Iron Promotes Hepatocyte Injury Induced By Alcohol

In all life forms, iron is a redox active metal involved in the oxidation-reductionreactions and regulation of cell proliferation, differentiation, oxygen transport, electrontransport and so on. However, excess iron, especially in the non-proteinbound, lowmolecular weight form, would induce the cellular injury by catalyzing the generation ofthe hydroxyl radical, the principal effector of oxidative damage in organs or body system.Iron plays a key role in the deveploment of alcohol liver diease. An increase in hepaticiron concentrations occurs in alcohol-dependent individuals and elevated hepatic ironuptake is seen in patients with alcohol-induced cirrhosis. Although animal experimentshave been investigated for the combined effect of alcohol and iron on iron homestasis,there is no cell model set up to analyze the interaction of alcohol and iron. Flavonoidsbelong to a group of natural substances with variable phenolic structures. These naturalproductswere known for their beneficial effects on health long before flavonoids wereisolated as the effective compounds. Although a lot of animal experiments have provedthat certain flavonoids have protective effect on the liver injury induced by acute orchronic alcohol administration, there is no cell model set up to analyze the protectiveeffect of flavonoids on hepatocyte injury induced by alcohol and iron.In this paper, we investigated the effect of different forms of iron on promotingoxidative stress in HepG2cells and then we used suitable iron to analyze the mechanismof iron in regulation of oxidative/nitrative stress induced by alcohol and the protectiveeffect of flavonoids in HepG2cell. The main results are as follows:(1) The effect of different forms of iron on promoting oxidative stress in HepG2cells.We used methylene blue assay, MTT assay, western blotting，flow cytometry andimmunofluorescence to analyze the iron-induced oxidative stress mechanisms in HepG2cells.The results are as follows: In vitro methylene blue assay, it showed that hemin andmetahemoglobin were more powerful agents than the other two forms of iron in chemicaloxidation of methylene blue, which means they has stronger capability in catalyzingFenton-like reaction to produce hydroxy radical. With the same concentration of iron,NaNO2and glucose oxidase (which could produce H2O2with glucose), different forms ofiron played different role in apoptosis, lipid peroxidation, protein expression and protein nitration. These results strongly suggested that iron porphyrin was the main form of iron incatalytic oxidation and there were various in mechanisms of iron reaction with RNS/ROSin HepG2cells.(2) Ferric citrate CYP2E1-independently promotes alcohol-induced apoptosis inHepG2cells via oxidative/nitrative stress which is attenuated by pretreatmentwith baicalin.We used MTT assay, flow cytometry, western blotting and immunofluorescence toanalyze the hepatocyte injury caused by the combination of alcohol and iron, and theprotective effect of baicalin. Compared with cells treated with100mM ethanol alone,ferric citrate enhanced the generation of reactive oxygen and nitrogen species, increasedthe occurrence of protein carbonylation/nitration and the levels of4-hydroxy-2-nonenal,changed the distribution of iNOS, upregulated the ERK1/2regulation and eventuallyresulted in apoptosis. The inhibition of iNOS could increase the level of apoptpsis inducedby iron and alcohol. However, pretreatment with baicalin inhibited the oxidative stressinduced by the combination of alcohol and iron. Our findings therefore suggest that ironcould CPY2E1-independently enhance the oxidative stress induced by alcohol, whichprobably contributes to the pathogenesis of alcoholic liver disease. In our cell model, NOprobably had protective effect and was a secondary messenger. Baicalin is a promisingphytomedicine for preventing alcoholic liver disease.(3) Mechanism of the protective effect of baicalin in hepatocyte injury induced byiron and alcohol.We used MTT assay, flow cytometry, ABTS assay and fluorescence labelling toanalyze the mechanism of the protective effect of baicalin in hepatocyte injury induced byiron and alcohol. It suggested that the protective effect of baicalin was mainly attributed toits property of chelating iron while the chelating iron property of flavonoid was not theguarantee for its protective effect in hepatocyte injury. DHE was the suitable fluorescentprobe to measure the superoxide anion in HepG2cells which could overcome thedifficulty in efficiently analyzing the effect of the flavonoids which could be used astrypsin inhibitor. Our cell model is suitable for the research in discovering efficientmedicine for ALD.