Conclusion:Evodiamine has a good preventive and therapeutic protective effect on different stages of canceration in mice model of inflammation-related colorectal cancer.The mechanism is related to reducing the level of inflammation and oxidative stress in the mice,inhibiting cancer cell proliferation and inflammatory cancer transformation,inhibiting STAT-3 signaling pathway excessive activation;at the same time,evodiamine can reduce the secretion of inflammatory factors in the intestinal inflammatory microenvironment,reduce the level of ROS,and inhibit STAT-3 phosphorylation;evodiamine is selective for the activity of intestinal epithelial cells and inhibits the colon cancer cell proliferation,by increasing the level of colon cancer cell ROS,blocking the cell cycle in G2 phase,regulating the expression of apoptosis-related proteins and promoting the apoptosis of colon cancer cell lines,but had no effect on the activity of normal colon epithelial cells.The mechanism is closely related to the inhibition of STAT-3 activation and nuclear entry and inhibition of the STAT-3 pathway.The mechanism of evodiamine in inhibiting STAT-3 phosphorylation was analyzed,and it was found that evodiamine inhibits STAT-3 Phosphorylation by up-regulating the expression of protein tyrosine phosphatase SHP-1. |
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