Study On Toxic Release Mechanism Of Modified Mycotoxin Zea Ralenone-14-glucoside

Z14G is one of the concealed mycotoxins that widely pollute food and feed in the world.It has been proved that it can degrade into its prototype ZEN in gut microbes and produce toxicity.Our study found that Z14G can form a self-assembled supramolecular gel for the first time.Based on the "self-assembly-self-release-selfdel ivery" characteristics of natural supramolecular gel Z14G,the self-assembly principle,self-release rule and self-delivery toxicity of Z14G were deeply studied.Based on the perspective of "gut microbe-metabolites-targets",the specific intestinal toxicity of Z14G and the mechanism of intestinal immunotoxicity caused by Z14G degradation to ZEN were explored through multiomics technology.1.The discovery,characterization and self-assembly principle of Z14G self-assembled gelIn this study,we found that Z14G can self assemble to form gel at a low concentration(2 mg/mL)in 20%methanol solution.We explored the self-assembly phenomenon and mechanism of Z14G through morphological characterization(morphological observation,optical microscope,SEM,TEM,Cryo-TEM and AFM),chemical characterization(Fourier infrared and ultraviolet characterization),rheological testing and molecular dynamics simulation.The morphological characterization results show that natural Z14G molecules can self assemble into onedimensional nanofibers based on temperature response,then further extend and cross link to generate three-dimensional fiber networks,and finally fix solvent molecules to form gel.Chemical characterization,rheological testing and molecular dynamics simulation further confirmed the excellent solid sample properties of Z14G gel,and found that the main driving forces of Z14G self-assembly were π-π interaction,hydrogen bond and van der Waals interaction.2.In vitro and in vivo release of Z14G self-assembled gel and its self-release ruleIn order to explore the release of Z14G self-assembled gel in vivo and in vitro,this part studied the release of Z14G in vivo and in vitro through the continuous metabolism in vitro model of the microbial key metabolic enzyme-Caco2 cells/HepG2 cells(MKME-CH)and the metabolic model ofrats,and the self-release law of Z14G in KGN cells was clarified by the validation experiment of Z14G key microbial metabolic enzymes and their inhibitors,combined with the determination of Z14G and its metabolites and the cytotoxicity of Z14G.In the MKME-CH model,the AUCZ14G-8h of ZEN is less than AUCZEN-8h,which indicates that Z14G administration plays a slow release role.In the rat metabolic model,oral administration of Z14G will continuously and slowly release ZEN in rats,which may be the result of the self-release characteristics of Z14G self-assembled gel.The metabolism experiment of Z14G in rats of PGF administration group also confirmed that the metabolism of Z14G in animals mainly depends on gut microbes.The KGN cell experiment of Z14G administration illustrates the objective law that Z14G can only be hydrolyzed into prototype ZEN byβ-glucosidase outside the cell and then enter the cell,while Z14G cannot enter the cell directly.3.In vivo toxicity enhancement of Z14G self-assembled gel and its self-delivery mechanismAt present,there are few reports on the toxicological study of Z14G in animals,based on clarifying the mechanism of self-assembly and self-release of Z14G gel,this study divided rats into control group,ZEN group,Z14G-L group and Z14G-H group through the rat toxicity model in vivo to study the toxicity of Z14G gel to various tissues of rats.HE staining was used to determine the toxicity of Z14G to various tissues of rats,and relevant indicators of highly toxic tissues,including ovary(LH and FSH)and liver(AST and ALT),were screened for analysis.The effects of ZEN and Z14G gel on protein expression in rat ovary were compared and verified by proteomics and western blot to explore the mechanism of toxicity enhancement of Z14G gel in rat ovary.The results showed that Z14G gel could release ZEN slowly in vivo,and lead to stronger tissue toxicity than ZEN by depletion of cytochrome P450.4.Specific intestinal toxicity of Z14G which mediated by gut microbesThe toxicity experiment of Z14G gel in rats showed that Z14G administration specifically led to abnormal proliferation of GALTs in rats.Rats were divided into control group,ZEN group,Z14G-L group,Z14G-H group and PGF-Z14G-H group.Metagenomic,metabolomic and proteomic analyses were performed on stool,serum and intestinal tract of rats in each group,and multi-omics association analysis was performed.Histopathological studies showed that Z14G resulted in significant dysplasia of GALTs compared to the ZEN group,which was eliminated by the inactivation of gut microbes in the PGF verification experiment.Fecal metapogenomic analysis showed that Z14G significantly promoted the proliferation of Bifidobacterium and Bacteroides compared with ZEN group.Serum metabolomics analysis showed that Z14G significantly reduced bile acid levels compared with ZEN group.Intestinal proteomic analysis showed that Z14G significantly reduced C-type lectin expression compared with ZEN group.Multitomic association analysis supports that Z14G is hydrolyzed by Bifidobacterium and Bacteroides into ZEN and promotes their cotrophic proliferation at the same time.When ZEN causes intestinal involvement,the high proliferation of Bacteroides will lead to the inactivation of lectin and lead to abnormal homing of lymphocytes.The result is abnormal proliferation of GALTs.5.The toxic mechanism of ZEN leading to intestinal immunosuppressionIn addition to the specific intestinal toxicity mechanism of Z14G different from ZEN,we also found a new mechanism of intestinal immunosuppression after Z14G was degraded into ZEN in the intestine through precise toxicology.The rats were divided into control group and ZEN group.At first the rats in each group were subjected to toxicological tests by tissue section staining,ELISA,western blot and other methods;Secondly,the serum and small intestine of rats in each group were analyzed by metabonomics and proteomics,and flow cytometry was used to verify the results in rat spleen lymphocytes;Finally,we use bioinformatics to explore the potential correlation between ZEN exposure and cancer.The results showed that ZEN exposure caused obvious pathological changes and immunosuppression in the intestine;ZEN exposure significantly increased the level of bile acid and decreased the level of omega-3 unsaturated fatty acid and lysophosphatidylcholine;In addition,ZEN exposure significantly decreased the level of ApoE and LXRs,significantly increased the level of intestinal MDSCs markers,and significantly decreased the level of intestinal 27-HC;In the rat spleen lymphocyte model,ApoE or LXRs agonists significantly alleviated ZEN-induced immunosuppression;Bioinformatics analysis shows that the downregulation of ApoE caused by ZEN exposure may increase the risk of colorectal cancer and other cancers.In conclusion,this study first found that the natural toxin Z14G can form gel by self-assembly.Based on the "self-assembly-self-release-self-delivery "characteristics of Z14G gel,it clarified the toxicity enhancement of Z14G gel in rat tissues,and explored the specific intestinal toxicity of Z14G based on gut microbiota and the new toxicity mechanism of its degradation to ZEN resulting in intestinal immunosuppression through precision toxicology.It fills the gap of natural selfassembled gel in the field of toxicology,provides toxicological basis for promoting Z14G and improving the limit standard of ZEN,provides theoretical basis for preventing acute and chronic poisoning of Z14G and ZEN,and provides direction for in-depth research on toxicology of mycotoxins.