Pharmacological activation of aldehyde dehydrogenase 2 by Alda-1 reverses alcohol-induced hepatic steatosis and cell death in mice

Abstract

Background & aims: Effective therapies for alcoholic liver disease are currently unavailable. The present study tested the efficacy of Alda-1, a specific aldehyde dehydrogenase 2 (ALDH2) activator, in treating alcoholic liver disease.

Methods: Male C57BL/6J mice were exposed to alcohol for a time-course study on aldehyde metabolism. The specificity and efficacy of Alda-1 on activating hepatic ALDH2 and aldehyde clearance were determined by acute treatments. Then, mice were fed alcohol for 8 weeks with Alda-1 administration for the last 10 days to test the therapeutic potential of Alda-1. Lastly, H4IIEC3 cells were treated with ethanol, acetaldehyde, or 4-hydroxynonenal to define the link between aldehydes and hepatotoxicity.

Results: Alcohol feeding for 8 weeks induced hepatic ALDH2 dysfunction and aldehyde accumulation. One dose of Alda-1 administration elevated hepatic ALDH activity, which was blocked by the specific ALDH2 inhibitor, daidzin. Alda-1 accelerated acetaldehyde clearance after acute alcohol intoxication. Alda-1 treatment in the 8-week alcohol feeding model reversed liver damage along with reduction of hepatic aldehydes. Alda-1 re-activated transcription factors, upregulated fatty acid oxidation enzymes, and reversed steatosis. Alcohol-induced endoplasmic reticulum stress and apoptotic cell death were also attenuated by Alda-1. Acetaldehyde or 4-hydroxynonenal treatment to H4IIEC3 cells inactivated transcription factors and induced endoplasmic reticulum stress and apoptosis, while ethanol per se showed limited effects.

Conclusions: Pharmacological activation of ALDH2 by Alda-1 reversed alcoholic steatosis and apoptosis through accelerating aldehyde clearance. This study indicates that ALDH2 is a promising molecular target and Alda-1 has therapeutic potential for treating alcoholic liver disease.

Keywords: Alcohol; Alda-1; Aldehyde dehydrogenase 2; Apoptosis; Steatosis.

Figure 1.. Chronic alcohol feeding failed to activate hepatic ALDH2 despite aldehyde accumulation.

(A) mRNA levels, (B) protein levels, and (C) activities of hepatic ALDH2 in mice fed alcohol for 2, 4, and 8 weeks. (D) Plasma and (E) hepatic acetaldehyde concentrations in mice fed alcohol for 8 weeks. (F) Immunohistochemical staining of hepatic 4-HNE in mice fed alcohol for 8 weeks. Significant difference indicated by * (Student’s t-test, P<0.05) or different letters (ANOVA, P<0.05). n=6 for PF, n=8 for AF. PF, pair-fed; AF, alcohol-fed.

Figure 2.. Activating hepatic ALDH2 alleviated chronic alcohol feeding-induced liver injury along with accelerated aldehyde clearance.

(A) Histopathological changes of liver. Scale bar, 50 μm. (B) Expression of hepatic ethanol and aldehyde metabolizing enzymes. (C) Hepatic ALDH2 activity. (D) Plasma and hepatic ethanol and acetaldehyde concentrations. (E) Immunohistochemical staining of hepatic 4-HNE. Significant differences indicated by different letters (ANOVA, P<0.05, n=6). CV, central vein; PV, portal vein.

Figure 3.. Alda-1 reversed alcohol-induced hepatic steatosis through modulating lipid metabolism, and reduced alcohol-induced hepatic apoptosis along with attenuation of ER stress.

(A) Hepatic lipid accumulation in mice. Scale bar, 50 μm. (B) Expressions of proteins involved in mediating lipid homeostasis in the liver. (C) Hepatic cell death (arrows) indicated by TUNEL. Scale bar, 50 μm. (D) Expressions of ER stress proteins and caspase-3. Significant differences indicated by different letters (ANOVA, P<0.05, n=6).

Figure 4.. Aldehydes induced transcription factor inactivation, ER stress and apoptosis in H4IIEC3 cells.

(A) Expressions of transcription factors and ER stress proteins after acetaldehyde treatment. (B) Expressions of transcription factors and ER stress proteins after 4-HNE treatment. (C) TUNEL staining of cellular apoptosis after aldehyde treatments. Significant difference indicated by * (Student’s t-test, P<0.05, n=6).