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

長鏈多元不飽和脂肪酸(Long-chain polyunsaturated fatty acids, PUFAs)包含AA(arachidonic acid)、EPA(eicosapentaenoic acid)及DHA(docosahexaenoic acid)，參與許多生理功能，包括腦部的發育、認知、生殖、發炎反應及身體衡定性。DHA是視網膜及腦部的細胞膜上磷脂質重要的組成份，在嬰兒早期發育過程中會快速累積於這些組織器官。DHA可直接由飲食中的脂肪提供，或者由它的代謝前驅物ALA(α-linolenic acid)或EPA合成而來。首先，ALA先在內質網中合成EPA、DPA(docosapentaenoic acid)及THA (tetracosahexaenoic acid)，接著THA會被運送到過氧化體，經過一次β-oxidation作用形成DHA。肝臟被視為PUFA代謝的重要器官，它會將新合成好的DHA送至視網膜及腦部以供利用；另一方面，有研究指出腦部會轉換ALA成EPA及DHA以維持其在細胞膜中的濃度。然而，不論是在肝臟或是腦部，從ALA或EPA轉換來的DHA量都非常低，但原因至今並不清楚。 動物細胞中，這些n-3PUFAs的含量和參與代謝的酵素活性有關，例如desaturases、elongase、acyl CoA oxidase (AOX)，分別在ALA及EPA轉換上扮演不同的角色。先前研究指出此代謝途徑在細胞中並不活躍，但其機制尚未被研究透徹，目前推論Δ-6 desaturase和AOX也許催化途徑中的速率決定步驟。在這個研究中我們使用了兩種細胞株，分別是HepG2 (human hepatoma cell line)和SH-SY5Y (human neuroblastoma cell line)，我們想知道給予ALA或EPA，在兩種細胞中n-3PUFA代謝情形，以及參與此代謝步驟重要酵素之基因表現情況。我們發現在HepG2，EPA、DPA和DHA的濃度並不會隨著ALA給予而增加；但若給予細胞EPA時，DPA的濃度會上升，並且有dose-response的情形，但DHA的含量並不會提高。在基因表現層次，不論是給予高濃度ALA或是EPA，HepG2細胞中Δ-6 desaturase的基因表現減少。然而，在SH-SY5Y中， DPA的濃度會隨著ALA或EPA給予而增加，而DHA的含量有些微下降，表示從ALA代謝到DPA的過程較不受限制，但卻代謝到DHA的量非常少。由mRNA表現來看，ALA或是EPA皆會明顯抑制AOX基因表現。接著我們想要了解SH-SY5Y隨著EPA處理時間延長，AOX之基因表現是否持續降低，time-course實驗結果顯示，SH-SY5Y中AOX會持續受EPA抑制至96小時。而我們利用Western blot發現當EPA添加濃度增加，AOX蛋白表現量也呈現減少的趨勢。我們想要探討此抑制作用是否透過AOX起動子上PPRE序列，將AOX-promoter Luciferase 報導基因轉染至SH-SY5Y中，在加入EPA或ALA，結果顯示EPA及ALA會降低luciferase的活性。但在HepG2中，我們發現EPA及PPAR agonist會促進AOX起動子的轉錄活性，而EPA也會提高AOX蛋白的表現量，顯示在HepG2中，PPAR會受EPA及agonist活化，促進AOX表現。因此，我們想繼續了解SH-SY5Y中AOX的基因轉錄是否和HepG2同樣受PPAR調控，以PPARα、γ、及δ的agonist分別添加到SH-SY5Y培養液中，發現三種PPAR agonist都會些微促進AOX蛋白表現；而以PPAR-luciferase報導系統的實驗顯示，EPA會透過到三種 PPARs提高Luciferase活性，但是相較於PPAR agonists，EPA活化PPARs的能力較差，因此EPA應該不是透過PPAR抑制AOX蛋白表現。綜合以上結果我們推測，HepG2細胞株中DHA合成率低的部分原因可能是因為ALA及EPA抑制了Δ-6 desaturase；而SH-SY5Y細胞株中由於ALA及EPA抑制了AOX表現，因此可能是導致DHA合成受阻的原因之一，我們的結果也顯示EPA對於AOX抑制作用並不是透過PPAR。

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.