The Effect Of Cholesterol On The Metabolism Of Liver Fat In Dairy Cows With Negative Energy Balance

Dairy cows are easily occur negative energy balance(NEB)in transition period.Fat mobilization is initiated to meet the energy needs of the transition cows,and produce great amounts of free fatty acids(NEFA),which may be esterified to form triglyceride accumulation in the liver.The triglyceride usually may be transported out of the liver to avoid accumulation by the main form of extremely low density lipoprotein(VLDL),which is mainly synthesized by cholesterol esters,phospholipids,and triglycerides.However,the mechanism of NEB leading to cholesterol metabolism in the liver of dairy cows is still not clear.The relationship between cholesterol and NEB is also not clear.According to this,this study through analysis of the cholesterol and other biochemical indexes in blood,clarify the influence characteristic of cholesterol on the health of dairy cows in perinatal period.By adding NEFA to calf hepatocytes in vitro to simulate the NEB condition,which are added into cholesterol,and then proteomics and metabolomics techniques are used to comprehensively illustrate the role of cholesterol in NEFA-mediated lipid deposition of calf liver cells,and reveal regulation mechanism of cholesterol on liver lipid metabolism in NEB status.It is of great significance to study the pathogenesis and control measures of fatty liver in perinatal dairy cows.This study included four tests that 1)the blood samples of cows from prepartum 2 weeks to14 days after delivery were collected clinically in an intensive dairy farm of Heilongjiang province.The test cattle were divided into a high blood cholesterol group(14 heads)and a low blood cholesterol group(14 heads)according to the blood cholesterol(TC)content of the prenatal 2 weeks.An independent sample T-test and Pearson correlation analysis were performed among groups on blood levels of energy,blood lipids,liver function and other indicators of the test cattle blood.2)Two-step collagenase perfusion method was used to separate calf liver cells in vitro culture.After 48 hours normal culture,starvation culture for 6 hours,added 1.2 m M NEFA,and the cells were further cultured for 12 hours to establish hepatocyte lipid deposition model.Using time gradient and density gradient method,the optimal concentration and the optimal time for cholesterol addition were determined by evaluating the hepatocyte lipid metabolism sensitive indicators,such as NEFA,TG,GLU,AST and GLO.3)After establishing the time and concentration of cholesterol supplementation,iTRAQ/MS technique was used todetect calf liver cell samples(CHOL vs.Con,N+CHOL vs.NEFA),obtain differential proteins among groups,and then perform GO enrichment and KEGG analysis to determine the metabolic pathway of differential proteins.4)GC/MS technology was used to detect calf liver cell samples(CHOL vs.Con,N+CHOL vs.NEFA),and obtain differential metabolites among groups,then determine metabolic pathways of differential metabolites by bioinformatics analysis.The results showed that 1)Blood levels of TC in high blood cholesterol group were significantly higher than those in low blood cholesterol group at 2 weeks prenatal,1 week prenatal,delivery day,7 days postpartum,and 14 days postpartum(P<0.01).The TG levels in high blood cholesterol group was significantly lower than that in the low blood cholesterol group(P<0.05)in the prenatal 2 weeks,prenatal 1 week,and on the delivery day;the TG was significantly lower in the postpartum 7 days compared with the low blood cholesterol group(P<0.01).The levels of HDL-C and LDL-C in the blood of the high blood cholesterol group were significantly higher than those in the low blood cholesterol group(P<0.01).NEFA levels were significantly lower(P<0.05)in the high blood cholesterol group than those in the low blood cholesterol group at prenatal 1 week,delivery day and 7 days postpartum.On the day of delivery,Mg levels in the high blood cholesterol group was significantly higher than that in the low blood cholesterol group(P<0.05).The AST levels was significantly lower in the high-blood cholesterol group than in the low-blood cholesterol group(P<0.05)at 7 days postpartum;the Ca was significantly increased(P<0.05)in the high blood cholesterol group than in the low blood cholesterol group at prenatal 1 week the delivery day,and 7 days postpartum.2)The results of adding different concentrations of cholesterol to NEFA-induced hepatocytes showed that the levels of TG,NEFA,and GLO were down-regulated,indicating that cholesterol alleviated the degree of NEFA-induced hepatic fat deposition,and the best effect was the incubation of50μm/dl cholesterol for 6 h.3)Through iTRAQ detection and Gene Ontology GO enrichment analysis,138 differential proteins were screened in the CHOL vs.CON group(ratio ≥ 1.2,ratio ≤0.83,P ≤ 0.05),and there were 50 up-regulated proteins and 88 down-regulated proteins in the CHOL group.In the N+CHOL vs.NEFA group,66 differential proteins were screened,41 of which were up-regulated and 25 were down-regulated in the N+CHOL.Up-regulation proteins were glyceraldehyde-3-phosphate dehydrogenase(GAPD),hexokinase(HK2),sorbitol dehydrogenase(SORD)and apolipoprotein C-III(APOC3).The down-regulated protein werefatty acid-binding protein(FABPS)etc.These differential proteins mainly involved in glycolysis and PPAR pathways.4)Through GC/MS detection and multivariate analysis,there were 28 differential metabolites in the CON vs.CHOL group.Among them,in the CON group there were15 decreased metabolites,such as alanine,valine,glycine,and methionine etc.,and 13 increased metabolites,such as lactic acid sugar and prostaglandin E2,etc.There were 17 different metabolites in the N+CHOL vs.NEFA group.In the N+CHOL group,the content of metabolites of salicylic acid cholesterol-3,5,6-triol metabolites decreased,and the contents of 16 metabolites increased,such as threonine,adenine,methyl linoleate,salicylic acid,and citrulline,etc.These differential metabolites mainly involved in gluconeogenesis and steroid anabolic pathway.Conclusion: High blood cholesterol levels in perinatal cows can reduce the incidence of postpartum hypocalcemia and negative energy balance and other related diseases.Cholesterol activates the Glycolysis pathway by upregulating glyceraldehyde 3-phosphate dehydrogenase(GAPD),hexokinase(HK2),and sorbitol dehydrogenase(SORD)to reduce NEB;as well as activate PPAR pathway by upregulating APOC3 and downregulating FABPS to promote VLDL synthesis,accelerate TG clearance,and reduce lipid deposition.