中文摘要 飲食油脂除了能夠提供生物體所需能量外，也是構成細胞膜磷脂質、合成前列腺素及白三烯素不可或缺的原料。許多研究顯示n-3、n-6多元不飽和脂肪酸（PUFA）可直接或間接調控基因表現，影響細胞生長、代謝及分化。本實驗室先前研究結果已知n-3及n-6 PUFA皆能抑制PB誘發CYP 2B1的表現，不論在protein或mRNA level中皆以花生四烯酸（Arachidonic acid）的抑制效果最強，但此一抑制作用機制仍不清楚。已知Arachidonic acid可經由環氧化（COX）代謝成PGE2；此外，PGE2可透過EP2 receptor誘發胞內cAMP增加，進而活化cAMP-dependent PKA達到抑制PB誘發CYP 2B1的作用機制也在先前實驗中被證實。因此，本研究目的在探討Arachidonic acid 抑制PB誘發CYP2B1的表現是否透過COX代謝成PGE2，進而活化cAMP-dependent PKA pathway。在PB誘發大鼠初代肝細胞表現CYP2B1實驗模式下，添加100 μmol/L Arachidonic acid觀察其對PB誘發CYP2B1的影響，並利用藥理模式添加adenylate cyclase抑制劑（SQ22536）及PKA抑制劑（H-89）觀察Arachidonic acid的抑制作用。此外，以RT-PCR觀察細胞中COX的表現。結果發現Arachidonic acid明顯抑制CYP2B1的表現，如預處理SQ22536或H-89則可逆轉此抑制效果。RT-PCR可偵測到肝細胞中有COX-1的表現但卻無法發現COX-2。綜合上述實驗結果，推測Arachidonic acid可能利用COX-1代謝成PGE2，進而活化cAMP-dependent PKA pathway達到抑制PB誘發CYP2B1的表現。
Abstract: Cytochromes P-450 are an important bioactivation-detoxification system in vivo. Its expression is regulated by foreign chemicals and dietary factors, and lipids have been found to regulate its gene expression. We showed previously that PGE2 decreases CYP2B1 expression in the presence of phenobarbital in rat primary hepatocytes through EP2 and that cAMP and the downstream cAMP- dependent PKA pathway are involved in this down-regulation. The objective of the present study was to determine the effects of n-3 and n-6 polyunsaturated fatty acids on PB inducible-CYP2B1 gene expression in rat primary hepatocytes and to determine whether the n-6 fatty acid-arachidonic acid regulated CYP2B1 expression in the presence of phenobarbital in rat primary hepatocytes through its metabolite-PGE2 and that cAMP and the downstream cAMP-dependent PKA pathway are involved in this down-regulation. We used a primary rat hepatocyte culture model in which EP2 was present to test our hypothesis. Both n-3 and n-6 PUFA were found to down-regulate the induction of CYP2B1 expression by phenobarbital. The cyclooxygenase inhibitor-indomethacin significantly reversed the down-regulatory effect of arachidonic acid (AA). In the present study, the PGE2 concentration in primary rat hepatocytes was significantly higher in hepatocytes treated with 100 ？Hgmol AA/L than hepatocytes treated with LA, EPA or DHA. Treatment with 100 ？Hgmol AA/L significantly increased formation of PGE2 than treatment with 1 or 10 ？Hgmol AA/L in primary rat hepatocytes, and 20 ？Hgmol indomethacin/L (COX inhibitor) decreased 50 % PGE2 formation of hepatocytes treated with 100 ？Hgmol AA/L. SQ22536, an adenylate cyclase inhibitor, reversed the down-regulation by AA, as did H-89, a protein kinase A inhibitor. These results suggest that EP2 and the downstream pathways of cAMP and protein kinase A are involved in the down-regulation of CYP2B1 expression by AA metabolite-PGE2 in the presence of phenobarbital.
