Network Pharmacological And Experimental Research On The Effect Of Pepper And Mint On Colon Cancer "Inflammatory Cancer Transformation"

Research Background:Colorectal cancer is still a malignant tumor that threatens human health,and the incidence and mortality ranks third and second among all tumor types worldwide,respectively.Inflammation,especially chronic inflammation in the intestine,is widely considered to be closely related to the occurrence of colorectal cancer.A large number of clinical studies at home and abroad have shown that the probability of ulcerative colitis patients eventually developing colon cancer is much higher than that of the general population,and this risk is significantly positively related to the course,scope and severity of inflammation.In addition,a large number of clinical observation studies,case-control studies,cohort studies,and meta-analysis have found that diet patterns(pro-inflammatory and anti-inflammatory diets)are significantly related to the degree of intestinal inflammation and the risk of colorectal cancer.Pepper and peppermint are common supplements or additives in our daily diet and daily necessities.However,it is not clear about their effect on colorectal cancer or related high-risk populations,especially the effect of colon cancer during the "inflammatory cancer transformation" stage and its related mechanism of action.With the iteration of sequencing technology,people have further understood the structure and function of the huge microbial community in the intestine.Among them,the research on intestinal flora and intestinal inflammation and intestinal tumors has become a hot spot in many studies.In addition,a large number of studies believe that diet can quickly cause significant changes in the structure and function of the intestinal flora.Pro-inflammatory diet(red meat,high fat,high protein,low fiber-based western diet)can promote the proliferation of pathogenic bacteria and the accumulation of intestinal toxic metabolites,which in turn promotes intestinal inflammation and colorectal cancer,but anti-inflammatory diets(high-fiber diets such as vegetables,fruits,and cereals)can promote the proliferation of probiotics and bacteria that produce short-chain fatty acids,thereby increasing the concentration of short-chain fatty acids in the intestine to inhibit intestinal inflammation and maintain intestinal health.In this study,we first identified the active ingredients in the "inflammatory-cancer transformation" pathway of colon cancer in pepper and peppermint by using bio-information analysis techniques and methods such as network pharmacology,and the pharmacological effects of active ingredients in pepper and mint were studied by literature mining methods.Furthermore,the AOM/DSS(AD)mouse model was used to continuously and dynamically observe the effects of capsaicin and menthol(the main components of pepper and mint)on the "inflammatory cancer transformation"process,and the role of intestinal flora and its metabolites,short-chain fatty acids.Research purposes:1.Based on the analysis methods and research strategies of network pharmacology,determine whether pepper and peppermint have certain effect on colon cancer "inflammatory-cancer transformation" and corresponding material composition;2.Through colitis-related colorectal cancer model(AD mouse),combined with relevant molecular biological detection methods,observe and verify the influences of the main effective components of capsicum and peppermint(capsaicin and menthol)for colon cancer"inflammatory-cancer transformation";using 16SrDNA sequencing technology and gas chromatography-mass spectrometry(GC-MS)to observe the effects of capsaicin and menthol on the intestinal flora and fecal short-chain fatty acid concentrations in AD mice;further clarify whether the molecular mechanism of capsaicin and menthol affecting colon cancer"inflammatory-cancer transformation" is related to intestinal flora.3.Validate and reveal the effect and the corresponding molecular mechanism of capsaicin and menthol diet on the "inflammatory-cancer transformation" of colon cancer through experiments,and provide an experimental basis for a reasonable diet for high-risk colon cancer patients.Research method and content:Network pharmacology research:we have applied the research methods and research ideas of bioinformatics such as network pharmacology,and using the TCMSP online database,we have collected and analyzed the active ingredients and action targets of pepper and mint,respectively;by analyzing differentially expressed genes in ulcerative colitis and colon cancer samples from the GSE68306 data set,we identified key target genes in colon cancer "inflammatory cancer transformation".Interaction network relationships of key genes of pepper,mint target and colon cancer "inflammatory cancer transformation" were constructed by cytoscape plugin "bisogenet".To further analyze the network topology,we screened the key targets of pepper and mint that are closely related to colon cancer "inflammatory cancer transformation",and GO and KEGG enrichment analysis of these key targets were used to determine the relevant biological processes and effects.Finally,the "component-target-pathway" network diagrams of the effect of pepper and mint on colon cancer "inflammatory cancer transformation" were constructed by cytoscape software.Experimental Study:we randomly divided the mice into:Control group(Ctrl group),AOM/DSS(AD)group,low dose of capsaicin(0.01%)group(AD/Cap 0.01%),medium dose of capsaicin(0.02%)+AD group(AD/Cap0.02%),Capsaicin high dose(0.03%)group(AD/Cap 0.03%),12 rats in each group.Control group(Ctrl group),AOM/DSS(AD)group,menthol low dose(1%)group(AD/Menl%),menthol medium dose(1.5%)group(AD/Men 1.5%),menthol high dose(2%)group(AD/Men2%),12 rats in each group.During the feeding period,mice were collected and observed for survival,intestinal inflammation,weight change and food intake.After 4 months,the mice were sacrificed and the liver and spleen,colon tissue and intestinal contents were collected.HE staining was used to observe the intestinal pathological changes.Immunohistochemistry and immunofluorescence were used to evaluate the intestinal tissue proliferative protein changes.To evaluate the gene expression of inflammatory cytokines(IL-6,TNF-?,IL-10 and MPO)in the terminal colon by RT-qPCR.Flow cytometry was used to observe the infiltration of leukocytes?neutrophils and MDSC cells in the spleen.16SrDNA sequencing technology was used to detect the effects of capsaicin and menthol on the distribution characteristics of intestinal flora of mice.GC-MS method was used to detect the effects of capsaicin and menthol on the concentration of short chain fatty acids in feces of AD mice.Research result:Chapter 1 of Part 1:42 active ingredients(arachidic acid,linolenic acid,capsaicin,etc.),9 targets(ESR1,AR,PPARG,GSK3B,BCL2,RB1,APP,LDHA,and MAPK8)and 113 signaling pathways(viral carcinogenesis Role,PI3K-Akt,MAPK,and IL-17 signaling pathways,etc.)have been identified as the molecular pathways in which pepper affects the "inflammatory cancer transformation" of colon cancer.Further digging the literature,we found that 26(62%)of these components have significant anti-inflammatory and anti-tumor effects,while only 4(10%)of the components have been studied to have a certain pro-inflammatory effect.Chapter 2 of Part 1:High-dose capsaicin can significantly reduce the survival rate of AD mice and reduce the weight of AD mice.Compared with mice in the AD group,mice in the capsaicin diet group had significantly more colon tumors and larger tumor volumes.The capsaicin diet can increase the inflammatory activity of colorectitis in AD mice in a dose-dependent manner.For example,the capsaicin diet group showed significantly higher DAI scores than the AD group mice.In addition,we also found that the capsaicin diet significantly promoted the intestinal tissue pathological damage and inflammatory infiltration in AD mice,which was reflected in the pathological score of colon tissue,pro-inflammatory cytokines(IL-6,TNF-?,and MPO)and the expression of ?-catenin and Ki67 protein in the capsaicin diet group compared with the AD group is significantly higher.The infiltration of leukocytes?neutrophils and MDSC cells in the spleen of mice in the capsaicin diet group was significantly higher than that in the AD group.In addition,the capsaicin diet can significantly affect the intestinal flora distribution characteristics of AD mice,which is specifically manifested in that the capsaicin diet group mice(AD/Cap)have higher ?diversity than the AD group mice.In addition,at the level of genus species,the histograms of the profiling of each group of sample species and the star chart analysis of top 10 species showed that the abundance of Allobaculum(butyric acid-producing bacteria)in feces of AD/Cap group mice was significantly lower than that of AD group mice;the LEfSe analysis results showed that the abundance of Faecalibaculum was significantly lower in the AD/Cap group than in the AD group.Further testing the concentrations of short-chain fatty acids in the feces of each group of mice,we found that compared with mice in the AD group,the concentrations of butyric acid,isobutyric acid,isovaleric acid,and valeric acid in the feces of the capsaicin diet group were reduced,and the concentration of butyric acid was the most significant.Chapter 1 of Part2:35 active ingredients(Thuja,naringenin,D-limonene,luteolin,etc.),16 targets(EGFR,MAPK1,JUN,CDK2,CDK4,TP53,MAPK14,GSK3B,HSP90AA1,etc.)And 108 signaling pathways(PI3K-Akt,MAPK,Wnt,VEGF,JAK-STAT,NF-?B,and TNF signaling pathways,etc.)were identified as molecular pathways in which peppermint acts on "inflammatory-cancer transformation".By digging the relevant literature for its active ingredients,32(91%)ingredients have significant anti-inflammatory and anti-tumor effects.Chapter 2 of Part 2:Menthol diet can significantly reduce the weight of AD mice,but has no significant effect on the survival rate and food intake of AD mice.Compared with mice in the AD group,the menthol diet significantly reduced the tumors number with a size of 1-3 mm,but had no significant effect on the tumors number with a size of 3-5 mm and greater.The menthol diet significantly reduced the inflammatory activity of colorectal inflammation in AD mice,which showed that compared with the AD group mice,the menthol diet group mice showed significantly lower DAI scores.Menthol diet significantly reduced intestinal histopathological damage and inflammatory infiltration,which was manifested in significantly lower colonic histopathological scores,expression of pro-inflammatory cytokines(IL-6,TNF-?,and MPO)and ?-catenin and Ki67 proteins in menthol diet group mice compared with AD mice.In addition,compared with AD mice,mice in the menthol diet group had significantly lower levels of leukocytes?neutrophils and MDSC cell in the spleen.In addition,the menthol diet significantly altered the intestinal flora of AD mice,specifically compared with the AD group mice,the menthol diet group mice(AD/Men)had a higher a diversity;the results of the principal coordinate analysis further showed significant differences in the distribution characteristics of the flora structure among the Ctrl group,the AD group,and the AD/Men group.In addition,at the level of genus species,the corresponding histogram of the profiling of each group of sample species and the star plot analysis of the top 10 species show that:the abundance of Allobaculum(butyric acid-producing bacteria)in feces of AD/Men group mice was significantly higher than that of AD group mice.The results of LEfSe analysis showed that the abundance of Roseburia and Intestinimonas was significantly higher in the AD/Men group than in the AD group.The GC-MS results in the feces of each group of mice showed that compared with the AD group,the concentrations of various short chain fatty acids in the feces of the AD/Men group mice were increased,among them,the concentrations of butyric acid,propionic acid,valeric acid and acetic acid increased most significantly.Conclusion:Although pepper does not contain direct pro-inflammatory and carcinogenic components,excessive intake of capsaicin has the risk of promoting "inflammatory cancer transformation" of colorectal cancer,which is related to capsaicin inhibiting the growth of butyric acid-producing bacteria(Allobaculum and Faecalibaculum)and reducing the concentration of butyric acid in feces of AD mice;peppermint contains a large number of anti-inflammatory active ingredients,and the menthol diet can significantly inhibit the "inflammatory cancer transformation" of colorectal cancer.which is related to menthol increasing the abundance of butyrate-producing bacteria(Allobaculum,Roseburia,and Intestinimonas)and the concentration of butyric acid in feces of AD mice.