| Fatty liver is a kind of nutritional metabolic disease which occurs frequently in perinatal dairy cows and seriously affects the production performance of dairy cows.Due to reduced dry matter intake(DMI)in the perinatal period,cows could not meet the energy needs of themselves and the fetus,leading to negative energy balance(NEB).In order to alleviate energy deficit,a large amount of adipose tissue is mobilized and excessive nonesterified fatty acids(NEFA)are released into the blood,which causes hypernefaemia in dairy cows.NEFA is mainly composed of oleic acid and palmitic acid.Part of NEFA is absorbed by the liver and oxidized and decomposed to provide energy,but excessive NEFA is esterified into triglycerides(TG)and deposit in liver tissue,leading to fatty liver.Existing studies have shown that NEFA is closely related to lipid metabolism disorders in hepatocytes,but it is not clear through which metabolites or metabolic pathways NEFA affects lipid metabolism disorders in hepatocytes.Therefore,this study aims to explore the effect of high concentration NEFA on lipid metabolism in hepatocytes by means of lipidomics methods and molecular biology techniques,clarify the pathogenesis of fatty liver in dairy cows,and provide theoretical basis for the prevention and control of fatty liver in dairy cows.A fatty liver cell model was constructed by adding NEFA to cultured mouse AML-12 cells in vitro.Except for the control group,the final concentrations of 0.3,0.6,1.2 and 2.4 mM NEFA were added to the remaining four groups,respectively.After treatment for 12 h,cell vitality was detected by cell counting kit-8(CCK-8),and intracellular lipid droplet content was determined by oil red O staining.The content of TG in cells of each group was determined by TG kit,and the optimal concentration of NEFA was explored,which was then used to construct the model of fatty liver cells.Quantitative polymerase chain reaction was used to detect the differences in the mRNA expression levels of lipid metabolism-related molecules such as carnitine palmitoyltransferase 1 A,hormone-sensitive lipase and fatty acid synthase in the cells of the control group and the experimental group.Finally,cell samples from the control group and the experimental group were collected for lipidomics analysis to further explore the differential metabolites and metabolic pathways regulated by NEFA.The results are as follows:(1)The results of CCK-8 experiment showed that compared with the control group,0.3 mM NEFA had no significant effect on the viability of hepatocytes,while0.6,1.2 and 2.4 mM NEFA-treated groups significantly decreased the viability of hepatocytes in a concentration-dependent manner.(2)The results of TG content determination showed that compared with the control group,0.3,0.6 and 1.2 mM NEFA could significantly promote the increase of TG content in hepatocytes in a concentration-dependent manner.At the same time,the results of oil red O staining showed that the liver cells in the control group were in good growth condition,with blue-purple nuclei and very few lipid droplets were available in the cells.However,after NEFA treatment,a large number of lipid droplets appeared in the cells,and with the increase of NEFA concentration,the number and volume of lipid droplets increased.The percentage of lipid drop area in the total area of cells treated with 0.3 mM NEFA and 0.6 mM NEFA was higher than that in the control group.Most of the cells in the 1.2 mM NEFA treatment group died and cell fragments were visible in the visual field,while all the cells in the 2.4 mM NEFA treatment group died and the cell structure could not be observed.These results suggested that 0.6 mM NEFA could significantly reduce the viability of hepatocytes and promote lipid accumulation in hepatocytes.Based on the above results,0.6 mM NEFA was used to construct the model of fatty liver cells.(3)The results of qPCR showed that the mRNA expression of lipogenesis-related molecules FASN was significantly higher and the mRNA expression of lipolysis-related molecules CPT1 A and HSL was significantly lower in the cells of 0.6 mM NEFA group compared with the control group.(4)The analysis of lipidomics results showed that the characteristics of lipidomics in hepatocytes treated with 0.6 mM NEFA were significantly different from that in the the control group.There were 107 kinds of significantly different metabolites,including77 up-regulated metabolites and 30 down-regulated metabolites.The metabolites with the most significant differences were LPE(18:2),LPE(18:3),LPE(18:1),PE(36:3),LPE(20:0),DAG(38:4),LPE(20:3),PE(34:1),and LPE(22:6).Further enrichment analysis of KEGG signaling pathway was conducted on 107 different metabolites.The results showed that five different metabolic pathways related to lipid metabolism were obtained by analysis,including glycerol phospholipid metabolism,glycosyl phosphatidylinositol anchored biosynthesis,triglyceride metabolism,sphingolipid metabolism,and inositol phosphate metabolism.In conclusion,0.6 mM NEFA could significantly reduce the viability,promote TG accumulation,promote mRNA expression of the lipogenic molecule FASN and inhibit mRNA expression of lipolytic molecules CPT1 A and HSL in hepatocytes.The results of lipidomics analysis showed that 0.6 mM NEFA could induce lipid metabolism disorders in hepatocytes by regulating metabolic pathways such as glycerol phospholipid metabolism,glycosyl phosphatidylinositol anchored biosynthesis,triglyceride metabolism,sphingolipid metabolism,and inositol phosphate metabolism. |
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