Abstract
Malay (macrolanguage) The metabolism of alcohol involves cytochrome P450 2E1 (CYP2E1)-induced oxidative stress, with the association of phosphatidylinositol-3-kinases (PI3K) and nuclear factor kappa B (NFκB) signalling pathways. CYP2E1 is primarily involved in the microsomal ethanol oxidising system, which generates massive reactive oxygen species (ROS) and ultimately leads to oxidative stress and tissue damage. Lauric acid, a major fatty acid in palm kernel oil, has been shown as a potential antioxidant. Here, we aimed to evaluate the use of lauric acid as a potential antioxidant against ethanol-mediated oxidative stress by investigating its effect on CYP2E1 mRNA expression and the signalling pathway in ethanol-induced HepG2 cells. HepG2 cells were firstly treated with different concentrations of ethanol, and subsequently co-treated with different concentrations of lauric acid for 24 h. Total cellular RNA and total protein were extracted, and qPCR and Western blot was carried out. Ethanol induced the mRNA expression of CYP2E1 significantly, but lauric acid was able to downregulate the induced CYP2E1 expression in a dose-dependent manner. Similarly, Western blot analysis and densitometry analysis showed that the phosphorylated PI3K p85 (Tyr458) protein was significantly elevated in ethanol-treated HepG2 cells, but co-treatment with lauric acid repressed the activation of PI3K. However, there was no significant difference in NFκB pathway, in which the normalised NFκB p105 (Ser933) phosphorylation remained constant in any treatment conditions in this study. This suggests that ethanol induced CYP2E1 expression by activating PI3K p85 (Tyr458) pathway, but not the NFκB p105 (Ser933) pathway in HepG2 cells. Metabolisme alkohol melibatkan tekanan oksidatif akibat cytochrome P450 2E1 (CYP2E1), dengan penglibatan phosphatidylinositol-3-kinases (PI3K) and faktor nuklear kappa B (NFκB). CYP2E1 memainkan peranan penting dalam sistem pengoksidaan etanol microsomal, yang akan menghasilkan spesies oksigen reaktif (ROS) dan akhirnya membawa kepada kerosakan tisu. Asid laurik, yang merupakan asid lemak utama dalam minyak isirong sawit, telah terbukti memperoleh potensi antioksidan. Kajian ini bertujuan untuk menilai penggunaan asid laurik sebagai antioksidan terhadap tekanan oksidatif yang dirangsangkan oleh etanol. Kami menyiasat kesan asid laurik terhadap ekspresi mRNA CYP2E1 dan laluan isyarat yang disebabkan oleh etanol dalam sel HepG2. Justeru, HepG2 dirawat dengan kepekatan etanol yang berbeza, dan seterusnya dirawat dengan kepekatan asid laurik yang berbeza selama 24 jam. Jumlah RNA dan jumlah protein telah diekstrak, dan qPCR dan blot western telah dijalankan. Etanol mendorong ekspresi mRNA CYP2E1 dengan ketara, tetapi asid laurik dapat mengimbangi ekspresi CYP2E1. Analisis blot western dan analisis densitometri menunjukkan bahawa tahap protein fosforilasi PI3K p85 (Tyr458) telah meningkat dengan ketara dalam sel-sel HepG2 yang dirawat etanol, tetapi rawatan bersama dengan asid laurik menindas pengaktifannya. Walau bagaimanapun, laluan NFκB tidak menunjukkan sebarang perbezaan. Tahap fosforilasi NFκB p105 (Ser933) kekal sama dengan sebarang keadaan rawatan dalam kajian ini. Ini menunjukkan bahawa etanol merangsangkan expresi CYP2E1 dengan mengaktifkan laluan PI3K p85 (Tyr458), tetapi bukan melalui NFκB p105 (Ser933) dalam sel HepG2. Asid laurik menindas rangsangan etanol dengan menghentikan pengaktifan laluan PI3K.