Effects Of DON And Its Acetylated Derivatives On Lipid Metabolism In Normal Human Hepatocytes Based On Multi-omics Studies

Backgrounds:Due to the high contamination rate of mycotoxins on crops(especially cereal crops)and the high exposure of humans through the intake of related foods,the prevention and control of mycotoxins has become an urgent task in the field of food safety.Trichothecenes family,which deoxynivalenol(DON)and its acetylated derivatives(3-ADON,15-ADON)belong to,as one of the most common classes of mycotoxins deserves deep research.Therefore,in this study,the effects of DON,3-ADON and 15-ADON on the RNA and lipid metabolites of normal human hepatocytes(L-02)were investigated by a multi-omics analysis method combining transcriptomics and lipidomics.Method:(1)DON and its acetylated derivatives solutions of eight different dose concentrations(0 μM,0.5 μM,1 μM,1.5 μM,2 μM,5 μM,10 μM,20 μM)were selected to expose L-02 for 24 hours.The changes of cell viability were detected by CCK-8 assay.Oil red O staining was performed on L-02 cells exposed to DON and its acetylated derivatives at a concentration of 2 μM for 24 hours,and RNA sequencing was used to determine the m RNA expression.verify.(2)Changes of lipid metabolites in L-02 cells exposed to DON,3-ADON and15-ADON at a dose of 2 μM for 24 hours were detected based on a non-targeted lipidomic method.Potential differential lipid metabolites in L-02 cells exposed to DON,3-ADON and 15-ADON were screened by multivariate statistical analysis method,and pathway enrichment analysis was performed.(3)Combined transcriptome results and lipid metabolism results by combined pathway analysis method,and use targeted lipidomics method to expose DON,3-ADON and 15-ADON solutions at 2 μM for 24 hours Qualitative and semi-quantitative analysis of potential differential metabolites in L-02 cells.Result:(1)The toxicity of DON,3-ADON and 15-ADON three toxins to L-02 cells was detected by CCK-8 experiment,and it was found that at the same dose,the toxicity ranking of the three toxins was: 15-ADON>DON>3-ADON.Oil red O staining showed that DON and 15-ADON had significant effects on the formation of lipid droplets in L-02 cells,while 3-ADON had less effect on the formation of lipid droplets in L-02 cells.The results of m RNA sequencing experiments showed that DON and 15-ADON regulate the transcriptome of L-02 cells very significantly.Compared with the untreated control group,the number of differentially expressed genes in the DON experimental group was 2286,and the number of significantly differentially expressed genes in the 15-ADON experimental group was 4177.In the KEGG analysis of differentially expressed genes,39 and 44 pathways were significantly enriched in DON and 15-ADON groups,respectively.Among them,glycerophospholipid metabolism pathway and phospholipase D signaling pathway were not found in the study of DON and its acetylated derivatives.report.(2)Untargeted lipidomics results 505 potential differential metabolites were preliminarily screened by multivariate statistical analysis.The pathway enrichment analysis of 505 potential differential metabolites was carried out.The results showed that the pathway enrichment results in positive and negative ion mode were basically the same.The effects of DON,3-ADON and 15-ADON on L-02 lipid metabolism were mainly through glycerophospholipid metabolism.pathway,glycosylphosphatidylinositol pathway,autophagy(animal)pathway,autophagy(other)pathway and sphingolipid signaling pathway.(3)15 potential differential metabolites were finally identified by combined analysis of transcriptomic and non-targeted lipidomic results.The 15 potential differential metabolites were qualitatively and semi-quantitatively analyzed by targeted lipidomics method,and 9 lipids were finally identified,namely:PC(15:0/16:0),PC(16:1/18:3),PC(18:1/22:6),PC(16:0/16:0),PC(16:0/16:1),PC(16:1/18:1),PC(14:0/18:2),PE(14:0/16:0)and PE(18:1/18:3),and 9 lipids were all down-regulated in the experimental exposure group.Conclusion:This study found that the glycerophospholipid metabolic pathway,which is related to the nucleic acid level and the metabolic level,has a decisive role in regulating lipid metabolism in L-02 cells exposed to DON,3-ADON and 15-ADON.This is achieved by PE-PC cycling.The main differential metabolites regulated are PC(15:0/16:0),PC(16:1/18:3),PC(18:1/22:6),PC(16:0/16:0),PC(16:0/16:1),PC(16:1/18:1),PC(14:0/18:2),PE(14:0/16:0)and PE(18:1)1/18:3).