Mechanism Analysis Of Orphan Nuclear Receptor RORγ In Regulating Ferroptosis In Porcine Hepatic And Intestinal Cells Induced By Mycotoxins

Mycotoxins are secondary metabolites produced by fungi during growth and reproduction,pose serious threats to the health of both human and animal.Long-term exposure to mycotoxins has been known to cause damage to the functional organs of piglets,such as the intestine,liver,and kidney,resulting in developmental retardation,diarrhea,and even death,leading to substantial economic losses in China’s swine industry.However,the potential regulatory targets and molecular mechanisms underlying the toxicity of mycotoxins on piglet hepatic and intestinal tissues remain barely.understood.In recent years,nuclear receptors have garnered widespread attention as crucial transcription factors regulating various biological processes and show potential for the development and application of disease-resistant breeding in livestock and poultry.Therefore,this study aims to explore the endogenous pathway involved in my cotoxin-induced piglet hepato-intestinal injury from the perspective of nuclear receptor transcriptional regulation,and screen potential targets against mycotoxins,and then provide essential scientific evidence and theoretical foundations for improving piglet health and exploring mycotoxin resistance.This study primarily investigates the growth performance and pathological injury pathway in piglets exposed to mycotoxins.Combined RNA-sequencing(RNA-seq)analysis with targeted pathway functional validation,it was found that ferroptosis mediates mycotoxin-induced hepato-intestinal injury in piglets.Key pathways involved in the regulation of ferroptosis were identified through cholesterol content,key ferroptosis enzyme activity measurements,and chromatin immunoprecipitation(ChIP)sequencing analysis.Subsequently,RNA-seq and small molecule compounds functional assays were used to screen for core nuclear receptors which regulate ferroptosis.Furthermore,quantitative real-time PCR(qRT-PCR),Western blot(WB),dual-luciferase reporter gene assay,and assay for transposase-accessible chromatin with high throughput sequencing(ATAC-seq)were performed to identify specific target genes regulated by the core nuclear receptors in the ferroptosis pathway,and explored the molecular mechanisms underlying the regulation.Finally,the mitigating effects and mechanisms of desmosterol(DES)on mycotoxin-induced hepato-intestinal injury in piglets at both cellular and individual levels were investigated by supplementing with the algal extract DES.The main findings are as follows:1.Mycotoxin-induced ferroptosis causes hepato-intestinal injury in pigletsTo explore the endogenous mechanisms of piglet resistance to mycotoxins,the study evaluated the growth performance,serum biochemical indices,and hepatointestinal pathological damage in mycotoxin-exposed piglets.Results found that mycotoxins significantly reduced piglet body weight and feed intake,increased alanine transaminase(ALT),aspartate amino transferase(AST),and alkaline phosphatase(ALP)levels in the liver,caused hepatocyte vacuolization,and induced intestinal villus atrophy and damage,indicating the model of mycotoxin-induced hepato-intestinal injury in piglets was successfully established.Furthermore,mycotoxins reduced mouse body weight and feed intake,induced changes in liver,kidney,and spleen organ indexes,increased inflammatory cell secretion levels,and elevated liver and kidney injury markers such as ALT,AST,ALP,and uric acid(UA),and exacerbated hepato-intestinal pathological damage by mouse feeding experiment.In addition,RNA-seq analysis,gene ontology(GO)functional annotation,and gene set enrichment analysis(GSEA)of differentially expressed genes was conducted on hepatic and intestinal cells exposed to mycotoxins.It was found that the ferroptosis pathway was significantly enriched after mycotoxin exposure.Specifically,the expression of ferroptosis-activating genes was significantly increased,while that of ferroptosis-resistant genes was significantly decreased.On this basis,qRT-PCR and Western blot results of key ferroptosis pathway genes further validated the results.In addition,rescue assays were carried out using ferroptosis pathway antagonists for deoxynivalenol(DON)-induced intestinal cell damage,and it was found that ferroptosis antagonists alleviated DON-induced cell damage.Finally,functional assays confirmed that ferroptosis antagonists effectively reduced DON-induced malondialdehyde(MDA)and reactive oxygen species(ROS)accumulation while reviving reduced glutathione(GSH)levels and inhibiting oxidized glutathione(GSSG)levels.These results indicate that ferroptosis mediates mycotoxininduced hepato-intestinal injury in piglets.2.Mevalonate pathway is involved in nuclear receptor RORγ-mediated regulation of ferroptosisTo investigate the key pathways involved in the regulation of ferroptosis,RNAseq analysis on my cotoxin-exposed intestinal porcine epithelial cell line(IPEC-J2)was performed and results showed that differentially expressed genes were significantly enriched in the cholesterol metabolism pathway.Furthermore,qRT-PCR and Western blot analyses further validated that the key gene expression of the mevalonate pathway(MVA)and the master regulator SREBP2 in cholesterol pathway was significantly downregulated after mycotoxin exposure.Subsequently,the key indicators of the MVAferroptosis pathway in mycotoxin-induced piglet liver were detected,and the results revealed that mycotoxin exposure significantly and simultaneously hepatic cholesterol and bile acid synthesis,decreased the content of MVA metabolic product squalene,and led to significant inhibition of key ferroptosis-regulated enzymes,including glutathione peroxidase 4(GPX4),glutathione synthetase(GSS),and squalene synthase(SQS).Nuclear receptor RORγ was found to directly regulate MVA pathway genes and block SREBP2-mediated cholesterol-negative feedback.Therefore,in this study,ChIPseq was utilized to investigate the effect of mycotoxins on the binding capacity of RORγto MVA pathway gene.Results revealed that mycotoxins were able to reduce the binding capacity of RORγ to the regulatory regions of MVA pathway gene.Further ChIP-qPCR analysis demonstrated that mycotoxin exposure diminished the enrichment of histone H3K27ac,H3K4me1,H3K4me2,and co-factors P300 and SRC-3 in the enhancer region of the HMGCS1 gene within the MVA pathway.These findings indicate that the MVA pathway is involved in mycotoxin-induced ferroptosis mediated by RORγ in the porcine liver.3.RORγ mediates mycotoxin-induced hepatic and intestinal ferroptosis in pigletTo identify key targets regulating ferroptosis in hepatic and intestinal cells,this study first used RNA-seq to comparatively analyze the expression of nuclear receptor family members.The results revealed that the expression differences of RORγ and REV-ERBα were the most significant under mycotoxins exposure.Furthermore,rescue assays were conducted on mycotoxin-induced porcine small intestinal cell damage using the small molecule agonist SR0987 and DES targeting RORγ,and the small molecule antagonist SR8278 targeting REV-ERBα.The results showed that DES exhibited the most significant protective effect against mycotoxin-induced cytotoxicity.qRT-PCR and Western blot detection revealed that RORγ expression was downregulated in response to various mycotoxins exposure.Based on these findings,bisulfite sequencing PCR(BSP)was performed to investigate the epigenetic modifications affecting RORγ expression,demonstrating mycotoxin induced an elevated DNA methylation level in the promoter region of RORγ in porcine small intestinal cells.Moreover,a significant negative correlation was observed between methylation at the mC-4 site and RORγ mRNA expression.ChIP-seq analysis further revealed that mycotoxins induced a genome-wide reduction in RORγ enrichment in porcine liver.These results indicate that RORγ is a key target of mycotoxin-induced ferroptosis in porcine hepatic and intestinal cells.4.RORy regulates ferroptosis pathway gene SLC7A11 to alleviate mycotoxininduced porcine intestinal cell damageIn order to investigate the potential mechanism of nuclear receptor RORγ in regulating the ferroptosis pathway,this study first established RORγ-overexpression cells.Through trypan blue staining,transmission electron microscopy observation,and ferroptosis phenotype identification,it was found that RORγ overexpression could alleviate the mycotoxins-induced decrease in survival of porcine small intestinal cells,restore the damaged mitochondrial structure,and simultaneously reduce total cellular iron and ferrous iron levels,decreased MDA and ROS accumulation,and increased GSH synthesis.Furthermore,qRT-PCR,Western blot analysis,and ATAC-seq were performed to reveal that mycotoxins induced downregulation of mRNA and protein expression and reduced chromatin accessibility in the promoter region of the key ferroptosis pathway gene SLC7A11.Subsequently,dual-luciferase reporter gene assays showed that RORγ overexpression could enhance SLC7A11 promoter activity,while the mutations of binding site led to the loss of RORy-mediated transcriptional regulation of SLC7A11.Based on these findings,ferroptosis phenotypes were measured in RORγ-overexpressing cell lines transfected with si-SLC7A11.The results showed that silencing SLC7A11 elevated total and ferrous iron levels,increased MDA and ROS accumulation,decreased GSH synthesis,and caused mitochondrial ultrastructural damage in mycotoxins-exposed cells.In addition,ChIP-qPCR detection revealed that mycotoxin exposure led to a decrease in the enrichment of co-factor P300 and histone marks H3K27ac,H3K4me1,and H3K4me2 in the SLC7A11 promoter region.In conclusion,these findings suggest that RORγ alleviates mycotoxin-induced ferroptosis in porcine small intestinal cells by regulating the SLC7A11 gene.5.RORγ activation by desmosterol alleviates mycotoxin toxicityIn this study,DES was successfully extracted from seaweed using ethyl acetate extraction and HPLC-MS analysis.Furthermore,DES was added to porcine small intestinal cells,and differentially expressed genes were screened by RNA-seq and analyzed for functional enrichment analysis.It was found that cholesterol homeostasis,oxidative phosphorylation,reactive oxygen species,and inflammatory response pathways were significantly enriched.Using enzyme-linked immunosorbent assays(ELISA)and ROS detection,it was observed that DES supplementation increased cytokine secretion and reduced ROS levels.Calcein/PI fluorescence and Caspase3/7 activity assays revealed that DES supplementation alleviated mycotoxins-induced apoptosis and cell viability.Flow cytometry showed that DES supplementation mitigated mycotoxins-induced increase in ROS levels and the reduction of mitochondrial membrane potential.In addition,the detection of ELISA and antioxidant system indicator demonstrated that DES supplementation reduced cytokine secretion and restore antioxidant enzyme activity.Based on the above results,DES was added to piglets’ diets,and the diarrhea rate and hepato-intestinal injury were measured.It was found that DES supplementation significantly reduced mycotoxins-induced the diarrhea in piglets,alleviated liver damage,and maintained the integrity of intestinal barrier.