The Antioxidatant Effect Of Daidzein In Vitro & The Protective Effect Of Daidzein On Ethanol-induced Hepatocytes Oxidative Injury

Objection: First we observe the antioxidation activity of Daidzein (DAI) in vitro. Furthermore we try to study the prophylaxis of DAI on ethanol-induced oxidative damage in rat primary hepatocytes.Methods:1. In order to detect the DAI's ability of eliminating·O2-, we imitated the xanthine and xanthine oxidase system to produce·O2-. For detect the DAI's ability of eliminating·OH, we imitated Fenton system to produce·O2-.2. 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 mM) for 8 h.3. By MTS method, we observed the role of ethanol in different doses on alcoholic oxidative damage of rat primary liver cells, and protective effect of DAI (1.0μM for 2h) pre-treatment in ethanol-induced oxidative damage.4. The ethanol-intoxicated hepatocytes were respectively pre-treated (2 h) with DAI while the time-dependent and dose-dependent (0.5-20μM) DAI pre-treatment were used in the present research. The parameters of alanine aminotransferase(ALT), aspartate transaminase (AST), malondialdehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD) were determined by spectrophotometer at the end of each treatment to address the alterations of cell damage and prooxidant-antioxidant state after DAI intervention.Result:1. DAI has a certain capability of cleaning·O2- and·OH. Compared with Quercetin (QE) and Vit C, the capability of cleaning·O2- and·OH is between QE and Vit C.2. As the dose of ethanol increasing, the ethanol-induced oxidative damage in rat primary hepatocytes was strengthened. The ethanol-induced oxidative damage was significantly reduced by the pre-treated of DAI (1.0μM for 2 h).3. Protection effect of DAI on ethanol-induced oxidative damage in hepatocytes is time-dependent and dose-dependent. The pre-treatment with 0.5 to 2.5μM DAI during 2 to 6 hours before ethanol exposure, caused a significant decrease of AST and ALT release in the supernatants as well as MDA formation in hepatocytes which compared with ethanol group (P<0.01). In addition, the depletion of GSH and the inactivation of SOD were suppressed significantly by pre-treatment with DAI (P<0.01).Conclusions:1. DAI has anti-oxidation effect by directly eliminating·O2- and·OH. in vitro.2. Pre-treatment with DAI could prevent rat primary hepatocytes from ethanol-induced oxidative damage by reducing GSH consumption, improving antioxidant enzyme activity and inhibiting lipid peroxidation reaction. Moreover the protective effect of DAI displayed dose-dependent and time-dependent relation.