Study On The Characteristics Of Lipid Deposition And Liver Injury Of Primary Hepatocytes In Calf With Different Fatty Acids

Perinatal period is the period of high incidence of nutritional metabolic disease from 3 weeks before delivery to 3 weeks after delivery.Some studies have shown that postnatal energy demand in cows is about twice as high as that before delivery,and at the same time,changes in body condition,metabolism and hormone secretion in cows after delivery have led to a decrease in dry matter intake in cows.It causes the cows to be in the negative energy balance(NEB)state.In order to meet the energy needs of dairy cows in the early lactation stage,the body mobilizes the energy reserve of adipose tissue during pregnancy.Under negative energy balance,when a large amount of NEFA enters the liver,it will cause ketosis,fatty liver and liver function damage because it can not be completely oxidized and re-esterified to produce a large amount of triglyceride,which will lead to ketosis,fatty liver and liver function damage.The severe oxidative stress of hepatocytes induced by high NEFA in perinatal dairy cows under negative energy balance has been confirmed.However,the characteristics of liver damage caused by different fatty acids in the blood of dairy cows are not clear.In this study,the fatty acids in serum and milk of healthy cows and negative energy balance cows were determined,and the fatty acids with the highest content in healthy cows and negative energy balance cows were selected.To investigate the effects of different fatty acids on endoplasmic reticulum stress,oxidative stress and adipose ab initio synthesis in primary calf hepatocytes,and to clarify the characteristics of hepatocyte injury mediated by different fatty acids.In this study,blood samples were collected 7 days before delivery,3 days,7 days,14 days,21 days after delivery,and milk samples were collected at 3d,7d,14 d and 21 d after delivery.According to the blood NEFA and BHBA levels at any sampling time postpartum,they were divided into control group(group C,n=5,NEFA<0.4 mmol/L,BHBA<0.5 mmol/L)and energy negative balance group(group K,n=5,BHBA>1.2 mmol/L,NEFA>0.4 mmol/L).The distribution characteristics of fatty acids in blood and emulsion samples of two groups of cows were analyzed by gas chromatography-mass spectrometry.The results showed that 16 kinds of fatty acids were detected in the serum.The levels of C16:0,C18:0,C18:1n9 and C18:2n6 were higher in the two groups;the levels of C18:0 and C18:1n9 in the K group were significantlyhigher than those in the C group.the levels of C20:0 and C22:1n9 were significantly lower than those in group C,while there was no significant difference in blood C16:0 levels between the two groups.Twenty fatty acids were detected in the milk samples,of which C16:0,C18:0,C18:1n9,C14:0,C16:1n7,C18:2n6 were higher.The levels of C18:1n9 in the milk of group K were significantly higher than those in group C,and the levels of C14:0 and C18:2n6 were significantly lower than those in group C,but the levels of C16:0,C16:1n7 and C18:1n9 were not significantly different between the two groups.The study finally selected C16:0(palmitic acid,PA),C16:1n7(palmitoleic acid,POA),C18:0(stearic acid,SA),C18:1n9(oleic acid,OA),C18:2n6(linoleic acid,LA)as a fatty acid that studies the effects of a single fatty acid on NEB cows.To screen fatty acid benign solvents,OA was dissolved in H2 O,0.1 mol/L KOH,alcohol,PBS,BSA(0.4%,0.8%,and 1.2%),DMSO(0.1%,0.4%,and 0.8%).And then the activity of the cells was detected by MTT method,and the triglyceride(TG)and total cholesterol(TC)in the cells were detected by the kit.The reactive oxygen species(ROS)was detected by fluorescence staining and the concentration of endoplasmic reticulum stress marker GRP78 m RNA was detected by fluorescence quantitative PCR.The results showed that the levels of TG and ROS in hepatocytes induced by KOH group and ethanol group were significantly higher than those of other solvent groups.In view of the fact that the addition of ethanol significantly increased the ROS level of hepatocytes.This study ultimately determined that KOH is the most suitable solvent choice for fatty acids in liver fatty disease research.In order to clarify the characteristics of lipid deposition and liver damage in primary calf hepatocytes by different fatty acids,the primary calf hepatocytes were isolated and cultured,and 1.2mmol/L PA,POA,SA,OA,LA and non-esterified fatty acids(NEFA)were added respectively,stimulated primary calf liver cells for 12 h.The intracellular lipid droplets were detected by Bodipy 493 / 503 fluorescence staining method,and the intracellular TG,very low density lipoprotein(VLDL),malonaldehyde(MDA)and hydrogen peroxide(H2O2)concentration were measured by commercial kit,and detection of intracellular ROS levels by fluorescence staining.Fluorescence quantitative PCR and Westernblot were used to detect the expression of m RNA and protein in adipose tissue ab initio synthesis-related index sterol regulatory element-binding protein 1c(SREBP1C),fatty acid synthase(FAS),acetyl coenzymeA carboxylase 1(ACC1),stearic acid coa desaturase 1(SCD1)and the expression of m RNA and protein in endoplasmic reticulum stress maker GRP78,C/EBP-homologous protein(CHOP)and the expression of m RNA and protein in indicators related to the Synthesis of VLDL such as sortilin 1(SORT1),proprotein transforming enzyme pcsk 9(PCSK9)and apolipoprotein B100(APOB100).The levels of VLDL and its associated secretory protein APOB100 in the SFAs group were significantly lower than those in the other groups,and the GRP78 and CHOP levels in the SFAs group were significantly higher than those in the other groups.It is indicated that the stimulation of SFAs leads to endoplasmic reticulum stress,which in turn reduces the secretion of VLDL.In summary,compared with unsaturated fatty acids OA,LA,and POA,saturated fatty acids PA and SA cause severe inflammatory and oxidative stress damage in hepatocytes,reducing the level of VLDL and its regulatory proteins in hepatocytes,and lead to the liver fat deposition.