Proteomics And Metabolomics Research On The Hepatic Lipidosis Of Dairy Cows With Choline

Dairy cows are often in state of negative energy balance(NEB)and immunosuppression.A great amount of non-esterified fatty acid(NEFA)is released from body fat mobilization during the NEB.Liver uptake of NEFA may lead to lipid metabolism injury in the liver because a lot of triglycerides accumulated in the liver.Very low density lipoprotein(VLDL)is the main form of triglyceride which is removed from the liver.Phospholipids,cholesterol esters and triglycerides are the major constituents of VLDL,and choline is an important precursor of phospholipids synthesis in liver.At present,the protected choline is often supplemented in the diet to improve liver lipid metabolism and immune function of dairy cows in transiton period.However,it is unclear that the choline regulates the detail pathway of liver metabolism.Therefore,the model of NEFA stimulation of NEB was used to induce lipid deposition in liver cell of calf.After adding choline chloride,lipids metabolic characteristics of liver cell were comprehensively analyzed using iTRAQ proteomics and GC/MS metabolomics to clarify the choline regulation on mechanism and main metabolic regulatory pathways of the liver lipid deposition caused by NEB.It will provide a basis of prevention and treatment of dairy fatty liver in future.In this study,calf liver cells were isolated and cultured by two-step collagenase perfusion method.After 48 hrs of normal culture,the cells were again cultured for 6 hours with good growth.1)Using Time gradient and density gradient method were used to determine the optimal time of NEFA-induced cells,the optimal concentration of choline chloride and the optimal time of choline chloride addition.By evaluating TG,TC,GLU,AST,GLO and NEFA and other liver lipid deposition sensitive indicators.2)After the aboved determination,iTRAQ technology was used to detect calf liver cell sample from two groups(NEFA vs.Con,N+CHO vs.NEFA),mass spectrometry analysis was used to obtain differential expression proteins between the two groups,and then GO and KEGG analysis were used to determine metabolic pathway of the differential expression of proteins.3)At the same time,two groups of cell samples were detected by GC/MS technique to obtain different metabolite spectra,differential metabolites of NEFA vs.Con and CHO+N vs.NEFA,and then to determine the metabolic pathways of the differential metabolites by biological analysis.The results showed that 1)Compared with the blank control group,the levels of TG,TC and NEFA were higher in NEFA-induced hepatocytes for 12 h,Indicating that the cells are in the state of hepatic fat deposition,determine the best time for 12h;The levels of TG,TC,NEFA and GLO were down-regulated by the addition of different concentrations of choline chloride induced hepatocytes to NEFA-induced hepatocytes,suggested that choline may alleviate NEFA-induced hepatic fat deposition,and 10?m/dl choline was the best and the optimal time was 6h.2)A total of 144 differential proteins were determined in NEFA vs.Con group using iTRAQ technique and GO analysis,including 89 down-regulated proteins,There are L-lactate dehydrogenase A chain,sorbitol dehydrogenase,fructose-diphosphate aldolase,phospholipid transport ATPase,oxysterin binding protein and hexokinase,etc.and 55 up-regulated proteins,There are actin,fatty acid binding proteins,histones,beta-casein,arginase and mitochondrial pyruvate carriers,etc.The differential proteins were mainly involved in the metabolic pathways of Steroid biosynthesis,glycolysis / gluconeogenesis,amino acid biosynthesis,RNA transport and carbon metabolism,and of 74 differential proteins in N+ CHO vs.NEFA were also identified,including 30 down-regulated proteins,There are Mitochondrial pyruvate carrier,mitochondrial input intimal transposon subunit,transmembrane protein and ribosomal protein L37,etc.and 44 up-regulated proteins,There are Isoleucine dehydrogenase,lipid-coated protein,carnitine O-palmitoyltransferase 1,apolipoprotein C-III,oxidized low density lipoprotein receptor,etc.The differential proteins are mainly involved in PPAR signaling pathway and RNA transport.3)Twenty-one differential metabolites were identified by GC/MS and multivariate statistical analysis,including 8 up-regulated metabolites,Lactose,O-phosphate ethanolamine,linoleic acid,lauric acid,?-mannosyl glyceride,20?-hydroxycholesterol,digoxigenin,6-aminopenicillanic acid and 13 down-regulated metabolites,There are adenine,?-tryptophan,succinic acid,heptadecanoic acid,2-hydroxypyridine,glycolic acid,pentadecanoic acid,gluconic acid,malonamide,etc.The metabolites mainly took part in linoleic acid metabolism and steroid biosynthesis.14 different metabolites were also determented in NEFA vs.N+CHO group,including 4 down-regulated metabolites,There are 3-hydroxypropionic acid,fosfomycin,cholestane-3,5,6-triol,threonic acid and 10 up-regulated metabolites,Lactose,stearic acid,adenine,arachidonic acid,2-glyceryl monopalmitate,malonamide,beta-mannosyl glyceride,salicylic acid,thymidine and citrulline.They mainly participated in unsaturated fatty acid biosynthesis and arachidonic acid metabolism and other metabolic pathways.Conclusions: iTRAQ proteomics and GC/MS metabolomics techniques were first applied,combined with multivariate statistical analysis and bioinformatics techniques,to obtain differential expression proteins and differential metabolites in the NEFA vs.Con and NEFA vs.N+CHO groups,and to reveal omics metabolism of NEFA-induced hepatocytes lipid deposition and regulation of choline on differential proteins and metabolites in liver cell with lipid deposition.It will lay a foundation for further studying the pathogenesis of fatty liver in dairy cows.