| Choline,as an essential nutrient for animal growth and development,plays an important role in the prevention and treatment of fatty liver disease in dairy cows,but the regulatory mechanism is μnclear.This study investigated the mechanism of choline regulating the size of lipid droplets in bovine hepatocytes from aspects of protein expression,lipid metabolism,and lipid droplet fusion by culturing cow liver cells in a choline deficient medium,followed by choline supplementation,in order to lay a foundation for clarifying the mechanism of choline prevention and treatment of cow fatty liver.The experiment first established a model of fatty liver cells in dairy cows.After adding linoleic acid at concentrations of 0,50,100,150,200,300,and 400 μmol/L to the culture medium for 24 hours,it was found that 150 μmol/L LA had a small inhibitory effect on the viability of bovine hepatocytes through CCK-8,oil red O,and Nile red methods,significantly increasing the accumulation of TG and the formation of lipid droplets in the cytoplasm of bovine liver.Therefore,in the subsequent experiment,150 μmol/L LA wasμsed to treat bovine hepatocytes to establish a fatty liver model.In the subsequent experiment,normal cells were μsed as control,and the cell culture medium was changed to a choline deficient medium for culture.Oil red O staining was performed,and changes in the size,number,and proportion of lipid droplets were analyzed μsing fluorescence microscope software.The results showed that compared with the control group,choline deficiency increased the average diameter of lipid droplets(1.59 μm vs 2.10 μm),and the proportion of small lipid droplets(<2 μm)decreased from 74% to 56.6%,while the proportion of large lipid droplets(>4 μm)increased from 5.3% to 15%.At the same time,choline deficiency increases the TG content in hepatocytes(0.39 μmol/mg vs 0.57 μmol/mg).Protein analysis revealed increased expression of sterol regulatory element binding protein(SREBP),sterol regulatory element binding protein cleavage activating protein(SCAP),fatty acid synthase(FASN),and peroxisome proliferator-activated receptor(PPARγ).μsing a live cell workstation to observe lipid droplets,it was found that the lack of choline treatment improved the fusion rate and fusion speed compared to control cells.Thin layer chromatography showed that compared to the control cells,the total phosphatidylcholine(PC)content of the cells decreased after choline deficiency treatment.Further liquid chromatography-mass spectrometry analysis showed that choline deficiency resulted in an increase in the content of 27 phosphatidylethanolamine(PE)compounds and a decrease in the content of 16 PC compounds compared to control cells.In order to further explore the mechanism of choline recovery on lipid droplet size,the effects of different choline concentrations on lipid droplet size were studied.After treating cells with 0,30,60,and 90μm/L choline chloride,it was found that with the addition of choline,the diameters of lipid droplets at 0,30,60,and 90 μmol/L were 2.37 μm,1.54 μm,1.43 μm,and 1.39 μm,respectively,and the size of lipid droplets decreased with the increase of concentration.The triglyceride content in lipid drops gradually decreased to 0.65 μmol/mg,0.45 μmol/mg,0.36μmol/mg,and 0.37 μmol/mg,respectively.At the same time,the content of PC increased.Detection of autophagic proteins revealed that the expression of the autophagic marker LC3 protein gradually increased,while the expression of P62 protein gradually decreased.This suggests that choline may increase autophagy levels to regulate the size of lipid droplets.The above results suggest that choline deficiency increases lipid droplet size and fusion,and affects cell phospholipid composition and protein expression.When choline is added,the autophagic activity of cells gradually increases,leading to a reversal of the phenotype of lipid droplets.This result confirmed that the supply of choline is necessary to alleviate the high incidence rate of fatty liver disease in cows during labor. |
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