Mechanism Of Steatosis Induced By Palmitic Acid In BRL 3A Cells And The Protective Effect Of Niacin

Nonalcoholic fatty liver is a lipid disorder metabolic syndrome characterized by steatosis and excessive lipid deposition in liver parenchyma cells.Palmitic acid is the highest content of saturated fatty acids in free fatty acids.A large number of free fatty acids can not be metabolized and accumulated in time to induce steatosis in hepatocytes,but the mechanism of steatosis induced by palmitic acid in hepatocytes is not clear.Niacin is transformed into bioactive nicotinamide in mammals and participates in carbohydrate,lipid synthesis and catabolism.Some studies have shown that niacin can reduce triglyceride synthesis and the degree of steatosis,but its mechanism is not clear.In our study,palmitic acid was used to induce steatosis in BRL 3A cells to establish a non-alcoholic fatty liver model in vitro.To reveal the molecular mechanism of steatosis in BRL 3A cells induced by palmitic acid,to explore the protective effect of niacin on steatosis and its protective mechanism,and to provide a new theoretical basis for the prevention and treatment of nonalcoholic fatty liver disease and liver lipid metabolism disorder in livestock and poultry.1.Effect of palmitic acid on toxic damage of BRL 3A cellsTo explore the toxic effect of palmitic acid on BRL 3A cells,CCK-8 method and real-time label free cell analysis technique were used to determined the proliferative toxicity,immunofluorescence staining was used to observe the morphological changes of nucleus and skeleton,and oil red O staining was used to observe the formation of lipid droplets and the content of TG,transmission electron microscope was used to observe the ultrastructural changes.The results showed that compared with the control group,0.2,0.4 mM palmitic acid had no effect on cell proliferation;0.6 mM palmitic acid inhibited cell proliferation.With the increase of palmitic acid concentration,the nucleus shrank,deformed and fragmented,the cytoskeleton was destroyed and the microfilament was broken.0.4 mM palmitic acid treatment for 24 hours,lipid droplets accumulated and obvious steatosis occurred.With the increase of palmitic acid concentration,the nucleus shrank,deformed and fragmented,the cytoskeleton was destroyed and the microfilament was broken.And the TG content increased significantly with the increase of palmitic acid concentration;the nuclear membrane was damaged,the nucleus wrinkled and deformed,the mitochondrial ridge was broken,and a large number of lipid droplets were formed in the cytoplasm.The results showed that palmitic acid could damage the morphology and induce lipid droplet accumulation.2.Study on the mechanism of steatosis induced by palmitic acidTo investigate the mechanism of BRL 3 A cells steatosis induced by palmitic acid;the cells were treated with different concentrations of palmitic acid(0.2,0.4,0.6 mM)for 24 h,and the ROS content was determined by flow cytometry.The cells were treated with 0.4 mM palmitic acid for 24 h,evaluated the nuclear translocation of ChREBP;and the transcription and expression of ChREBP,Acaca/ACC,Fasn/FAS,Dgatl/DGAT1 and Dgat2/DGAT2 were determined by qRT-PCR and Western blot.The results showed that the intracellular ROS level increased significantly(P<0.05 or P<0.01)with the increase of palmitic acid concentration;and the ratio of ChREBP fluorescence in intracellular/nuclear increased significantly after treatment with 0.4 mM palmitic acid for 24 h.And the transcription and expression of ChREBP,Acaca/ACC,Fasn/FAS,Dgatl/DGAT1 and Dgat2/DGAT2 were increased significantly(P<0.05 or P<0.01).At36 after si-ChREBP transfection,the transcription level of ChREBP and the protein expression of ACC,FAS,DGAT1 and DGAT2 decreased significantly(P<0.05 or P<0.01).The results showed that palmitic acid could increase the expression of ACC/FAS/DGAT1/DGAT2,promote TG synthesis and induce steatosis by up-regulating the expression of ChREBP and improving the efficiency of nuclear translocation.3.The role of niacin in palmitic acid-induced BRL3A cells injury and steatosis and its mechanismTo investigate the role and mechanism of niacin in toxic damage and steatosis of BRL 3 A cells induced by palmitic acid.CCK-8 and RTCA were used to screen niacin concentration;IF was used to observe the effect of palmitic acid and niacin co-treatment on nucleus and skeleton;and determined nuclear translocation of ChREBP.And observed lipid droplet production and cell ultrastructural changes;determined the TG content;the transcription and expression of ChREBP,Acaca/ACC,Fasn/FAS,Dgatl/DGAT1 and Dgat2/DGAT2 were determined by qRT-PCR and Western Blot.The results showed that compared with the control group,1.0 mM niacin had no effect on cell proliferation for 24 h.The intracellular ROS level in co-treatment group was significantly lower than palmitic acid group.The degree of nuclear damage,microfilament breakage and cytoskeleton integrity,lipid droplet production and TG content in co-treatment group were significantly lower than palmitic acid group.The ratio of ChREBP fluorescence in co-treatment group was significantly lower than palmitic acid group,the transcription and expression levels of ChREBP,Acaca/ACC,Fasn/FAS,Dgatl/DGAT1,Dgat2/DGAT2 in co-treatment group were significantly lower than palmitic acid group.The results showed that 1.0 mM niacin could alleviate the damage of BRL 3 A cells induced by palmitic acid,reduce the lipid droplet production of BRL 3A cells induced by palmitic acid,reduce the content of TG,and down-regulate the expression of ChREBP protein and nuclear translocation of ChREBP,thus down-regulate the expression of ACC,FAS and DGAT2 proteins and alleviate the degree of steatosis of BRL 3 A cells induced by palmitic acid.