The Mechanism Of BRL 3A Cell Steatosis Induced By Oleic Acid And The Protective Effect Of Biotin

As we know,dairy cow fatty liver is a disease that often occurs in the perinatal period,and during this disease,oleic acid is the most monounsaturated free fatty acid produced in the blood.Biotin is a water-soluble vitamin,and many studies have shown that oleic acid can induce steatosis in a variety of cells,but biotin has the effect of alleviating steatosis,in addition,the under mechanism is not clear.In order to investigate the mechanism of oleic acid-induced cell steatosis and the role of biotin in it,so in this study,the BRL 3 A cell line was used as a research model to investigate the role of MAPK/TLR4/PPARy signaling pathway and the protective effect of biotin in oleic acid-induced BRL 3 A steatosis process.Furthermore,this study will provide a theoretical basis for further elucidation of oleic acid-induced hepatic steatosis,and provide a new direction for the treatment of animal fatty liver.1.Effect of oleic acid on BRL 3 A cell damageIn order to investigate the effects of oleic acid on proliferation,morphology,lipid droplet formation and ATP content of BRL 3 A cells,the CCK-8,RTCA and oil red O staining were used to establish a model of BRL 3 A cell steatosis.And then the effects of oleic acid on cell surface structure and ultrastructure were observed by scanning electron microscopy and transmission electron microscopy.The changes of nuclei,DNA and cytoskeleton were also observed by immunofluorescence staining.In addition,kits were used to detect changes in TG and ATP levels.The results showed that compared with the control group,1.8 mM oleic acid treatment cells had no significant effect on cell proliferation,and the damage of cytoskeleton,nucleus and cell ultrastructure were concentration-dependent.Meanwhile,TG content increased significantly with increasing oleic acid concentration(P<0.05),but the ATP content decreased significantly with the increase of oleic acid concentration(P<0.05).The results showed that oleic acid can cause cell morphological damage and fatty degeneration.2.Study on the mechanism of BRL 3A cell steatosis induced by oleic acidIn order to investigate the mechanism of oleic acid-induced BRL 3A cell steatosis,cells were treated with different concentrations of oleic acid(0,1.2,1.8,2.4 mM)for 24 hours and then the content of MDA,ROS and the activities of antioxidant enzymes GSH-PX,T-SOD,CAT,T-AOC were detected.Besides,the phosphorylation of MAPK/TLR4/PPAR? pathway protein and the expression of FAS and SREBP-lc were also detected.In addition,to further explore the effect of MAPK pathway on TLR4 protein,1.8 mM oleic acid was co-treated with MAPK signaling pathway inhibitors or TLR4 inhibitors,then the TLR4,FAS,SREBP-1c expression and the phosphorylation level of PPARy were detected.The results showed that compared with the control group,the content of MDA and ROS and the activity of CAT increased significantly with the increase of oleic acid concentration(P<0.05),while the activities of GSH-PX,T-AOC and T-SOD decreased significantly(P<0.05).In addition,the phosphorylation levels of fat synthesis-related proteins and MAPK signaling pathway-related proteins were significantly or significantly increased(P<0.05 or P<0.01).The expression of TLR4 was significantly or significantly decreased after co-treatment with oleic acid and MAPK inhibitor(P<0.05 or P<0.01).Moreover,the expression of fat synthesis-related protein was significantly decreased after co-treatment with oleic acid and TLR4 inhibitor(P<0.05).The results showed that oleic acid can cause oxidative stress in BRL 3A cells and activate MAPK signaling pathway,thereby activate TLR4 and PPARy and then to increase the expression of FAS and SREBP-lc,and aggravate the degree of steatosis.3.The role of biotin in BRL 3A cytotoxic injury induced by oleic acidIn order to explore the role of biotin in oleic acid-induced BRL 3A toxic damage,the CCK-8 method,RTCA,oil red O staining,electron microscopy,immunofluorescence and detection kits were used to detect the effects of biotin on cytotoxic damage induced by oleic acid.The results showed that compared with the oleic acid co-treatment group,the cell proliferation index increased and the number of lipid droplets decreased,in addition,the nucleus and cytoskeleton became more intact.The results indicate that biotin can significantly improve the cytotoxic damage caused by oleic acid.4.Protective effect of biotin in BRL 3A cell steatosis induced by oleic acidIn order to explore the protective mechanism of biotin in oleic acid-induced BRL 3A steatosis,co-treatment of cells with 80 ?M biotin and 1.8 mM oleic acid for 24 h,and then the content of MDA and ROS and the activities of antioxidant enzymes GSH-PX,T-SOD,CAT,T-AOC were detected;furthermore,the expression of FAS,SREBP-lc and the phosphorylation level of MAPK/TLR4/PP-ARy pathway-related proteins were also detected.In addition,to further verify the role of PPARy in it,the cells were co-treated with oleic acid and the PPAR gamma agonist ROZ or the inhibitor T007,then detected the expression of fat synthesis-related proteins.The results showed that the levels of MDA and ROS were significantly lower in the biotin and oleic acid co-treatment groups than in the oleic acid alone treatment group(P<0.05),besides,the activities of antioxidant enzymes GSH-PX,T-SOD,CAT and T-AOC were increasedsignificantly or extremely significant(P<0.05 or P<0.01);futhermore,western blot results showed that the expression of TLR4 and the phosphorylation level of PPARy and MAPK pathway-related protein were significantly or extremely significant decreased(P<0.05 or P<0.01).In addition,compared with the oleic acid alone treatment group,the oleic acid and PPARy agonist ROZ co-treatment group significantly increased the expression of FAS and SREBP-lc,but the oleic acid and PPARy inhibitor T007 co-treatment group significantly reduced the expression of FAS and SREBP-1c in fat synthesis related proteins(P<0.05).The results indicate that biotin can enhance the antioxidant capacity of cells and reduce the production of ROS.At the same time,it can significantly reduce the expression of liposynthesis-related proteins and reduce the degree of cell steatosis.