Hepcidin Regulation Through Endoplasmic Reticulum Stress And Quercetin Protection On Alcoholic Liver Iron Overload

Objective:1. To investigate the potential protective effect of quercetin against the oxidative injury in alcoholic liver iron overload of mice.2. To investigate the role of endoplasmic reticulum stress on regulation of hepcidin of mice with alcoholic iron overload and the protective effect of quercetin.Methods:One hundred and twenty male C57BL/6J mice were randomly divided into eight groups by body weight:normal control, ethanol control, iron control, ethanol+iron, quercetin+ethanol, quercetin+ethanol+iron, quercetin+iron and quercetin control. The mice were pair-fed with Lieber-DeCarli liquids diets containing ethanol (30%of total energy), carbonyl iron (0.2%) and/or quercetin (100mg/kg. bw) for15weeks to induce alcoholic liver iron overload. Subsequently, all mice were sacrificed after overnight fast. And blood samples were collected to prepare serum for the assay of ALT and AST level. Intact fresh livers were stripped immediately and weighed to calculate liver iron index. The content of hepatic total iron was determined by atomic absorption spectrometry. ROS generation of hepatic sample was measured with DHE assay by using a fluorescence microscope. The mRNA expression of hepcidin, XBPS/XBPT and GRP78were measured by real-time PCR. Moreover western blot were used to estimate the protein expression of X-box binding protein1(XBP1) and glucose regulated protein78(GRP78) in liver.Results:1. Following energy matching feeding for15weeks, the body weight of all ethanol-treated mice was lower than non-ethanol mice significantly despite quercetin intervention. Iron or quercetin itself had no effect on final body weight.2. Compared to normal control, both ethanol and iron exposure increased the level of liver index and the level of serum ALT and AST. What’s more, the liver index, as well as the level of serum ALT and AST was further elevated when fed with ethanol plus iron. Quercetin treatment evidently decreased liver index and serum aminotransferase level of ethanol and/or iron-dosed mice as compared with corresponding control.3. Compared to normal control group, ethanol or iron significantly increased hepatic total iron content and content and ROS level, which was efficiently inhibited by quercetin. Both parameters were further induced when treated by ethanol and iron, which was also suppressed by quercetin. Quercetin itself had no effect on hepatic total iron and ROS level.4. Expression of hepcidin mRNA. Hepcidin mRNA is down-regulated by alcohol and up-regulated by iron. The combined treatment of ethanol and iron decreased the mRNA expression of hepcidin compared with iron control in spite of higher expression than normal control. Quercetin intervention suppressed the down-regulated of hepcidin mRNA by ethanol and the induction of hepcidin mRNA by iron.5. Endoplasmic reticulum stress (ERS):Chronic ethanol and especially iron exposure increasing evidence of ERS makers, such as the mRNA and protein expression of XBPS/XSPT and GRP78. ERS was aggravated by co-treatment of ethanol and iron. Quercetin decreased ERS maker by ethanol and especially by iron or/plus ethanol. Quercetin itself had no effect on ERS in contrast with intact normal control.Conclusions:Quercetin protected mice from ethanol and/or iron induced hepatic oxidative damage and iron overload. Alcohol led to ERS down-regulated hepcidin mRNA expression; iron caused worse ERS whereas up-regulated hepcidin mRNA expression. ERS was further exacerbated when co-treated with ethanol and iron, but hepcidin mRNA expression fell in between normal control and iron control. Quercetin alleviated ERS and down-regulation of hepcidin mRNA expression induced by alcoholic and/or iron. These findings suggest that ERS may participate in the regulation of hepcidin expression when dosed with iron, but may not participate in the regulation of alcohol on hepcidin.