The Protective Effect And Mechanism Of Hemeoxygenase-1/carbon Monoxide System On Ethanol-induced Hepatocytes Oxidative Injury

Objective: To illustrate the prophylaxis of carbon monoxide(CO)orginated from heme on ethanol-induced oxidative damage in rat primary hepatocyte. Then the potential mechanism of CO protection was investigated in the present study to hightlight the prospects of nutritional interventaion for alcohol liver diseases (ALD) by occurring naturally phytochemicals.Methods:1. Sprague-Dawley rat primary hepatocytes were isolated using a two-step collagenase perfusion technique. The intoxicant damage model was made by exposing hepatocytes to ethanol (100 mmol/L) for 24 hours.2. The ethanol-intoxicated hepatocytes were respectively treated with quercetin, hemin and hemoglobin for 24 h. To address the alternations of cell damage and quercetin protection involving HO-1 induction, the parameters of lactate dehydrogenase (LDH), aspartate transaminase (AST), malondialdehyde (MDA), glutathione (GSH) were determined by spectrophotometer at the end of each treatment.3. The ethanol-intoxicated hepatocytes were treated with different dose of CO release molecule(CORM-2,5～50μmol/L) only or cotreated with quercetin, ZnPPⅨand CORM-2; Meanwhile, The ethanol-intoxicated hepatocytes were treated with iCORM, hemin, DADS respectively. All groups were detected AST, LDH, MDA and CYP2E1 activity.Results:1. There are significant differences in rat primary hepatocytes between ethanol-insulted group (100 mmol/L ethanol exposure for 24 h) and normal control group: the release of cellular AST and LDH reduced while MDA elevation and GSH depletion were inhibited.2. Quercetin (100μmol/L) which added to the ethanol-intoxicated hepatocytes caused a significant decrease of AST and LDH release in the supernatants as well as MDA formation in hepatocytes compared with ethanol groups. Hemoglobin (100μM), a potent scavenger of CO, completely abolished quercetin-elicited cytoprotection and aggravated ethanol-induced hepatotoxicity compared with data generated from ethanol plus quercetin.3. CORM reduced the release of cellular AST and LDH induced by ethanol in a dose-dependent manner (within 20μM). Furthermore, we observed a parallel inhibition of MDA elevation and GSH depletion following CORM treatment for ethanol-incubated hepatocytes. In contrast, the inactive form of CORM (iCORM) did not show any beneficial effect on ethanol-elicited oxidative damage, and CORM itself had no influence on any such measured parameters.4. Ethanol virtually triggered the enzymatic activity of CYP 2E1. The inactivation of CYP2E1 by classic inhibitor diallyl disulfide (DADS, 50μM) partially protected hepatocytes from ethanol-induced oxidative stress. Quercetin, hemin and CORM all inhibited CYP2E1 activty; Hemoglobin (100μmol/L) completely abolished quercetin-elicited inhibition and increased CYP2E1 activity. The inhibition of both quercetin and CORM on CYP2E1 activity were weakened by ZnPPⅨ. The inactive form of CORM (iCORM) did not show any beneficial effect on CYP2E1 activity.Conclusions:1. Quercetin prevented rat primary hepatocytes from ethnol-induced oxidative damage by inducing HO-1.2. CO, a HO-1 metabolite, played an important role aganist ethanol-induced oxidative damage. This protection was involved in its inhibition of CYP2E1 activity.