Study On The Mechanism Of PhIP-Induced Colon Injuryand The Preventive Effect Of Grape Seed Extract

Heterocyclic amines(HCAs)are a class of common carcinogenic and mutagenic compounds in human diets produced during the thermal processing of protein-rich foods at temperatures above 150°C.2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine(PhIP)is one of the most abundant heterocyclic amines,mainly produced during the thermal processing of red meat and fish.PhIP has mutagenic effects on bacteria,and long-term intake of PhIP is carcinogenic to rodents,especially inducing the occurrence of colon cancer.However,there are few studies on the harm of short-term intake of PhIP on the body,and the influence of PhIP on intestinal flora and colonic metabolic balance is still unclear.Therefore,it is of great significance to explore the mechanism of short-term exposure to PhIP to induce colonic injury and to search for beneficial dietary components to prevent early harm of PhIP to the body.Grape seed extract(GSE)is an antioxidant substance rich in proanthocyanidins,which has an effective role in preventing cancer and is a good dietary supplement.Whether GSE can prevent PhIP-induced colon injury in vivo,however,is unknown.Therefore,this study intends to use Wistar rats as an animal model to explore the mechanism of short-term intake of PhIP to induce colon damage and the preventive effect of GSE on PhIP-induced colon damage.The results of this research are as follows:Short-term exposure to PhIP significantly induced colonic injury in rats.PhIP significantly restricted the growth of rats,especially the weight gain was significantly lower than that in the control group.PhIP induced colonic oxidative damage by increasing the levels of malondialdehyde and nitrotyrosine and reducing the activities of superoxide dismutase and glutathione peroxidase,and induced colonic DNA damage by increasing the levels of γH2AX and DNA oxidation.In addition,the results of blood routine examination showed that PhIP affected on the immune regulatory system.PhIP induced intestinal flora disturbance and changes in fecal metabolite levels.The16 S r RNA gene sequencing results showed that PhIP significantly induced intestinal flora disorders by reducing the relative abundance of Lactobacillus and Ruminococcus and increasing the relative abundance of Clostridium＿sensu＿stricto＿1,ASF356 and Prevotellaceae＿UGG-001.Changes in the intestinal flora are also accompanied by changes in the function of the flora.The results of function prediction in this study showed that PhIP significantly inhibited the lipid metabolism and amino acid metabolism of the intestinal flora.The lipid metabolism functions affected by PhIP mainly include synthesis and degradation of ketone bodies,glycerophospholipid metabolism,sphingolipid metabolism and linoleic acid metabolism.The amino acid metabolism functions affected by PhIP mainly include D-glutamine and D-glutamate metabolism,alanine metabolism,taurine and hypotaurine metabolism,glutathione metabolism and tyrosine metabolism.The results of metabolomics further showed that PhIP exposure affected lipid metabolism and amino acid metabolism,especially inducing up-regulation of dihydrowyerone acid,Lyso PC(15:0),PC(22:2(13Z,16Z)/14:1(9Z)),PC(22:4(7Z,10 Z,13Z,16Z)/16:0),aminoadipic acid,creatinine and shikimate 3-phosphate,and down-regulation of cortisol,taurocholic acid,cortisone acetate,sciadonic acid,urocanic acid,ketoleucine,N-acetylglutamic acid.Among them,Lyso PC(15:0),PC(22:2(13Z,16Z)/14:1(9Z))and PC(22:4(7Z,10 Z,13Z,16Z)/16:0)are related to glycerolipid metabolism and linoleic acid metabolism pathways,and aminoadipic acid,shikimate 3-phosphate,ketoleucine and N-acetylglutamic acid are related to amino acid biosynthesis pathway.PhIP significantly affected gene expression in colon tissue.The results of RNA-seq sequencing and bioinformatics analysis showed that PhIP induced up-regulation of 644 genes and down-regulation of 466 genes.GO enrichment analysis results showed that PhIP exposure significantly affected 27 biological processes,15 molecular functions,and 19 cellular components.Moreover,we annotated all genes to the KEGG database,with a total of 7761 genes receiving annotation information.The pathways with the highest enrichment of differentially expressed genes between the PhIP group and the control group are metabolic pathways,cytokine-cytokine receptor interaction,chemokine signaling pathway,transcriptional misregulation in cancers,NF-κB signaling pathway,etc.Gene set enrichment analysis(GSEA)results showed that the genes affected by PhIP are significantly enriched in pathways related to lipid metabolism and amino acid metabolism.PhIP exposure inhibited steroid hormone biosynthesis,fatty acid elongation,fatty acid degradation and glycerolipid metabolism,as well as tryptophan metabolism,beta-alanine metabolism,glutathione metabolism,and valine,leucine and isoleucine degradation.We found that PhIP affected both lipid metabolism and amino acid metabolism in colon tissue and intestinal flora,which was consistent with the results of fecal metabolite determination.Therefore,PhIP affected the metabolic homeostasis of the colon.In addition,the RT-q PCR method was used to further verify the expression of some genes in the above metabolic pathways,and the results were consistent with RNA-seq results.The dietary supplementation of GSE can effectively prevent colon damage,intestinal flora disturbance and colon m RNA expression disorder induced by PhIP.In the GSE intervention group,rats were fed a diet supplemented with GSE for 2 weeks before administering PhIP.Then GSE and PhIP were ingested together for 4 weeks.The results showed that GSE significantly prevented oxidative damage and DNA damage induced by PhIP in the colon.The 16 S r RNA gene sequencing results showed that GSE significantly prevented PhIP-induced intestinal flora disorder by up-regulating Lactobacillus and Ruminococcus and down-regulating Clostridium.Moreover,GSE ameliorated PhIP-induced intestinal flora dysfunction.In particular,GSE improved PhIP-induced decline in lipid metabolism and amino acid metabolism of the intestinal flora.The results of metabolomics also indicated that GSE can ameliorate the metabolite disorder induced by PhIP.More importantly,transcriptome results showed that GSE has an effective preventive effect on PhIP-induced gene expression disorders.Notably,related genes in the NF-κB signaling pathway,steroid synthesis pathway,and glycerolipid metabolism pathway are involved in the process of GSE preventing PhIP-induced colon injury.The verification results of special genes showed that GSE may prevent PhIP-induced colonic injury by down-regulating the expression of the pro-inflammatory gene Tlr4 and up-regulating the expression of genes Srd5a1,Aldh1b1 and Dgat2 related to lipid metabolism and detoxification.