Study On Oleoylethanolamide Ameliorates Fatty Acid-induced Lipid Accumulation And Oxidative Stress In Hepatocytes

When people consumed with high-fat diet,it is often accompanied by abnormal fatty acid metabolism which will cause lipid accumulation and further oxidative stress.Non-alcoholic fatty liver disease(NAFLD)is considered a metabolic disease that closely related to lipid accumulation and oxidative stress.Oleylethanolamide(OEA)is an endogenous biological lipid signaling molecule with multiple physiological functions.However,the mechanism by which OEA regulates lipid metabolism and oxidative stress thus regulates NAFLD has not been fully elucidated.This study explored the effect and mechanism of OEA on fatty acid-induced Hep G2 and L02 cells from lipid metabolism and oxidative stress to provide some scientific basis and theoretical reference for the prevention and treatment of NAFLD.Firstly,an in vitro cell lipid accumulation model was established by oleic acid and p almitic acid(2:1).HepG2 and L02 cells were treated with different concentrations of FFAs for 24 h.The results indicated that there was no significant inhibitory effect on cell activity when the cells treated with 0.2 mM or 0.4 mM FFAs.However,when the FFAs concentration increased to 0.6 mM,the cell activity of L02 cells decreased to 69.33% compare with the blank.In addition,the number of red lipid droplets and intracellular TG content increased in a concentration-dependent manner with the FFAs in HepG2 and L02 cells.When the cells treated with 0.4 mM FFAs,a large amount of red lipid droplets could be observed and the intracellular triglyceride(TG)content increased significantly compared with the blank group(p < 0.01).Therefore,we confirmed that 0.4 mM FFAs could successfully induce lipid accumulation in HepG2 and L02 cells.Secondly,the effect of OEA on fatty acid-induced lipid accumulation in HepG2 and L02 cells was investigated.The cells were treated with 0.4 mM FFAs and different concentrations of OEA for 24 h.The results indicated that 0.1-25 μM OEA has no toxic effect to HepG2 and L02 cells.In addition,intracellular TG content reduced in a dose-dependent manner by OEA.Further researchs found that OEA up-regulated the expression of fatty acid oxidation regulators: peroxisome proliferators-activated receptor alpha(PPAR-α)and carnitine palmitoyltransferase 1(CPT-1),while down-regulated the expression of key factors of lipogenesis,such as sterol regulatory element binding protein-1c(SREBP-1c)and fatty acid synthase(FAS)from the transcription and translation levels.Next,the effect of OEA on FFAs-induced oxidative stress was investigated in HepG2 and L02 cells.The cells were treated with 0.4 mM FFAs and different concentrations of OEA for 24 h.The results indicated that OEA inhibited increased generation of reactive oxygen species(ROS)incuced by FFAs in a concentration-dependent manner.Thereby reduced the content of lipid peroxidation product: malondialdehyde(MDA).Additionly,OEA significantly enhanced the activities of antioxidant enzymes,such as superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GPx).Cell mitochondrial membrane potential was also increased.Further researchs founded that the regulation of OEA on oxidative stress was achieved by activating the expression of nuclear factor erythroid 2-related factor 2(Nrf2)and heme oxygenase 1(HO-1).Finally,the expression of the total regulatory factor AMPK was examined.It was found that OEA could significantly enhance the phosphorylation of AMPK while had no significantly effect on total AMPK.To verify whether AMPK regulated lipid metabolism and oxidative stress,the HepG2 and L02 cells were treated with OEA and Compound C,an AMPK inhibitor for 24 h.Compound C inhibited the expression of AMPK thus reduced phosphorylation.Thereby blocked p-SREBP-1c induced by OEA.Unphosphorylated SPEBP-1c entered the nucleus and regulated the expression of FAS.On the other hand,Compound C blocked AMPK phosphorylation and inhibited the upregulation of Nrf2 and HO-1 caused by OEA.In summary,OEA reduced cellular lipid accumulation through activating PPAR-α and inhibiting SREBP-1c pathways,and ameliorated oxidative stress through Nrf2 pathway.OEA regulated cellular lipid metabolism and oxidative stress may be partially achieved by AMPK phosphorylation.These data provide some scientific support that OEA may be an effective treatment strategy for NAFLD.