Effects Of Alpha - Linolenic Acid On Fatty Acid Oxidation And Synthesis In Rat Liver And HepG2 Cells

Objectives:Utilizing n-3PUFA in prevention of hyperlipidemia through diet had been paidhigh attention abroad. Some researchers have proposed that adding a reasonableamount of n-3PUFA in dietary can effectively reduce the economic burden ofobesity in the world. In domestic daily ediable oil, the level of n-3PUFA is low.Moreover,the proportion of linseed oil, perilla oil and fish oil with high ratio ofn-3PUFA in the diet is also low. Clinical and animal experiments showed thatn-3PUFA can reduce serum lipids, but the effect which different doses of ALAhave on the mechanism is still unkown.We believe that n-3PUFA regulation of serum lipids may be derived from theregulation of lipid metabolism-related gene expression, such as the expression ofgenes related tocholesterol metabolism, fatty acid oxidation and synthesis.Geneexpression levels may be associated with n-3PUFA dose.This study was to investigate the effect of ALA on animal and cell. Fatty acidoxidation and synthesis andcholesterol synthesis-related gene expression wasstudiedto revealregulation mechanism for lowering blood lipids and providescientific data for the use of ALAin the prevention and treatment of dyslipidemia.Contents：In this study, high ratio perilla oil was given to SD rats, biochemical indexes,mRNA and protein expressionlevel of key genes such as key genes of fatty acidoxidation PPARα and CPT1A, key genes of fatty acid synthesis SREBP1C, FASand ACC, cholesterol synthesis genes SREBP2and HMGCR were analysed.Basedon animal experiments, cellular reseach aimed to analysefurther into these genes.HepG2cells were treated with stearic acid andthen stearic acid was replaced bydifferent levels of ALA, mRNA and protein expressionwere measured to make theeffect of ALA onmechanisms of fatty acid oxidation and synthesis clear in ratsliver and HepG2cells.Methods:16SD rats were divided into two groupsrandomly, normal controlgroup(NC),n-3PUFA group (TUF),16weeks later, take the measurement of serum totalcholesterol (TCH),triglycerides (TC), low density lipoprotein lipid (LDL-c), highdensity lipoprotein lipids (HDL-c) and free fatty acid (FFA) levels, quantitativereal-time PCR and Western blotting were used to detectmRNA and proteinexpression of genes related to fatty acidoxidation and synthesis.HepG2cells were divided into two groups, normal control group (NC) andhigh fat group (HF) in which cells were firstly cultured for36h in the medium contained0.5mmol/L stearic acid. Real-time quantitative PCR was taken to detectmRNA and protein expression of genes related to fatty acidoxidation andsynthesis.After that, there are significant differences in fatty acidoxidation andsynthesis, and10%,20%,50%,70%,100%ALA took the place of stearic acid,36h later, real-time quantitative PCR and Western blotting were taken to detectmRNA and protein expression of genes related to fatty acidoxidation and synthesisResults:1. Rat liver(1) Perilla oil group was significantly decreased serum TG, TC and HDL-c levels(P<0.001); there were no significant difference in serum LDL-c, FFA betweenPerilla oil and the normal control group;(2) Quantitative PCR results showed that gene CPT1A of perilla oil groupshowed no difference in relative expression level to the normal control group,while PPARα is significantly higher (P=0.0016); SREBP1C, FAS and ACCrelative expression levels were significantly increased (P=0.0006, P=0.0228, P=0.0028); SREBP2relative expression level was significantlyhigher (P=0.007); HMGCR was not significantly different;(3) Result of protein expression showed perilla oil group CPT1A expressionincreased and PPARα decreased. ACC, FAS and SREBP1C were lower thanthe normal control group, while HMGCR was higher than the normal controlgroup. SREBP2expression showed no significant difference.2. HepG2cells(1) Comparing with the HF group, genes PPARα and CPT1A mRNA expressionof ALA substitution groups showed no significant difference. Fatty acidoxidation genes CPT1A protein expression of70%ALA group and100%ALA group increased, the rest of the groups were not significantly different,100%ALA group PPARα reduced, and the rest showed no significantdifferences;(2) SREBP1C mRNA expression of ALA substitution groups were significantlylower than the high-fat group (P <0.001).The FAS of100%ALA group and70%ALA group were significantly lower than the high-fat group (P<0.001).Level of ACC genes mRNA was not significantly. SREBP1C and FAS proteinexpression were significantly lower than the high-fat group, but ACC showedno significant difference;(3) HMGCR mRNA level of20%ALA,50%ALA,70%ALA and100%ALAgroups were significantly higher than HF group. And there were nostatistically significant differencesinSREBP2mRNA level between HF groupand ALA substitution groups. HMGCR protein expression was significantly increased.While SREBP2proteinshowed no significant difference.Conclusions:1. Rat liver(1) Prolonged, high-dose intake of perilla oil significantly reduced serum lipids;(2) Excessive intake of perilla oil enhanced fatty acid oxidation gene mRNAexpression in rat liver, while that varied when it came to protein expression;(3) Intaking high doses of perilla oil promoted fatty acid synthesis gene mRNAexpression in rat liver and inhibit protein expression. Key gene of fatty acidsynthesis in rat liver didexist post-transcriptional regulation;(4) Excessive intake of perilla oil promoted SREBP2gene mRNA expression inrat liver and enhanced HMGCR protein expression. Perilla oil is not by thesynthesis pathway to lower serum cholesterol.2. HepG2cells(1) After the acid treatment, key genes mRNA expression of fatty acid oxidationand synthesis in HepG2cells wereenhanced.Key genes mRNA expressions ofcholesterol synthesis weredecreased;(2) High proportion ALA promoted fatty acid oxidation of HepG2cells byenhancing CPT1A expression, which doesn’t depend on PPARα;(3) As the dose of ALA substitution increased, inhibitionof key geneexpressionsoffatty acid synthesisenhanced;(4) High-dosesubstited ALA promote cholesterol synthesis.