Iron-Mediated Lysosomal Membrane Permeabilization in Ethanol-Induced Hepatic Oxidative Damage and Apoptosis: Protective Effects of Quercetin

Abstract

Iron, in its free ferrous states, can catalyze Fenton reaction to produce OH∙, which is recognized as a crucial role in the pathogenesis of alcoholic liver diseases (ALD). As a result of continuous decomposition of iron-containing compounds, lysosomes contain a pool of redox-active iron. To investigate the important role of intralysosomal iron in alcoholic liver injury and the potential protection of quercetin, male C57BL/6J mice fed by Lieber De Carli diets containing ethanol (30% of total calories) were cotreated by quercetin or deferoxamine (DFO) for 15 weeks and ethanol-incubated mice primary hepatocytes were pretreated with FeCl3, DFO, and bafilomycin A1 at their optimal concentrations and exposure times. Chronic ethanol consumption caused an evident increase in lysosomal redox-active iron accompanying sustained oxidative damage. Iron-mediated ROS could trigger lysosomal membrane permeabilization (LMP) and subsequent mitochondria apoptosis. The hepatotoxicity was attenuated by reducing lysosomal iron while being exacerbated by escalating lysosomal iron. Quercetin substantially alleviated the alcoholic liver oxidative damage and apoptosis by decreasing lysosome iron and ameliorating iron-mediated LMP, which provided a new prospective of the use of quercetin against ALD.

Figure 1

The effect of quercetin and DFO on the mice liver oxidative damage delivered by chronic alcohol consumption. The liver MDA (a) and the ratio of GSH/GSSH (b) were detected by spectrophotometry. Each value represents the mean ± SD (n = 12). Liver ROS (c) was detected and visualized with fluorescence microscope (×100). Fluorescence intensities in randomly selected areas of the images were quantified with IPP 6.0 image analysis software (d). C: control; E: ethanol; EQ: ethanol plus quercetin; ED: ethanol plus DFO; Q: quercetin; D: DFO. A: P < 0.05 versus the control; B: P < 0.05 versus the ethanol group.

Figure 2

Quercetin extenuated hepatocytes apoptosis elicited by ethanol. TUNEL staining was used to assay hepatocytes apoptosis (a). The TUNEL-positive nucleus showed brown (black arrow) and the negative nucleus showed blue (blue arrow). (b) Quantitation of data obtained in (a) by using the IPP image analysis software. The activation of caspases-3 was analyzed by Western blotting (c, n = 3) and quantified by Image J software (c). Blotting with anti-β-actin was used as a protein loading control. The activity of caspase-3 was determined by assay kit (Beyotime) and standardized by protein concentration measured by the Bio-Rad protein assay kit (d). Results were presented as mean ± SD or mean ± SE. C: control; E: ethanol; EQ: ethanol plus quercetin; ED: ethanol plus DFO; Q: quercetin; D: DFO. A: P < 0.05 versus the control; B: P < 0.05 versus the ethanol group.

Figure 3

Quercetin decreased liver total iron and labial iron pool (LIP) as well as DFO. Mice liver total iron (a) and LIP (b) were determined by flame atomic absorption spectrophotometry. Each value represents the mean ± SD (n = 12). Ft-L expression was measured by Western blotting and the band densities were measured by Image J software (c, n = 3). C: control; E: ethanol; EQ: ethanol plus quercetin; ED: ethanol plus DFO; Q: quercetin; D: DFO. A: P < 0.05 versus the control; B: P < 0.05 versus the ethanol group.

Figure 4

Quercetin decreased intralysosomal redox-active iron and it induced ROS in lysosome as well as DFO. The mice primary hepatocytes were isolated according to a two-step collagenase method and treatment for 6 hours. The intralysosomal iron was measured by SSM ((a) the exposure time was 50 min in the dark, n = 3) and quantitated by flame atomic absorption spectrophotometry (b). The cells were incubated with a combination of LysoTracker (to detect lysosomes) and H₂DCFDA (to detect ROS production) for 30 min and immediately observed by confocal microscopy with sequential recording in green (oxidized product of H₂DCF) and red (LysoTracker). All photomicrographs were taken at ×200 (c, n = 3). C: control; E: ethanol; EQ: ethanol plus quercetin; ED: ethanol plus DFO; Q: quercetin; D: DFO. A: P < 0.05 versus the control; B: P < 0.05 versus the ethanol group.

Figure 5

Quercetin attenuated ethanol-induced lysosomal membrane permeabilization (LMP) and mitochondria membrane permeabilization (MMP). The alterations of LMP were assayed by AO staining with flow cytometry, as evident by decrease in FL3 red fluorescence and increase in FL1 green fluorescence (a and b). Cathepsin B reconstitution (c) and activity (d) were determined by Western blot and assay kit. The red mitochondrial fluorescence stained with TMRM of 10,000 cells per sample was determined by flow cytometry by using the FL3 channel (e). Cytochrome C release (f) was evaluated by Western blotting and the band densities were measured by Image J software (g, n = 3). The blotting with GAPDH and VDAC1 was used as a protein loading control. C: control; E: ethanol; EQ: ethanol plus quercetin; ED: ethanol plus DFO; Q: quercetin; D: DFO.

Figure 6

The role of lysosomal iron in ethanol-induced hepatocytes damage and the protective effect of quercetin. Mouse primary hepatocytes grown on coverslips were treated with ethanol (100 mmol/L, 24 hours), bafilomycin A1 (100 nmol/L, 4 hours), FeCl₃ (60 μmol/L, 4 hours), DFO (1 mmol/L, 4 hours), and quercetin (100 μmol/L, 24 hours). The intralysosomal redox-active iron content (a) and lysosome status (b) were measured by SSM (the exposure time for the FeCl₃ group was 20 min; other groups were 50 min) and AO-uptake technique, respectively. All photomicrographs were taken at ×200 (n = 3). The leakage of AST and LDH from hepatocytes (c, d, n = 12), together with cellular ROS production (e, n = 12), was detected by spectrophotometry. MMP (f and g) and apoptosis (h) were measured by flow cytometry (n = 3). Each value represents the mean ± SD. C: control; E: ethanol; EI: ethanol plus FeCl₃; EB ethanol plus bafilomycin A1; ED: ethanol plus DFO. A: P < 0.05 versus the control; B: P < 0.05 versus the ethanol group.