The Preventive Effect And Mechanism Of Kaempferol On Intestinal Barrier Injury Induced By Deoxynivalenol

Deoxynivalenol(DON),as a highly toxic class B trichothecenes mycotoxin produced by the genus Fusarium,is widely distributed in cereal crops.Even if various measures can be used to reduce the degree of its contamination,the exposure of food-borne DON is still inevitable,and the prevention and intervention of its in vivo toxic effects is important to the public health.Toxicological studies have shown that DON can severely disrupt the intestinal barrier homeostasis leading to a variety of toxic effects.Therefore,the intestinal barrier homeostasis may be an important target for the prevention and intervention of DON 's toxicity.Although many studies have found that phytochemicals can enhance the homeostasis of the intestinal barrier,there are few reports on the selection of appropriate phytochemicals for prevention or intervention of the damage caused by DON.The phytochemical kaempferol(KAM)is a natural polyphenol compound widely distributed in fruits and vegetables,in addition to its anti-cancer,anti-oxidant,and anti-inflammatory activities,it also has the ability to enhance the integrity of the Caco-2 cell model.Therefore,a nutritional approach will be used to study the effect and mechanism of KAM pretreatment on the improvement of intestinal barrier function adversely affected by DON.The specific research content and results are as follows:1.The establishment of Caco-2 cell model simulating human intestinal epithelial barrier.The monlayer integrity,permeability and cell polarity were used to verify the successful construction of Caco-2 cell monolyer.Seven phytochemicals including hesperetin,quercetin,kaempferol,resveratrol,berberine,catechin,and naringenin were screened and found that KAM was the best candidate to protect the integrity of Caco-2 cell monolayer.Based on the impacts of DON and KAM on the Caco-2 cell proliferation inhibition rate and model permeability,5 ?M DON and 100 ?M KAM were selected as the working concentration.Cell viability,oxidative stress and cell junction of Caco-2 model were studied.The results showed that KAM pretreatment has a protective effect against the damage of cell tight junctions caused by DON.2.Exploration of the effects of DON and KAM on the cell junctions of Caco-2 cell model at different stages of proliferation and differentiation.In order to simulate the rapid proliferation and differentiation of intestinal epithelial cells,the cells from the 3rd,11 th and 21 st days during the modeling process were selected for the research.The results showed that the effects of DON and KAM on the integrity and tight junctions of Caco-2 cell model were highly dependent on the stages of proliferation and differentiation.During the period of rapid proliferation stage(3rd day),both DON and KAM significantly slowed down the increase of TEER of the monolayer while the TEER of Caco-2 cell model at the 11 th day was increased.For cells at the 21 st day representing the maturation of Caco-2 cell monolayer,DON reduced the integrity of the cell monolayer,while KAM pretreatment significantly improved the barrier integrity.Changes in the expression of tight junction proteins at different stages of proliferation and differentiation demonstrated that claudin 4 was closely related to DON,and the up-regulation of claudin 3 was the function of KAM,indicaiting the claudin proteins are the target of both DON and KAM.3.In view of the inhibitory effect of DON on protein synthesis,proteomics was used to explore the protective effect and mechanism of KAM on the barrier integrity of the Caco-2 cell model.GO analysis showed that the cell adhesion molecule binding(MF),cell junction(CC)and cell junction assembly(BP)were significantly enriched(p<0.05)in KAM,DON and KAM+DON groups.The hierarchical clustering of the proteins enriched in KEGG analysis showed that KAM pretreatment has a significant effect on tight junction,adherens junction and cytoskeleton-related proteins.Further pathway and WB analysis indicated that PKA pathway and the MAPK/ERK pathway were responsible for KAM's function in improving the DON-induced barrier dysfunction of Caco-2 cell model through increasing the expression and assembly of tight junction and adherens junction proteins,respectively.4.In order to fully understand the relationship between the intestinal barrier function and other physiological processes after DON and KAM treatment,metabolomics analysis was performed on treated Caco-2 cell model,followed by omics correlation analysis with proteomics.The results showed that the number and types of metabolites were significantly different between the treatment groups.The KAM+DON group had the largest number of differential metabolites,and the phospholipid metabolites changed significantly.In addition to the effects on purine metabolism and fatty acid biosynthesis,the KAM group also had extensive effects on amino acid metabolism related pathways.The metabolic pathways of the KAM+DON group and DON alone are similar,both of them had great influence on amino acid metabolic pathways,and the aminoacyl-t RNA biosynthesis pathway is the most significant pathway,which is closely related to protein synthesis.The correlation analysis of metabolomics and proteomics showed that KAM and DON mainly affect the aminoacyl-t RNA biosynthesis pathway through the effect on the expression of t RNA ligase protein,and affect glycerophosphatide through the regulation of aldehyde dehydrogenase,acylglycerol kinase,fructose diphosphate aldolase,alcohol dehydrogenase and acylglycerol lipase.5.Finally,the BALB/c mouse model was used to verify the in vivo preventive effect of KAM pretreatment on intestinal damage caused by DON,and to study the relationship between intestinal microbiota and intestinal barrier homeostasis.The results showed that KAM pretreatment significantly improved the intestinal villi damage caused by DON,reduced the content of FD-4 and zonulin in the serum,increased the content of c NOS,and the concentration of TC and HDL-C,reflecting it's positive effect on intestinal barrier dysfunction and the low degree of inflammation induced by DON.In addition,DON has significant adverse effects on short-chain fatty acids(SCFAs)metabolism and intestinal microbiota.The increased abundance of Erysipelotrichaceae and a decrase of Lachnospiraceae and Ruminococcaceae were likely associated with intestinal injury caused by DON.KAM pretreatment increased the reduction of species abundance caused by DON,and reduced the facultative anaerobes and intestinal inflammation with improved intestinal homeostasis by reducing the abundance of Erysipelotrichaceae.