α-次亞麻油酸和二十碳五烯酸抑制人類神經瘤細胞株SH-SY5Y之acyl CoA oxidase基因表現

Abstract

Very long-chain polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA, C20:4n-6), eicosapentaenoic acid (EPA, C20:5n-3), and docosahexaenoic acid (DHA, C22:6n-3) are essential for a variety of biological functions including brain development, cognition, reproduction, inflammatory responses, and homeostasis. DHA is an important component of membrane phospholipids in the retina and brain and accumulates rapidly in these tissues during early infancy. DHA is directly provided by dietary fats or is synthesized from its metabolic precursors, α-linolenic acid (ALA, C18:3n-3), and EPA. The conversion of ALA, the essential dietary precursor, occurs in the endoplasmic reticulum (ER), producing the long-chain metabolites EPA, DPA and tetracosahexaenoic acid (THA, C24:6n-3). Then THA is transferred to the peroxisomes to be shortened to DHA through one cycle of β-oxidation. Liver has been considered the primary site for the production of long chain PUFAs, and ALA conversion in liver is a major source of newly synthesized DHA for supply to the periphery. In addition, ALA conversion in brain may be important for maintaining membrane EPA and DHA concentrations. However, formation of DHA from ALA or EPA is very low in both liver and brain, and the mechanisms responsible for the low conversion of ALA and EPA to DHA remains unclear. The content of n-3PUFAs in mammalian cells depends on the activity of the desaturases, elongase, and acyl CoA oxidase (AOX), these enzymes also involved in the synthesis of DHA from ALA or EPA. In general, the Δ-6 desaturase and AOX were considered as the rate limiting steps in the conversion. In this study we used HepG2 (human hepatoma cell line) and SH-SY5Y (human neuroblastoma cell line) as in vitro models to examine the conversion of n-3 fatty acids after supplementation of ALA or EPA. We also quantified the mRNA levels of gene encoding these enzymes. We found that EPA, DPA, and DHA were not increased when HepG2 cells were cultured with increasing amount of ALA. In addition, only DPA was linearly increased when EPA was supplemented in the medium. The Δ-6 desaturase mRNA abundance decreased in response to supplementation of ALA or EPA in HepG2 cells. Different from that in HepG2, the content of DPA was increased when SH-SY5Y cells were cultured with addition of ALA or EPA, but DHA was decrease slightly. The results suggest that synthesis of DPA from ALA or from EPA was an active process in SH-SY5Y cells. Our data further show that AOX mRNA was down regulated by both ALA and EPA. Time-course study showed AOX mRNA expression was inhibited by EPA up to 96h. In addition, our data also showed decrease of AOX protein levels by EPA in SH-SY5Y cells. We then investigated the effect of EPA on AOX expression in cells transfected with AOX promoter-Luciferase reporter plasmid. The result showed that luciferase activity was decreased by EPA and ALA in SH-SY5Y cells. But in HepG2 cells, AOX-Luciferase was induced by EPA and by agonist of PPARα, γ or δ. The AOX protein level was also up regulated by EPA in HepG2 cells. The result support that EPA and PPARs induce AOX expression in HepG2 cells. We further tested if the effect of PPAR agonists on AOX in SH-SY5Y cells. The results show that all agonists of the PPARα, γ and δ isoforms induce AOX protein expression in SH-SY5Y. These results suggest that EPA down regulates AOX expression is not through a PPAR dependent pathway. Taken together, our data showed that expression of Δ-6 desaturase was inhibited by ALA and EPA in HepG2 cells, and may responsible for the low conversion rate of ALA to longer n-3 fatty acids. On the other hand, inhibition of AOX mRNA and protein levels by ALA or EPA in SH-SY5Y cells, may be the reason, at least in part, for the low synthesis of DHA from ALA or DHA. Our results further suggest that the inhibitory effect of EPA on AOX expression is probably through an PPAR independent pathway.