Mitochondria-targeted ubiquinone (MitoQ) enhances acetaldehyde clearance by reversing alcohol-induced posttranslational modification of aldehyde dehydrogenase 2: A molecular mechanism of protection against alcoholic liver disease

Abstract

Alcohol metabolism in the liver generates highly toxic acetaldehyde. Breakdown of acetaldehyde by aldehyde dehydrogenase 2 (ALDH2) in the mitochondria consumes NAD⁺ and generates reactive oxygen/nitrogen species, which represents a fundamental mechanism in the pathogenesis of alcoholic liver disease (ALD). A mitochondria-targeted lipophilic ubiquinone (MitoQ) has been shown to confer greater protection against oxidative damage in the mitochondria compared to untargeted antioxidants. The present study aimed to investigate if MitoQ could preserve mitochondrial ALDH2 activity and speed up acetaldehyde clearance, thereby protects against ALD. Male C57BL/6J mice were exposed to alcohol for 8 weeks with MitoQ supplementation (5mg/kg/d) for the last 4 weeks. MitoQ ameliorated alcohol-induced oxidative/nitrosative stress and glutathione deficiency. It also reversed alcohol-reduced hepatic ALDH activity and accelerated acetaldehyde clearance through modulating ALDH2 cysteine S-nitrosylation, tyrosine nitration and 4-hydroxynonenol adducts formation. MitoQ ameliorated nitric oxide (NO) donor-mediated ADLH2 S-nitrosylation and nitration in Hepa-1c1c7 cells under glutathion depletion condition. In addition, alcohol-increased circulating acetaldehyde levels were accompanied by reduced intestinal ALDH activity and impaired intestinal barrier. In accordance, MitoQ reversed alcohol-increased plasma endotoxin levels and hepatic toll-like receptor 4 (TLR4)-NF-κB signaling along with subsequent inhibition of inflammatory cell infiltration. MitoQ also reversed alcohol-induced hepatic lipid accumulation through enhancing fatty acid β-oxidation. Alcohol-induced ER stress and apoptotic cell death signaling were reversed by MitoQ. This study demonstrated that speeding up acetaldehyde clearance by preserving ALDH2 activity critically mediates the beneficial effect of MitoQ on alcohol-induced pathogenesis at the gut-liver axis.

Keywords: Alcoholic liver disease; Aldehyde dehydrogenase 2; MitoQ; Posttranslational modification.

Fig. 1

Effects of MitoQ on hepatic nitrosative and oxidative stress in alcohol-fed mice. (A) Immunohistiochemistry of hepatic 3-NT. (B) Immunohistiochemistry of hepatic 4-HNE. (C) Plasma hydrogen peroxide concentrations. (D) Hepatic GSH concentrations. (E) Hepatic total NAD concentrations. (F) Hepatic NAD⁺ concentrations. (G) Hepatic NADH concentrations. (H) Hepatic NAD⁺/NADH ratio. (I) Western blot of mtETC complex subunits, NOX4 and SOD2 in the liver. Scale bar: 50 µm. * P < 0.05 vs PF, ** P < 0.01 vs PF, # P<0.05 vs. AF, ## P<0.01 vs. AF, § P<0.05 vs. PF/MitoQ, §§ P<0.01 vs. PF/MitoQ.

Fig. 2

MitoQ administration alleviated alcohol-impaired ALDH activity along with accelerated acetaldehyde clearance. (A) Hepatic ethanol and acetaldehyde concentrations. (B) Plasma ethanol and acetaldehyde concentrations. (C) Hepatic ALDH activity. (D) Expression of hepatic ethanol and aldehyde metabolizing enzymes. ** P < 0.01 vs PF, ## P<0.01 vs. AF.

Fig. 3

MitoQ enhanced ALDH activity in alcohol-fed mice through modulating ALDH2 post- translation modifications. Hepatic ALDH2 proteins from 4 groups were purified and subjected to immunoblot analysis with the specific anti-ALDH2, anti-S-nitrosocysteine antibody, anti-3-NT antibody or 4- HNE antibody respectively (A-C). (D) Hepatic nitrite contents. * P < 0.05 vs PF, ** P < 0.01 vs PF, # P<0.05 vs. AF, ## P<0.01 vs. AF. For in vitro studies, hepatocytes were pretreated with BSO (500 μmol) for 16 h. BSO was then replaced with either medium or SNAP (1 mM) for 8 h in the absence or presence of 0.5 μM MitoQ. (E) Nitrite concentrations. (F-G) Immunoblot analysis for immunopurified ALDH2 proteins from Hepa1c1c7 cells with anti-ALDH2, anti-S- nitrosocysteine antibody or anti-3-nitrotyrosine antibody. (H) Hepatocyte ALDH activity. (I) Flow cytometry to measure cell death. (J) Apoptosis rate in Hepa1c1c7 cells. ** P < 0.01 vs control, # P<0.05 vs. BSO plus SNAP, ## P<0.01 vs. BSO plus SNAP.

Fig. 4

MitoQ reversed alcohol-induced gut barrier disruption along with enhanced intestinal ALDH activity. (A) Intestinal ALDH activity (B) Bar graphs shown mRNA levels of Claudin 1 determined by RT-qPCR. (C) Bar graphs shown mRNA levels of Occludin determined by RT- qPCR. (D) Immunofluorescent staining of ZO-1 in the ileum. Scale bar: 10 µm. Red: ZO-1; blue: 4′,6-diamidino-2-phenylindole counterstaining of the nuclei; white trigangle: disassembled tight junction proteins. * P < 0.05 vs PF, ** P < 0.01 vs PF, # P<0.05 vs. AF.

Fig. 5

Effects of MitoQ on endotoxemia and TLR4-NF-κB signaling pathway activation. (A) Plasma LPS levels. (B) Immunoblot bands of hepatic TLR4 and NF-κB. (C) Bar graphs shown mRNA levels of hepatic Mcp-1. (D) Bar graphs shown mRNA levels of hepatic KC. (E) Bar graphs shown mRNA levels of hepatic TNFα. (F) Immunofluorescent staining of CD11b⁺ in mouse liver. Scale bar: 50 µm. Arrows: CD11b⁺ cells; blue: 4′,6-diamidino-2-phenylindole counterstaining of the nuclei. * P < 0.05 vs PF, # P<0.05 vs. AF.

Fig. 6

MitoQ ameliorated alcohol-induced liver injury and lipid metabolism alterations. (A) Levels of plasma ALT and AST in each group. (B) Liver histopathological changes in each group shown by H&E staining (arrows: lipid droplets, arrowheads: inflammatory cells) Scale bar: 50 µm. (C) Oil red O staining of neutral lipids. Scale bar: 20 µm. (D) Levels of proteins involved in lipid metabolism. (E) Liver TG content. (F) Liver FFA concentration. (G) Plasma TG content. (H) Plasma FFA concentration. Proteins levels were quantitated by NIH image J. All values are denoted as means ± SD. * P < 0.05 vs PF, ** P < 0.01 vs PF, # P<0.05 vs. AF, ## P<0.01 vs. AF, §, P<0.05 vs. PF/MitoQ.

Fig. 7

MitoQ reversed alcohol-induced hepatic ER stress along with attenuation of apoptosis. (A) Western blot of proteins involved in ER stress (B) Western blot of proteins involved in apoptosis. (C) Expression of hepatic LC3I/II. * P < 0.05 vs PF, ** P < 0.01 vs PF, # P<0.05 vs. AF, ## P<0.01 vs. AF, § P<0.05 vs. PF/MitoQ.

Fig. 8

Depicting the possible molecular mechansims by which MitoQ prevents alcoholic liver disease through modulating ALDH2 posttranslational modifications. After chronic alcohol abuse, oxidative and nitrosative stress were increased, which impaired mitochondrial ALDH2 activity via cysteine S-nitrosylation, tyrosine nitration and 4-HNE adducts formation. Acetaldehyde accumulated in the system result in ileal tight junction disruption and increased plasma endotoxin and subsequently inflammatory response in the liver. Excess acetaldehyde accumulation in the liver leads to enhanced lipotoxicity, ER stress and cell apoptosis pathway activation. MitoQ supplementation prevented alcohol-induced ALDH2 posttranslational modifications and accelerated acetaldehyde clearance, which against alcohol-induced pathogenesis at the gut-liver axis.