Study On Mechanism Of Choline Regulating Lipid Metabolism In Cow Hepatocytes

Negative energy balance（NEB）is a common physiological state of perinatal cows,and the body fat stores would be mobilized to meet the energy requirements.NEB can induce the increase of non-esterified fatty acids（NEFA）concentration,and dramatically increase the prevalence of metabolic diseases（Fatty liver disease and ketoacidosis）in cows.Moreover,NEB decreases liver function,productive performance,and physiological health of perinatal cows.As a common nutritionally essential component,Choline is widely used in dairy farming which can decrease fat deposition in liver and improve production performance.In previous studies,the mechanisms of Choline mainly focus on the activation of AMPK signaling pathway and choline one-carbon unit cycle pathway,but the gene expression analysis was not comprehensive enough.The aim of this study was to explore new regulatory targets and theoretical basis of Choline regulatory function in dairy cows.Additionally,the mechanisms of Choline in alleviating the negative energy balance state and improvement of the health and production performance were investigated in perinatal cows.Exp.1 Effects of NEFA on liver cell viability,lipid deposition and inflammatory response of Dairy cowsIn this experiment,the primary bovine hepatocytes were taken as the research object.By adding different concentrations of NEFA,the effects of NEFA on the viability,lipid deposition and oxidative stress of bovine hepatocytes were studied,and then the appropriate concentration was selected for the follow-up experiment to simulate the high NEFA metabolic environment of bovine hepatocytes in the state of negative energy balance.Bovine primary hepatocytes were isolated by three-step perfusion collagenase digestion method.The concentrations of NEFA in the treatment group were 0,0.6,1.2 and 2.4 mmol/L,3 repetitions for each concentration,and the treatment time was 12 h.Cell viability,lactate dehydrogenase（LDH）activity,triglyceride（TG）content and the gene expression of TNF-αwere used as evaluation index.The results showed that 2.4 mmol/L NEFA significantly decreased cell viability（P<0.05）,significantly increased LDH enzyme activity（P<0.05）,and 0.6-2.4mmol/L NEFA significantly increased intracellular TG content（P<0.05）and m RNA expression of TNF-α（P<0.05）.This experiment shows that high concentration of NEFA can cause damage to cow hepatocytes,reduce cell viability,produce inflammatory reaction and cause fat deposition.According to the comprehensive consideration of the study,1.2 mmol/L NEFA was selected as the hepatocyte model concentration of negative energy balance body fat mobilization in dairy cows.Exp.2 Effects of choline on liver cell viability,lipid deposition and inflammatory response of Dairy cowsOn the basis of Experiment 1 NEFA cell model,the effects of choline on the viability,lipid deposition and oxidative stress of bovine hepatocytes under NEFA treatment were studied by adding different concentrations of choline chloride,and the appropriate concentration of choline was selected for subsequent experiments.The customized choline free medium was used.On the basis of adding 1.2 mmol/L NEFA,choline chloride was set 6 concentration gradients of 0,25,50,75,100 and 150μmol/L,3 repetitions for each concentration,and the treatment time was 12 h.Cell viability,LDH enzyme activity,TG content and the gene expression of TNF-αwere used as evaluation index.It was found that 75-150μmol/L choline significantly increased the viability of hepatocytes treated with NEFA（P<0.05）,25-150μmol/L choline significantly reduced TG content（P<0.05）,100 and 150μmol/L choline significantly decreased the m RNA expression of TNF-αThere was no effect on LDH enzyme activity in each choline treatment groups（P>0.05）.This experiment showed that in the 1.2mmol/L NEFA induced bovine energy negative balance body fat mobilization hepatocyte model in vitro,high concentration of choline could improve cell viability,improve cell inflammatory injury and reduce hepatocyte fat deposition.Exp.3 Study on the mechanism of choline on lipid metabolism of bovine hepatocytes under NEFA condition based on transcriptomeBased on the concentration screening in the first two chapters,this study systematically analyzed the effect of choline on the gene expression profile of bovine hepatocytes under NEFA conditions by using transcriptome sequencing technology,and verified the mechanism of lipid metabolism by using RT q PCR and Western blot,so as to provide a theoretical basis and research basis for comprehensively revealing the mechanism of choline on lipid metabolism in bovine liver.The experiments were blank control group（T₀ group）,NEFA addition group（T_N group,NEFA concentration of 1.2 mmol/L）and NEFA+choline group(T_NC group,NEFA concentration of 1.2 mmol/L;choline concentration of 75μmol/L),5repetitions per treatment,treatment time of 12 h,and Illumina transcriptome sequencing.Transcriptome analysis showed that compared with T_N group,657 differentially expressed genes were screened in T_NC group,of which 377 were up-regulated and 280 were down regulated.The differentially expressed genes were enriched,mainly including complement and coagulation cascade,pertussis,leishmaniasis,leukocyte endothelial migration,toxoplasmosis,endocrine resistance,cellular senescence,hepatitis B,nitrogen metabolism,Parathyroid hormone synthesis,secretion and function,phenylalanine metabolism,protein export and other pathways.Through subsequent verification,it was found that compared with T_N group,T_NC group significantly promoted p38 protein phosphorylation（P<0.05）,significantly increased the gene expression of PPARα,CPT1A,Apo B100 and MTTP（P<0.05）,and significantly reduced the expression of SREBP1c and FAS gene（P<0.05）.The results show that choline can promote the phosphorylation level of p38 protein,promote fatty acid oxidation,promote fatty acid transport and reduce fatty acid synthesis to reduce fat deposition in hepatocytes.In conclusion,the results in this study suggested that high concentration of NEFA could induce liver cell injury,reduce cell viability,produce inflammatory reaction and cause fat deposition in dairy cows.The addition of 1.2 mmol/L NEFA in cell culture medium can be used as a cell model for in vitro study of body fat mobilization in NEB state of dairy cows;Choline can improve the viability of bovine hepatocytes under NEFA,alleviate cellular inflammatory injury and hepatocyte fat deposition;Choline can increase the phosphorylation level of p38 protein,promote fatty acid oxidation,induce fatty acid transport,reduce fatty acid synthesis,and decrease fat deposition in hepatocytes under NEFA conditions.