Study On Inhibitory Effect Of Pinolenic Acid On Lipid Accumulation In HepG2 Cells Induced By Oleic Acid

Korean pine nut oil,a natural oil,is obtained from pine nuts,which contains a large amount of unsaturated fatty acid such as linoleic acid,oleic acid and pinoleic acid and has various pharmacological activities.Especially,pinoleic acid is a unique polyunsaturated fatty acid in pine nut oil,which has significant activities such as weight loss and lipid lowering.In this study,we first find that pinoleic acid inhibite lipid accumulation induced by oleic acid in HepG2 cells and its molecular mechanism.In addition,L-carnitine is a kind of vitamin extracted from red meat,which has a significant effect on weight loss and fat consumption.In order to achieve the best lipid-lowering effect,this study studied the optimal lipid-lowering concentration ratio of pinoleic acid/L-carnitine compound.The main research results as follows:1.Pinoleic acid inhibited the accumulation of lipid in HepG2 cells.The MTT assay showed that 0.5 mM oleic acid had no significant inhibitory effect on the survival rate of HepG2 and L02 cells,and oil red O staining showed that a large amount of lipid droplets were produced in the cells treated with 0.5 mM oleic acid for 24 h.Therefore,HepG2 cells were cultured with 0.5 mM oleic acid for 24 h,which were successfully induced lipid accumulaton.The MTT assay showed that when the HepG2 and L02 cells were treated with pinoleic acid for 12 h,survival rate was higher.There was no significant inhibitory effect on the HepG2 and L02 cells grouth when the pinoic acid below at 40 ?M concentration.After pretreatment with pinoleic acid(10,15,20,25 ?M)for 12 h,the levels of intracellular lipid droplets,triglyceride and cholesterol were significantly decreased(compared with the model group),indicating that pinoleic acid inhibits lipid accumulation.2.Pinotonic acid improved oleic acid-induced lipid accumulation by regulating oxidative stress in HepG2 cells.HepG2 cells were incubated with pinoleic acid(10,15,20,25 ?M)or saline for 12 h,and then treated with 0.5 mM oleic acid for 24 h.Pinacid acid pretreatment promoted the expression of superoxide dismutase(SOD)and glutathione peroxidase(GPX),and inhibited the expression of lipid peroxidation product lipid peroxidation(MDA).In addition,Pinacid acid could significantly block the production of intracellular reactive oxygen(ROS)caused by oleic acid.Meanwhile the protein levels of Nrf2 and heme oxygenase-1(HO-1)were significantly increased in the cells pretreated with pinoleic acid(compared with the oleic acid group).It was indicated that pinoleic acid could inhibit the lipid accumulation by inhibiting oxidative stress damage.3.Pinoleic acid inhibits lipid accumulation in HepG2 cells by regulating the AMPK/SIRT1 pathway.The Western blot showed that compared with the blank control,oleic acid treatment significantly increased the levels of fatty acid synthase(FASN),sterol regulatory element binding protein 1(SREBP1c)and stearoyl-CoA desaturase 1(SCD1)in HepG2 cells.But pretreatment with pinoleic acid(10,15,20,25 M)was remarkable lower than the oleic acid treatment group with a dose-dependent effect.The activation of AMPK and SIRT1 proteins is closely related to the promotion of fatty acid oxidation.Oleic acid stimulation inhibited intracellular AMPK phosphorylation and decreased levels of SIRT1.But after pretreatment with pinotonic acid,the level of AMPK phosphorylation,SIRT1 and PPARa in the cells recovered.After treatment with AMPK inhibitor compound C,AMPK phosphorylation was inhibited,and sirtuin 1(SIRT1)and Peroxisome proliferator-activated receptor a(PPAR)prion protein levels were also decreased to some extent,suggesting pinoleic acid improve intracellular lipid accumulation by activate AMPK/SIRT1 pathway.4.Pinoic acid/L-carnitine compound reduced oleic acid-induced lipid accumulation in HepG2 cells.HepG2 cells were pretreated with pine nut oil containing 50%pinoleic acid or L-carnitine for 12 h.The MTT assay showed that there was no significant cytotoxicity to cells when pinoleic acid below 50 ?M,and the L-carnitine below 500 ?M had a lower inhibition rate on cell growth.The oil red O staining showed that the optimal lipid-lowering concentration of pinoleic acid was 6.25 ?M,and the optimal lipid-lowering concentration of L-carnitine was 250 ?M.The optimal lipid-lowering concentration of pinoic acid/L-carnitine compound was 1 p.M for pinoic acid and 40 ?M for L-carnitine.It is indicated that the pinoic acid/L-camitine compound has drug use less and better effect in reducing lipid metabolism of HepG2 cells.In summary,pinoleic acid pretreatment can inhibit lipid peroxidation and the levels of promote fat synthesis proteins such as FASN,SREBP1c,SCD1.Meanwhile,pinoic acid can activate the phosphorylation of AMPK and its downstream SIRT1/PPARa signaling pathway to achieve lipid-lowering effects.In addition,optimize the optimal lipid-lowering ratio of the pinoleic acid/L-camitine compound,which provides a scientific basis for the development of high-quality products for weight loss and lipid-lowering.