Multi-omics Study Of The Toxicological Effects And Intervention Mechanisms Of Long-term Exposure To Mycotoxins In High-fat Dietary Patterns

Mycotoxin exposure is a long-term concern for human health.Aflatoxin B1(AFB1)and zearalenone(ZEN)are globally contaminated and widely present in everyday foods(e.g.,heavily consumed cereal products),with fats and oils and their products being an important source of mycotoxin exposure in the diets of the population.Since both AFB1 and ZEN have been reported to have toxic effects on the liver and the intestinal barrier is inevitably affected by mycotoxins,it is necessary to assess the "enterohepatic axis" of mycotoxin exposure under different dietary patterns and to understand the mechanisms of toxicity.Several population studies have found an association between food patterns characterized by high-fat diet(HFD)choices and the prevalence of obesity.Populations with dietary patterns characterized by HFD appear to be more likely to be exposed to high levels of AFB1 and ZEN,but combined exposure to fungal toxins and HFD has not been reported.Therefore,it becomes particularly important to investigate the toxic effects and mechanisms of long-term exposure to mycotoxins under both common and high-fat dietary patterns.This research investigated the effects of AFB1 and ZEN on liver and intestinal tissues and organs under different dietary conditions,and initially investigated the mechanisms of damage and sought corresponding interventions.In this study,C57 male mice and Hep G2 cells were used as exposure models to investigate the mechanism of damage to the "intestineliver axis" through a multi-omics approach.It was found that long-term exposure to mycotoxins caused metabolic disorders in the "gut-liver axis" in mice fed a common diet,and that long-term exposure to AFB1 and ZEN in the HFD mouse model resulted in a combined exposure risk.The mechanism of fungal toxin-induced metabolic disorders,and the mechanism of fungal toxin exposure exacerbating liver damage and lipid metabolism disorders in a high-fat dietary pattern were revealed through multi-omics.An antibiotic model was constructed to confirm the key flora induced by AFB1 and its role in the disruption of the "gut-liver ".Further,Bifidobacterium breve BAA-2849 and astilbin were used to successfully establish an intervention model for high-fat diet and aflatoxin exposure,and to analyze the intervention effect and metabolic improvement mechanism.The main contents are as follows.1.A multi-omics study was conducted to investigate the "gut-liver axis" damage induced by chronic exposure to the fungal toxins AFB1 and ZEN in mice with HFD and normal dietary patterns.It was found that chronic exposure to the hypotoxins induced and exacerbated liver damage and lipid metabolism disorders in ND and HFD mice.A long-term AFB1,ZEN combined with normal and high-fat diet C57 mouse exposure model was constructed,and the combination of phenotypic and histological indices revealed that toxin exposure induced liver injury,lipid accumulation and serum lipid abnormalities in normal diet(ND)mice,and further exacerbated lipid metabolism disorders in high-fat mice.Liver lipidomics revealed that AFB1 and ZEN exposure altered liver lipid composition,increased saturated triglyceride(TAGs)lipid levels and decreased acylcarnitines and other lipids in mice on both diets,and that the combination of high-fat diet and fungal toxins further exacerbated specific classes of liver lipid disorders.The results of gut microbiota sequencing showed that AFB1 caused the increase of Rikenellaceae RC9 gut group,Helicobacter,and a decrease in the abundance of Akkermansia and Catenibacterium and a change in the genus of short chain fatty acids(SCFAs)producing bacteria.The combined effect of HFD and AFB1 will further lead to the abnormal increase of Lachnospiraceae UCG-006,NK4A136 group and UCG-001.Chronic exposure to AFB1 disrupted and exacerbated the barrier function of intestinal tissues in ND and HFD mice,causing a decrease in fecal acetic and propionic acid levels.Liver and serum metabolomes revealed that exposure induced ND,HFD mice gluconeogenesis,tryptophan-indole and other pathways,and further exacerbated related pathway disorders and abnormal isoleucine metabolism in HFD mice.Glutamate-cysteineglutathione pathway metabolic pathways,isoleucine metabolism,and hypotaurine metabolism play important roles in AFB1-induced changes in the metabolome of liver and correlate with the abundance of characteristic intestinal flora,which may be an important toxic pathway for phenotypic changes in the liver of mice.Toxin exposure altered the composition of the dietary gut microbiota and intestinal damage was also verified in ZEN-exposed mouse models,with the main signature microbiota being Enterobacter,Enterococcus,Helicobacter and Deferribacteraceae etc.and significantly disrupted and exacerbated damage to the intestinal barrier in ND and HFD mice,causing a decrease in feces acetic acid.The key serum metabolites are indole-3-propionic acid and 2-hydroxybutyric acid.ZEN exposure attenuated indole-3-propionic acid and β-alanine metabolism and enhanced 2-hydroxybutyric acid metabolism,which was positively correlated with the abundance of Prevotellaceae UCG-004,UCG-001 and NK3B31 groups.2.The role of fungal toxin-induced intestinal flora in the "gut-liver axis" of metabolic disorders in the antibiotic model.A long-term antibiotic(1.0 mg/m L ampicillin and 0.5 mg/m L neomycin)intervention model for AFB1 dietary co-exposure was developed.The antibiotics significantly reduced the total number and diversity of intestinal species in AFB1 and HFD + AFB1 mice,and alleviated AFB1 and HFD + AFB1-induced liver damage and lipid accumulation by reducing the abundance of "harmful bacteria" such as Rikenellaceae RC9 gut group and Helicobacter and increasing the abundance of beneficial bacteria such as Parabacteroides and Akkermansia in the intestine.The antibiotic intervention significantly increased the abundance of acylcarnitine and other lipids and reduced the abundance of FAs in mouse liver.Decreased the level of saturated TAGs in the liver under HFD + AFB1 co-exposure.Improved product accumulation of arachidonic acid induced by AFB1 exposure,HFD + AFB1-induced disruption of isoleucine metabolism and the metabolite profile associated with the cysteine-glutathione pathway.However,antibiotics further exacerbated the damage to the barrier in the intestinal tissues of mice after ND,HFD and AFB1 exposure,causing a decrease in the levels of butyric and valeric acid in the feces.3.Intervention effect and mechanism analysis of astilbin and Bifidobacterium breve BAA-2849 on the combined toxicity of HFD and mycotoxins.In order to achieve interventions for toxin exposure,intervention models were established by gavage of C57 mice for 9 consecutive weeks with loxoside and probiotic BAA-2849,respectively.It was found that long-term intervention of both means with single and combined toxic effects of HFD and AFB1 reduced body weight gain and total energy intake,improved glucose tolerance levels,and alleviated liver damage and lipid accumulation in mice.The intervention effects improve the barrier function of intestinal tissues and increase the abundance of SCFAs.Improves systemic levels of inflammation in mice.Bifidobacterium breve BAA-2849 intervention was able to reduce the increase in FAs induced by AFB1 and the level of saturated TAGs under all three exposures.Increased the levels of PS,LPS and other lipids in the liver of mice.The intervention remodeled the gut microbiota,decreased the ratio of Firmicutes to Bacteroidetes under HFD,HFD + AFB1 exposure,decreased the concentration of harmful bacteria such as GCA-900066575 in the gut,and increases the concentration of beneficial bacteria such as Akkermansia.A liver metabolomics study found that Bifidobacterium breve intervention mainly altered amino acid metabolism,β-alanine metabolism,and nicotinamide metabolic pathway under HFD,AFB1,and HFD + AFB1 exposure,and significantly attenuated the product accumulation status of the cysteine-glutathione pathway.Lipid and hepatic lipidomic results showed that loxsin intervention only improved the abnormalities of blood lipids and hepatic saturated TAGs in HFD mice,and was not effective in AFB1,HFD + AFB1 group mice co-exposed.Long-term intervention of astilbin increased the total number of intestinal species,increased the abundance of Ruminiclostridium 9 and Muribaculum in the intestine of HFD mice,and decreased the abundance of Coriobacteriaceae UCG-002 and others.Increase the abundance of Ruminococcus UCG-014 and decrease the abundance of Odoribacter,Rikenellaceae RC9 gut group,etc.However,the effect on AFB1,the disruption of cysteine-glutathione pathway caused by HFD + AFB1,was not obvious.Further validation by in vitro assays revealed that intervention of AFB1 or ZEN toxin-exposed Hep G2 cell models in vitro using lapsigargin was able to alleviate toxin cytolipotoxicity.In summary,this study constructed long-term AFB1,ZEN and ND,HFD co-exposure models,respectively,and found that AFB1 and ZEN exposure to common diet mice caused flora disorders,damaged intestinal barrier,caused liver damage and metabolic abnormalities.When the fungal toxin and HFD acted together,it further aggravated intestinal damage,systemic inflammation and liver and serum lipid disorders,and disrupted the tryptophanindole metabolic pathway and cysteine-glutathione metabolic pathway in mice.The antibiotic model showed that by influencing the abundance of Rikenellaceae RC9 gut group,Helicobacter and Akkermansia in the intestine,it could alleviate the liver injury and lipid disorder caused by AFB1 exposure.Bifidobacterium breve BAA-2849 was found to protect the gut by regulating intestinal flora,significantly affecting the nicotinamide metabolic pathway,the cysteine-glutathione pathway,and providing better relief of lipid disorders in the liver and blood compared to astilbin.This study provides important theoretical support for aflatoxin exposure pathways and risk prevention.