Effects And Regulatory Mechanisms Of PEDV On Lipid Peroxidation And Cholesterol Metabolism In The Liver Of Piglets

Porcine epidemic diarrhea virus(PEDV)enrolled in the coronaviridae family of alphacoronaviruses could lead to acute diarrhea and dehydration,resulting in high mortality in newborn piglets.PEDV-infection could inhibit the barrier function of the intestine in piglets.However,current studies have found that there is an interaction between the intestine,liver and blood metabolites,and this interaction was called "gut liver axis".Organisms need liver to remove external antigens,bacteria,and endotoxin that enter the bloodstream from the gut to avoid causing serious damage to the body.Because of this,many diseases are often associated with liver damage.The liver is rich in mitochondria,where is the main site for producing reactive oxygen species(ROS),so it makes liver easier to be attacked by ROS than other organs,causing oxidative stress and promoting the accumulation of lipid peroxides.Lipid peroxide is the key mediator of cell proliferation and death.Therefore,the role and regulation mechanism of endogenous lipid peroxides metabolism in PEDV infection need to be elucidated.In this study,we found that the enzyme activities of antioxidant enzymes SOD,CAT,mitochondrial complex I,mitochondrial complex III and mitochondrial complex V,as well as the contents of mitochondrial ATP and the antioxidant glutathione in the liver of PEDVinfected piglets were significantly decreased.In contrast,the contents of lipid peroxidation biomarkers MDA and ROS were significantly increased in liver.In addition,transcriptome analysis showed that PEDV infection inhibited the peroxisome metabolic pathway,and the down-regulation of antioxidant gene expression such as GPX4,CAT,SOD1,SOD2,GCLC and SLC7A11 was further verified by qRT-PCR and western blotting.The mevalonate(MVA)pathway is essential for lipid peroxidation metabolism,and the nuclear receptor RORy is a new driver of MVA.We have provided new evidences that RORy could also regulate genes related to peroxisome metabolism in PEDV-infected piglets.The results of ChIP-seq and ChIP-qPCR analysis showed that RORγ could directly bind to key antioxidant genes,and PEDV infection strongly inhibited the binding enrichments.The occupancies of histone active markers such as H3K9/27ac and H3K4me1/2,as well as active cofactors p300 and polymerase II,were significantly reduced in the loci of these genes.Importantly,it was found by ChIP-reChIP analysis that PEDV infection disrupted the physical association between RORy and NRF2,a major regulator of lipid peroxidation,promoting the downregulation of antioxidant genes at the transcriptional level.Given that the MVA pathway is also involved in cholesterol biosynthesis,and in vitro coronavirus infection requires cholesterol,it is necessary to elucidate the role and mechanism of endogenous cholesterol metabolism in regulating porcine epidemic diarrhea virus infection.In this study,we found the contents of cholesterol and bile acid in the liver of PEDV-infected piglets were increased significantly compared with controls.Consistently,the expressions of key genes involved in cholesterol metabolism were significantly increased in the livers of piglets.Transcriptome analysis showed that cholesterol homeostasis pathway was one of the most abundant pathways.Surprisingly,the expressions of key genes in the cholesterol metabolic pathway were down-regulated at the mRNA level but up-regulated at the protein level.The major transcription factors of cholesterol metabolism including SREBP2 and FXR,were upregulated in both mRNA and protein levels after PEDV infection.Further ChIP-qPCR analysis showed that PEDV infection significantly inhibited the binding of these transcription factors to key gene loci in the cholesterol metabolic pathway.It was also observed that occupancies of histones H3K27ac and H3K4mel at the HMGCR and HMGCS1 loci of cholesterol metabolizer genes were inhibited.In conclusion,RORy may be a potential factor regulating the expressions of antioxidant genes in liver of PEDV-infected piglets by interacting with NRF2 and histone modifications.Meanwhile,PEDV inhibited SREBP2/FXR mediated transcription through epigenetic inhibition,leading to abnormal regulation of cholesterol metabolism genes.This provides a potential antiviral target for fighting PEDV and other coronaviruses.