Effect Of Quercetin On The Oxidative Damage Induced By Alcohol-iron And Expression Of Iron-metabolism Related Genes

Objective: To investigate the potential protective effect of quercetin against alcoholic liver disease (ALD), ethanol-incubated rat primary hepatocytes were employed in our study, and changes of oxidative damage related indices and libile iron pool (LIP) in hepatocytes were determined following iron and ethanol treatment by enzymatic and biochemical assay.Methods:1. Rat primary hepatocytes were isolated using two-step collagenase perfusion technique and treated with different doses of exogenous nitrilotriacetic acid amine (Fe-NTA, 5 ~ 100μmol / L) for 24 h. Cellular leakage of lactate dehydrogenase (LDH)and aspartate transaminase (AST), as well as redox indices were measured by spectrophotoemtry, which included the level of cellular malondialdehyde (MDA), ROS, glutathione (GSH), and the activity of superoxide dismutase (SOD).2. Rat primary hepatocytes were co-treated by ethnol (200 mmol/L) and Fe-NTA (50μmol/L) based on the dose-response of Fe-NTA, and the cellular leakage of LDH and AST and levels of redox indices were assayed with or without quercetin intervenetion.3. Rat primary hepatocytes were treated with ethanol (200 mmol/L), Fe-NTA (50μmol/L) and/or quercetin (100μmol/L) for 24 h, and the cellular content of (LIP-Fe) was measured by fluorospectrophotometry using calcein-AM as probe.Results:1. Fe-NTA dose-dependently (5 ~ 100μmol/L) induced a dramatic leakage of cellular of AST and LDH, GSH depletion and SOD inactivation, and increased the levels of ROS and MDA in compared with normal control.2. Compared to hepatocytes (normal control), ethanol (200 mmol/L) and Fe-NTA (50μmol/L) exposure respectively resulted in a dramatic leakage of cellular of AST and LDH, GSH depletion and SOD inactivation, and increased ROS production and MDA level. Compared with ethanol and Fe-NTA co-treated group, intense leakage of AST and LDH, obviously ROS production and high MDA level, and markedly GSH depletion and SOD inactivation, were the results of quercetin intervention.3. Compared with normal control, the cellular LIP-Fe level significantly elevated by ethanol (200 mmol/L) and/or Fe-NTA (50μmol/L). In addition, the production of LIP-Fe was obviously decreased with quercetin treatment.Conclusions:The iron aggravated oxdative stress of ethanol-induced rat primary hepatocytes injuries, in a dose-dependent manner. Significant oxidative damage and high level of cellular LIP-Fe were derived by ethanol and iron treatment, which were inhibited by quercetin. Objective: To explore the potential mechanism of quercetin counteracting iorn overload elicited by ethanol, the mRNA expression of iron-metabolism related genes were evaluated after ethanol-incubated hepatocytes treatment with Fe-NTA and/or quercetin.Methods:Rat primary hepatocytes were incubated with ethanol (200μmol/L), Fe-NTA (50μmol/L) and/or quercetin (100μmol/L) for 24 h, then the expression of the hepcidin, ferroportin, transferrin receptor 1( TfR1) and transferrin receptor 2(TfR2) mRNA were detected by real-time PCR technique.Results:1. Compared with normal control, ethanol (200 mmol/L) incubation respectively resulted in decrease of hepcidin and TfR2 expression, as well as elevation of TfR1 and ferroportin expression. Fe-NTA (50μmol/L) obviously increased mRNA expression of hepcidin, TfR1and TfR2, while ferroportin expression was quiet, compared to normal control.2. Treatment with ethanol and Fe-NTA, hepcidin expression was no change, expression of ferroportin and TfR1 were elevated obviously, and TfR2 expression was a slight decrease compared to normal control; Expression of hepcidin and ferroprotin were enhanced significantly, while TfR2 mRNA level was no change in contrast with ethanol group; obvious decreases of hepcidin ferroportin expression compared with Fe-NTA treatment group were also observed.3. There were no differences in mRNA expression of hepcidin, ferroportin and TfR2 between quercetin and normal control groups. We found that a dramatic elevation of ferroportin and TfR2 expression and no obvious change of hepcidin and TfR1 expression in quercetin and ethanol/Fe-NTA group compared to those of ethanol and Fe-NTA group. Conclusions:Hepcidin expression was inhibited by ethanol while a significant elevated expression with iron loading. There was an opposite relationship between the hepcidin and ferroportin expression. Quercetin was able to exert the beneficial effects on iron and ethanol cotreatment-induced rat primary hepatocytes injuries, the underlying mechanism is at least due to regulation the iron metabolism related genes expression.