Epigenetic Regulation And Mechanism Of Histone Modification On Porcine Hepato-Intestinal Injury Induced By DON

Mycotoxins are secondary metabolites produced by filamentous fungi and can cause acute or chronic toxicity in humans and animals.Deoxynivalenol(DON),also known as vomitoxin,is one of the most widely distributed mycotoxins.DON can cause growth retardation,vomiting,diarrhea,and even death by damaging the intestine,kidney,liver,and immune system,which causes serious harm to livestock and poultry production.Pigs are the most susceptible farm animals to DON,and DON exposure can cause oxidative stress to the intestine and liver of piglets,accompanied by an inflammatory response.Studies have shown that oxidative stress genes are susceptible to epigenetic modification regulation.Histone modification is one of the important epigenetic regulatory mechanisms,and histone methylation and acetylation play a crucial role in DON-induced toxicity and damage to livestock tissues and organs.The liver and intestine are important organs for defense against toxin attack.However,the targets and potential regulatory mechanisms of DON-induced hepato-intestinal inj ury in piglets are still unclear.Therefore,this study focused on exploring the key pathways and targets of DON-induced hepato-intestinal injury in piglets,and elucidating the epigenetic regulatory mechanism,providing a reliable theoretical basis for the prevention of DON poisoning in pigs.In this study,we preliminarily excavated histone demethylase UTX as a potential target for DON-induced oxidative damage in porcine intestinal cells through transcriptome sequencing(RNA-seq)and small molecule inhibitors rescue experiments.On this basis,we conducted an in-depth analysis focusing on UTX function and regulatory mechanisms.Firstly,a systematic functional validation of the relationship between UTX expression levels and DON resistance was performed.Secondly,RNA-seq,assay for transposase accessible chromatin with high-throughput sequencing(ATAC-seq),and chromatin immunoprecipitation sequencing(ChIP-seq)were used to screen essential regulatory pathways and key target genes.Subsequently,the regulation of UTX on target genes was explored by dual-luciferase reporter gene assays and quantitative real-time PCR(qRT-PCR).Finally,the effect of UTX on DONinduced intestinal injury in vivo was analyzed using a mouse model.In addition,a piglet liver DON poisoning model was constructed in this study,and the molecular mechanism by which sodium butyrate(NaBu),a histone deacetylase inhibitor in alleviating liver injury was explored.Firstly,HE staining,serum biochemical indicators,and RNA-seq analysis of liver tissues were used to determine that DON caused liver injury,and differentially expressed genes and signaling pathways associated with DON toxicity were screened.Secondly,qRT-PCR,western blot(WB),and ChIP-seq analysis were used to explore the epigenetic regulatory processes involved in the pathway.Finally,STRING prediction,ChIP-qPCR,and dual-luciferase reporter gene assay were used to reveal the regulatory role of key regulators within the pathway in DON-induced porcine liver injury.The main findings are as follows:1.Effect of histone demethylase UTX on DON-induced oxidative injury in porcine intestinal cellsThis study performed RNA-seq on intestinal porcine epithelial cell(IPEC-J2),ileal porcine epithelial cell(IPI-2I),and porcine jejunal tissues exposed to aflatoxin B1(AFB1),and found that gene expression of lysine demethylase(KDM)family members was significantly upregulated after mycotoxin exposure.To further investigate the role of histone methylation in DON-induced porcine intestinal cell damage,small molecule inhibitors of KDM family genes were used to rescue DON-induced intestinal cell injury.The results showed that the addition of KDM inhibitors GSK-J1,GSK-J4,and CPI-455 alleviated the impact of DON on cell number and viability,with GSK-J1 demonstrating the most significant protective effect.Furthermore,GSK-J1 significantly ameliorated DON-induced apoptosis and cell cycle arrest.Flow cytometry and Caspase 3/7 activity assays further confirmed that GSK-J1 could mitigate DON-induced apoptosis,thereby restoring cell viability.Since GSK-J1 specifically targets KDM6A(encoding UTX protein),we further explored the regulatory role of UTX on DON-induced oxidative stress.Oxidative stress indicator assays revealed that UTX knockdown could suppress the decrease in mitochondrial membrane potential and the increase in reactive oxygen species(ROS)levels caused by DON,and restore the reduced antioxidant enzyme activity and related gene expression.2.Mechanism analysis of UTX-regulated AP-1 pathway in alleviating DON-induced oxidative injury in porcine intestinal cellsTo explore the potential molecular mechanism underlying UTX regulation of DONinduced oxidative stress in porcine intestinal cells,RNA-seq was performed on cells treated with DON,KDM6A inhibitor,and KDM6A-siRNA.By intersecting differentially expressed genes across groups,conducting gene ontology(GO)functional annotation enrichment,and gene set enrichment analysis(GSEA),we identified that the activating protein-1(AP-1)pathway was involved in UTX-mediated resistance to DON.STRING predicted that UTX interacted with AP-1 pathway genes.Analysis of the AP-1 pathway gene expression profiles showed that DON significantly activated the AP-1 pathway,while inhibiting UTX leading to the downregulation of pathway gene expression.CRISPR/Cas9 was further utilized to knockout UTX,and the results showed that UTX knockout suppressed the mRNA and protein expression of DON-activated AP-1 pathway genes.AP-1 pathway inhibitors were added and cell viability and oxidative stress indicators were measured,and the result showed that inhibition of AP-1 pathway restored the reduced cell viability and increased oxidative stress levels induced by DON.UTX has been found to act as a histone demethylase and mediate oxidative stress damage by catalyzing histone demethylation.To investigate the regulatory mechanism of UTX on the AP-1 pathway,ATAC-seq results found that the chromatin accessibility in the promoter regions of AP-1 pathway genes increased after DON exposure,while UTX inhibition led to reduced chromatin accessibility.Concurrently,ChIP-seq analysis of histone markers H3K27ac and H3K4me2 revealed a significantly increased enrichment at the promoter region of the key AP-1 pathway protein JUN upon DON exposure,which could be reduced by adding GSK-J1.Furthermore,dual-luciferase reporter gene assays demonstrated that UTX overexpression significantly enhanced JUN promoter transcriptional activity.Finally,in vivo validation result in mice showed that UTX inhibitors could restore reductions in body weight,food intake,villus height,and crypt depth caused by DON,as well as alleviate DON-induced oxidative stress and inflammatory response in intestinal cells.3.Effect of histone deacetylase inhibitor NaBu on DON-induced oxidative stress and inflammation in porcine liverAs another crucial histone modification,histone acetylation can activate gene transcription and participate in the regulatory processes of DON resistance.NaBu is an important histone acetylation modifier,and studies have shown that it can significantly alleviate liver damage in piglets.In this study,28-day-old crossbred piglets were used to construct a DON-induced liver toxicity model and investigate the protective mechanism of NaBu on liver damage.The results demonstrated that DON exposure caused liver injury,characterized by mononuclear cell infiltration in the tissue,elevated serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),and alkaline phosphatase(ALP),and significant reductions in total protein(TP)and albumin(ALB)levels,while NaBu supplementation ameliorated these abnormal indicators induced by DON.To further explore the molecular mechanism,RNA-seq was performed and differentially expressed genes were analyzed using GO annotation and GSEA.Result found that the ROS and tumor necrosis factor-α(TNF-α)pathways were significantly activated after DON exposure.Antioxidant gene expression was markedly decreased in the DON group,while NaBu addition restored it to normal levels.Consistent with gene expression changes,NaBu alleviated the reduction in antioxidant enzyme activity caused by DON exposure.In addition,the mRNA expression and protein secretion of pro-inflammatory cytokines were examined,and the results revealed that NaBu could inhibit the increased mRNA expression and protein secretion of pro-inflammatory cytokines induced by DON.To further investigate the protective mechanism of NaBu against DON-induced liver damage,ChIP-seq analysis of the histone acetylation marker H3K27ac was performed.Results showed that DON exposure significantly increased H3K27ac enrichment in the promoter regions of ROS and TNF-α pathway genes,while NaBu supplementation significantly reduced H3K27ac enrichment.The nuclear receptor NR4A2 is a critical target in the TNF-α signaling pathway and is associated with oxidative stress pathway.Therefore,we further investigated whether NR4A2 is involved in NaBu-regulated ROS and TNF-α pathways.STRING analysis predicted that NR4A2 interacted with key genes in the ROS and TNF-α pathways.Both mRNA and protein expression levels of NR4A2 were significantly increased in the DON group and downregulated by NaBu.NR4A2 binding motif(AAAGGTCA)was identified in the promoters of the key oxidative stress gene GPX4 and the inflammatory gene IL-4 by sequence alignment analysis.Furthermore,ChIP-qPCR result confirmed that the downregulation of ROS and TNF-α pathway gene expression by NaBu was associated with transcriptional repression of NR4A2.4.Mechanism analysis of NaBu reversing pathological cholesterol metabolism disorder and alleviating liver injury induced by DON in pigletsOxidative stress injury is closely related to pathological cholesterol metabolism dysregulation.To explore whether NaBu ameliorates DON-induced oxidative stress injury by affecting cholesterol biosynthesis,RNA-seq,qRT-PCR,and western blot assays were performed,and it was found that cholesterol biosynthesis gene expression was significantly upregulated in the DON exposure group,while NaBu supplementation led to a marked downregulation.Biochemical analysis revealed that cholesterol,bile acid,and triglyceride levels were significantly elevated in the DON-exposed group,while NaBu effectively inhibited the pathological cholesterol metabolism dysregulation induced by DON.As NaBu is an important histone acetylation modifier,ChIP-seq analysis was performed to examine the chromatin genome-wide enrichment profile of the histone marker H3K27ac in pig liver tissue.Results showed that DON exposure significantly increased the genome-wide enrichment of H3K27ac,while NaBu supplementation markedly reduced the enrichment.Further analysis of the ChIP-seq result revealed that NaBu significantly decreased the enrichment of H3 K27ac on the promoter and enhancer regions of cholesterol biosynthesis genes in the DON-exposed liver tissue.Moreover,ChIP-qPCR results showed that NaBu effectively suppressed the increased enrichment of H3K27ac and H3K9ac on cholesterol biosynthesis genes upon DON exposure.In addition,the novel cholesterol regulator RORy was significantly enriched in hyperacetylated regions,and both its mRNA and protein expression levels were increased in the DON group,while significantly reduced upon NaBu supplementation.NaBu also hindered RORy transcriptional binding on these activated cholesterol biosynthesis genes(HMGCR,SQLE,and DHCR24).Furthermore,dual-luciferase reporter gene assay was performed,and it was found that the nuclear receptor RORγ directly binds to the promoter regions of these target genes,participating in the histone acetylation regulation process mediated by NaBu,In summary,the results contribute to dissecting the regulatory role of UTX and NaBumediated histone modifications on DON-induced hepato-intestinal injury in piglets,providing new perspectives and ideas for the prevention of mycotoxin poisoning from an epigenetic point of view.