Effects And Mechanisms Of RNA Methylation In Animal Steatohepatitis And Liver Fibrosis

The liver is the most important metabolic center of the body and the largest substantial detoxification and immune organ.In livestock and poultry breeding,various exogenous toxins,pathogenic infections,and endogenous metabolic disorders can lead to liver damage.Fatty liver disease,also known as Metabolic associated fatty liver disease(MAFLD)or non-alcoholic fatty liver disease(NAFLD),is the most common liver disease,seriously endangering human health and animals welfare.The MAFLD process includes simple fatty liver,steatohepatitis,liver fibrosis,and liver cirrhosis,and the terminal stage can develop into hepatocellular carcinoma.At present,there is no effective drug for the treatment of MAFLD in the world,mainly because the pathogenesis of MAFLD is still unclear.The course of MAFLD is complex and involves abnormal metabolism,activation,and interaction of hepatocytes,Kupffer cells(KC)and hepatic stellate cells(HSC)in the liver.Studies have shown that epigenetic modifications,especially RNA m~6A modification,are involved in and regulate the pathogenesis of fatty liver and hepatocellular carcinoma.However,the role and the target transcripts of m~6A in hepatocytes,KC,and HSC during the MAFLD process are unclear.Therefore,this study established a chicken fatty liver model and rat hepatitis and liver fibrosis model by feeding high-fat diets,respectively,to explore the role and mechanism of m~6A modification in the process of simple fatty liver,steatohepatitis,and liver fibrosis.At the same time,LPS-activated KC and TGF-β1-activated HSC cell line models were used to further reveal the role and mechanism of m~6A modification in KC and HSC activation.1 The effect and mechanism of RNA m~6A modification in fatty liver hemorrhage syndrome in laying hensForty-eight Hy-Line Variety Brown laying hens(260days of age,1.69±0.09kg in body weight)were raised in the animal house of Nanjing Agricultural university,with the room temperature at approximately 24°C,and the light regime of 16L: 8D.Three hens were housed in each cage(60cm×46cm×44cm)equipped with a nipple drinker.Hens were randomly divided into control(CON,twenty-four chickens in eight cages)and high-energy low protein diet(HELP,twenty-four chickens in eight cages)groups,fed control diet(2,610kcal/kg metabolizable energy,16.9% crude protein)and HELP diet(3,100kcal/kg metabolizable energy,12.1% crude protein),respectively,for 12weeks.The ingredient and calculated composition of the diets used in the current study are presented in Table Table1.1.Hens were subjected to feed restriction(110g per hen per day)with free access to water throughout the experiment.After 12weeks of dietary treatment,1 hen from each cage was randomly selected and killed by rapid decapitation that is considered acceptable for euthanasia of birds according to American Veterinary Medical Association(AVMA)Guidelines for the Euthanasia of Animals: 2013 Edition.The results showed that the total triglyceride content in the liver of the HELP group was significantly higher than that of the CON group,and at the same time,the genes related to hepatic lipogenesis were significantly up-regulated(P< 0.05).The level of m~6A modification of total RNA in the liver of laying hens was significantly decreased in the HELP group,accompanied by a significant increase in the mRNA and protein levels of m~6A demethylase FTO(P < 0.05),while the mRNA and protein levels of m~6A reading protein YTHDF2 were significantly decreased(P< 0.05).The SELECT method was used to analyze the m~6A modification at specific sites of lipid synthesisrelated genes FASN,SREBP1 and SCD mRNA,and it was found that the m~6A modification level of these transcripts specific sites were significantly decreased(P < 0.05)in the HELP group.Given the significant up-regulation of GR mRNA and protein expression in the liver of HELP group(P < 0.05),we further studied the transcriptional regulation of GR on m~6A-related enzyme genes and found that the binding of GR to FTO gene promoter in HELP group was significantly increased(P < 0.05)in HELP group(P < 0.05).In the control group.These results implicate a possible role of GR-mediated transcriptional regulation of m~6A metabolic genes on m~6A-depenent post-transcriptional activation of lipogenic genes and shed new light in the molecular mechanism of FLS etiology in the chicken.2 RNA m~6A modification is involved in high-fat diet-induced hepatitis and fibrosis in ratsRats were fed a high-fat diet for 16 weeks to establish hepatitis and fibrosis model,and to explore the changes of m~6A modification and the expression of related metabolic enzymes in the process of metabolic hepatitis and fibrosis.The results showed that high-fat diet feeding significantly increased the blood endotoxin concentration and the enzymatic activities of ALT and AST in rats(P< 0.05),and at the same time,the expressions of cytokines such as IL-1β,TNF-α,and TGF-β1 in the liver were significantly up-regulated(P < 0.05),the phosphorylation level of NF-κB p65 in the classical inflammatory pathway was significantly increased(P < 0.05),indicating the occurrence of liver inflammation.At the same time,the content of collagen in liver sections was significantly increased by Sirius red and Masson staining(P< 0.05).Western blot also showed that the content of liver fibrosis marker protein α-SMA was significantly increased(P< 0.05),indicating the occurrence of liver fibrosis.Liver Dot blot and m~6A high-performance liquid chromatography-mass spectrometry detection(LC-MS/MS)showed that mRNA m~6A level in the high-fat diet group was significantly increased(P< 0.05),and the expressions of m~6A methyltransferases METTL3 and METTL14 in the liver were significantly increased(P< 0.05),while the expression of m~6A demethylase FTO was significantly decreased(P< 0.05).These results suggest that m~6A modification is involved in the process of high-fat diet-induced metabolic hepatitis and fibrosis in rat liver.3 RNA m~6A modification promotes liver inflammation and fibrosis by regulating the translation of TGF-β1 in Kupffer cellsThe mouse Kupffer cells(KC)line was treated with LPS to explore the role and mechanism of m~6A modification in the activation of KC.The results showed that LPS treatment significantly increased the expression of pro-inflammatory factor IL-6 and pro-fibrotic factor TGF-β1 in KC culture medium and intracellularly(P<0.05).Using the KEGG method to analyze the RNA-seq data,it was found that the up-regulated transcripts after LPS treatment were significantly enriched in TLR/NF-κB-related inflammatory signaling pathways,while the mRNA level of IL-1β,IL-6,TNF-α and TGF-β1 was significantly increased(P< 0.05),indicating that LPS treatment led to the activation of KCs.At the same time,the level of m~6A modification of total RNA of KC activated by LPS was significantly increased(P< 0.05),and the expression of m~6A methyltransferase METTL3/14 was significantly increased(P< 0.05),while the expression of demethylase FTO was significantly increased(P< 0.05).decreased(P< 0.05).Using ChIP-PCR and dual-luciferase reporter system,combined with inhibitor treatment,it was found that NFκB p65 could directly activate METTL3 and METTL14(P< 0.05).Using Me RIP-seq and SELECT methods,specific m~6A modification sites on the 5’UTR of TGF-β1 mRNA were found to be significantly increased in activated KCs(P< 0.05).Simultaneous knockdown of METTL3 and METTL14 by si RNA could significantly alleviate LPS-induced KC activation,as well as the increase the level of total RNA m~6A modification and the up-regulation of TGF-β1 expression(P<0.05).Hepatic stellate cells were cultured with LPS-treated KC medium after knockdown of METTL3/METTL14,and it was found that the activation of hepatic stellate cells was significantly inhibited(P< 0.05).Conversely,METTL3 overexpression caused KC to upregulate TGF-β1 expression without LPS induction(P< 0.05).These results suggest that m~6A modification regulates KC activation and TGF-β1 expression,which is involved in the activation process of hepatic stellate cells.Knockdown of the Capdependent translation initiation factor e IF4 E did not affect the LPS-induced increase in TGF-β1 protein expression in KC,suggesting that LPS-induced increase in TGF-β1 expression exists in a Cap-independent translational manner.Using a luciferase reporter system containing the TGF-β1 5’UTR sequence,it was found that the mutation of the m~6A site on the 5’UTR could block the LPS-induced increase in luciferase activity(P< 0.05),indicating that LPS-induced TGF-β1 High expression depends on m~6A modification at this site.Injecting LPS or CCl4 into mice with specific knockout of METTL14 in macrophages to create models of hepatitis and liver fibrosis,respectively,the livers were collected and KCs were isolated.The m~6A modification level and TGF-β1 protein content of TGF-β1 mRNA 5’UTR-specific sites in the generation KC were also significantly decreased(P< 0.05).These results suggest that LPS transcriptionally activates METTL3/14 through NF-κB p65,increases m A levels at specific sites in the 5’UTR of TGF-β1 mRNA,and leads to TGF-β1 through Cap-independent but m~6A-dependent translation,which promotes KC activation and the transformation of hepatitis to liver fibrosis.4 DNA 5mC and RNA m~6A modifications successively regulate the initiation and perpetuation stages of hepatic stellate cell activationIn this experiment,the TGF-β1-activated HSC cell line JS1 was used as a model,and dynamic sampling was used to distinguish the initiation and perpetuation stages of HSC activation,and to study the changes and stage-specific effects of DNA 5mC and RNA m~6A modifications during HSC activation.The results showed that the morphology of JS1 changed significantly within 0-4 h after TGF-β1 treatment,showing fibroblast-like characteristics.Nile red staining and intracellular triglyceride content detection showed that the lipid droplet content was significantly decreased(P< 0.05).RT-PCR detection showed that the gene expression level of myofibroblast(MFB)marker protein α-SMA was significantly increased(P< 0.05),while immunofluorescence and RT-PCR detection showed that the expression of COL1A1,an important component of the ECM,was not expressed.Significant changes occurred.JS1 maintained fibroblast-like morphological characteristics within 4-24 h after TGF-β1 treatment,the intracellular triglyceride content and the gene expression level of MFB marker protein α-SMA did not change significantly,while the expression of COL1A1 was gradually increased(P< 0.05).According to the above phenotypes,the activation process of HSC cells was divided into an initiation stage(0-4 h)and a perpetuation stage(4-24 h).In the initial stage of HSC activation,the modification level of total DNA 5mC in cells was significantly increased,and the mRNA and protein levels of DNA methyltransferase DNMT3 A were also significantly increased(P < 0.05).5mC levels and DNA methyltransferase expression remained unchanged.In contrast,the level of RNA m~6A modification and the mRNA and protein levels of RNA methyltransferase METTL3/14 remained unchanged during the initiation stage but increased significantly during the perpetuation stage(P < 0.05).Using 5mC inhibitor 5-aza,m~6A inhibitor Cycloleucine(Cyc)or m~6A methyltransferase METTL3 si RNA to treat cells in the initiation and perpetuation stages,respectively,it was found that 5-aza treatment significantly inhibited the TG content of cells in the initiation stage the expression of COL1A1 in the perpetuation stage was reduced(P < 0.05),thereby alleviating the up-regulation of COL1A1 expression in the perpetuation stage(P < 0.05).The above results indicated that the 5mC/m~6A modification was successively involved in the initiation and perpetuation stages of HSC activation.5 DNA 5mC and RNA m~6A regulate hepatic stellate cell activation through SOCS3/STAT3/HIF-1α pathwayIn this experiment,cells at the initiation and perpetuation stages of HSC activation were taken as the research objects,the target genes/target transcripts modified by 5mC and m~6A were further screened,and the related signaling pathways and molecular mechanisms were analyzed.The results showed that the expression levels of aerobic oxidation key enzymes PCK1 and FBP1 were significantly decreased(P < 0.05),and the expression levels of glycolysis key enzymes HK2,PFK2 and PKM2 were significantly increased(P < 0.05),while lactate-related enzymes LDH,PDK3 and MCT1/4 were significantly increased(P < 0.05),suggesting that metabolic reprogramming from aerobic oxidation to glycolysis occurred at the initiation stage of HSC activation.In addition,along with the loss of lipid droplets at the initiation stage,the expression of the lipid synthesis-related gene PPARγ was significantly downregulated(P < 0.05),which was associated with the hypermethylation of its promoter.In addition,SOCS3,a negative feedback regulator of STAT3,is also inactivated by hypermethylation of its promoter,and STAT3 is thus hyperactivated and transcriptionally activates HK2,leading to metabolic reprogramming.Through bioinformatics analysis of the Me RIP-seq database published on the Internet,combined with Me RIP-PCR and SELECT methods,the collagen family members COL1A1,COL1A2,COL3A1,COL5A2 and COL6A2 were screened and verified as the target transcription of m~6A modification in the perpetuation stage Book.The level of m~6A modification at specific sites in the mRNA of these collagens was significantly increased during the perpetuation stage(P < 0.05)and was accompanied by a specific increase in the expression of the reader protein YTHDF1(P < 0.05).ChIP analysis showed that the binding of STAT3 to the promoter sequence of the HIF-1α gene was significantly increased during the perpetuation stage,thereby transcriptionally activating HIF-1α,which in turn transcriptionally activated YTHDF1,resulting in m~6Amediated collagen production.The above results suggest that the hypermethylation of the promoter leads to transcriptional repression of PPARγ and SOCS3 genes during the initiation stage of HSC activation.Downregulation of PPARγ results in lipid droplet loss,whereas downregulation of SOCS3 relieves the repression of the STAT3 pathway,resulting in HK2 transcriptional activation and metabolic reprogramming;during the perpetuation stage,m~6A modification of collagen family member mRNAs increase,and recruitment of YTHDF1 increases the stability of these collagen mRNAs,thereby causing overproduction and secretion of ECM.STAT3 promotes the process of HSC activation through transcriptional activation of HIF-1α,which in turn transcriptionally activates YTHDF1.In conclusion,this study established a chicken fatty liver model and a rat hepatitis and liver fibrosis model by feeding a high-fat diet,respectively,to explore the role of m~6A modification in the process of simple fatty liver,steatohepatitis and liver fibrosis.At the same time,LPS-activated KC and TGF-β1-activated HSC cell line models were used to further reveal the role and mechanism of m~6A modification in KC and HSC activation.The results of the study will expand the understanding of the epigenetic mechanism of the occurrence and development of MAFLD,provide cell-and stagespecific regulatory targets for the process of steatohepatitis and liver fibrosis,and provide new drug targets for the prevention and treatment of fatty liver disease in livestock and humans.