花生四烯酸及二十二碳六烯酸調控苯乙基巴比妥酸誘發大鼠初代肝細胞細胞色素P-450 2B1表現之機制探討

Abstract

Rat hepatocyte cytochrome P-450 2B1 (CYP 2B1) expression is modulated by dietary fatty acids and prostaglandin E2 (PGE2). Individual n-6 and n-3 polyunsaturated fatty acids (PUFAs), specifically, arachidonic acid (AA), linoleic acid (LA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), were used to determine their effects on CYP 2B1 expression induced by phenobarbital (PB) in rat primary hepatocytes. PB-induced CYP 2B1 expression was down-regulated by n-6 and n-3 PUFAs, especially AA and DHA. PGE2, a fatty acid metabolite, but not PGE3, down-regulated CYP 2B1 expression induced by PB in rat primary hepatocytes through the EP2 receptor. PGE2 increased the intracellular cAMP level and led to the activation of protein kinase A (PKA). It has been shown that increased cAMP suppresses the induction of CYP 2B1 by PB in rat primary hepatocyte cultures. Suppression of cAMP production by SQ22536, an adenylate cyclase inhibitor, and inhibition of PKA by H-89, resulted in reversion of the down-regulation of CYP 2B1 expression by AA and PGE2 in the presence of PB. PGE2 and the cAMP-dependent PKA pathway are thus involved in the down-regulation of PB-induced CYP 2B1 expression by AA. The mechanism for the down-regulation of PB-induced CYP 2B1 expression by AA has been delineated; however, the mechanism for this down-regulation by DHA was previously unknown. Constitutive androstane receptor (CAR) was shown to play a crucial role in metabolizing enzyme expression. PB induction of human CYP 2B6 and mouse cyp 2b10 was shown to mediated by CAR. In addition to prostaglandin and the downstream pathways of cAMP and PKA, CAR may be involved in the down-regulation of PB-induced CYP 2B1 expression by PUFAs. PB triggered the translocation of CAR from the cytosol into the nucleus in a dose-dependent and time-dependent manner, and CAR distribution in rat primary hepatocytes was significantly affected by DHA. DHA treatment decreased PB-inducible accumulation of CAR in the nuclear fraction and increased it in the cytosolic fraction. The down-regulation of CYP 2B1 expression by DHA occurred in a dose-dependent manner, and a similar pattern was found for the nuclear accumulation of CAR. The results of immunoprecipitation showed a CAR/RXR heterodimer bound to nuclear receptor binding site 1 (NR-1) of the PB-responsive enhancer module (PBREM) of the CYP 2B1 gene. The EMSA results showed that PB-induced CAR binding to NR-1 was attenuated by DHA. These results suggest that attenuation of CAR translocation and subsequent binding to NR-1 are mechanisms for the down-regulation of PB-induced CYP 2B1 expression by DHA. In summary, PB-induced CYP 2B1 expression was down-regulated by n-6 and n-3 PUFAs through different pathways. PGE2, EP2, and the cAMP- dependent PKA pathways are involved in the down-regulation of CYP 2B1 expression by AA, whereas the down-regulation by DHA is through attenuation of CAR translocation.