Aldehyde dehydrogenase 2 deficiency ameliorates alcoholic fatty liver but worsens liver inflammation and fibrosis in mice

Abstract

Aldehyde dehydrogenase 2 (ALDH2) is the major enzyme that metabolizes acetaldehyde produced from alcohol metabolism. Approximately 40-50% of East Asians carry an inactive ALDH2 gene and exhibit acetaldehyde accumulation after alcohol consumption. However, the role of ALDH2 deficiency in the pathogenesis of alcoholic liver injury remains obscure. In the present study, wild-type and ALDH2(-/-) mice were subjected to ethanol feeding and/or carbon tetrachloride (CCl4 ) treatment, and liver injury was assessed. Compared with wild-type mice, ethanol-fed ALDH2(-/-) mice had higher levels of malondialdehyde-acetaldehyde (MAA) adduct and greater hepatic inflammation, with higher hepatic interleukin (IL)-6 expression but surprisingly lower levels of steatosis and serum alanine aminotransferase (ALT). Higher IL-6 levels were also detected in ethanol-treated precision-cut liver slices from ALDH2(-/-) mice and in Kupffer cells isolated from ethanol-fed ALDH2(-/-) mice than those levels in wild-type mice. In vitro incubation with MAA enhanced the lipopolysaccharide (LPS)-mediated stimulation of IL-6 production in Kupffer cells. In agreement with these findings, hepatic activation of the major IL-6 downstream signaling molecule signal transducer and activator of transcription 3 (STAT3) was higher in ethanol-fed ALDH2(-/-) mice than in wild-type mice. An additional deletion of hepatic STAT3 increased steatosis and hepatocellular damage in ALDH2(-/-) mice. Finally, ethanol-fed ALDH2(-/-) mice were more prone to CCl4 -induced liver inflammation and fibrosis than ethanol-fed wild-type mice.

Conclusion: ALDH2(-/-) mice are resistant to ethanol-induced steatosis but prone to inflammation and fibrosis by way of MAA-mediated paracrine activation of IL-6 in Kupffer cells. These findings suggest that alcohol, by way of acetaldehyde and its associated adducts, stimulates hepatic inflammation and fibrosis independent from causing hepatocyte death, and that ALDH2-deficient individuals may be resistant to steatosis and blood ALT elevation, but are prone to liver inflammation and fibrosis following alcohol consumption.

Fig. 1. ALDH2^−/− mice have lower levels of serum ALT and hepatic steatosis after ethanol feeding than do WT mice

WT and ALDH2^−/− mice were fed a control or ethanol diet for 10 days, followed by a single gavage of maltose or ethanol. The mice were euthanized 9 h after gavage. (A) Body weight after ethanol feeding. (B) Hepatic acetaldehyde levels. (C) Serum ALT and AST levels. (D) Representative hematoxylin and eosin (H&E) staining and Oil Red O staining of liver tissues. (E) Hepatic triglyceride (TG) and cholesterol (Chol) levels. The values represent means ± SD. (n=4 in pair-fed WT or KO group, n=8 in ethanol-fed WT or KO group) *P < 0.05.

Fig. 2. ALDH2 deficiency accelerates the ethanol-induced liver inflammatory response (eg. IL-6 production) via MAA-mediated activation of Kupffer cells. (A)

WT and ALDH2^−/− mice were fed a control or ethanol diet for 10 days, followed by a single gavage of maltose or ethanol, respectively. Hepatic cytokine expression was measured using real-time PCR analyses. (n=4 in pair-fed WT or KO group, n=8 in ethanol-fed WT or KO group) (B) Expression of IL-6 and TNF-α mRNA in precision-cut liver slices (PCLS) ex vivo. (n=3 each group). (C) Hepatocytes, Kupffer cells, and HSCs were isolated from pair-fed or chronic-binge ethanol-fed mice and subjected to real-time PCR analyses. (n=3 each group). (D) Hepatic MAA levels from pair-fed or chronic-binge ethanol-fed mice were measured. (E) Kupffer cells from WT mice were isolated and treated with 25 μg/ml HSA or MAA-HSA in the presence or absence of LPS (1 ng/ml). After stimulation with MAA for 2 h, cell culture media were analyzed for IL-6 levels. (n=3 each group). The values represent means ± SD. *P < 0.05, **P < 0.01.

Fig. 3. ALDH2^−/− mice exhibit higher levels of hepatic STAT3 activation after chronic-binge ethanol feeding than do WT mice

WT and ALDH2^−/− mice were subjected to pair-fed or chronic-binge ethanol feeding. The mice were euthanized 9 h after alcohol or maltose gavage. Liver tissues were collected for Western blot or real-time PCR analyses. (A) Western blot analyses. (B) Real-time PCR analyses of fat metabolism-associated genes. (C) Western blot analyses of antioxidant proteins. The densities of STAT3, Ref-1 and MnSOD were quantified using densitometry. The values represent means ± SD. (n=4 in pair-fed WT or KO group, n=8 in ethanol-fed WT or KO group) *P < 0.05.

Fig. 4. An additional deletion of STAT3 in hepatocytes increases ethanol-induced serum ALT elevation and hepatic steatosis but suppresses inflammation in ALDH2^−/− mice

WT, ALDH2^−/−, and ALDH2^−/−STAT3^Hep−/− mice were fed an ethanol diet for 10 days, followed by a single gavage of ethanol. The mice were euthanized 9 h after gavage. (A) Western blot analysis. (B) Serum ALT levels. (C) Hepatic triglyceride (TG) levels. (D) Representative hematoxylin and eosin (H&E) staining and Oil Red O staining of liver tissues. (E) The hepatic expression of cytokines was measured using real-time PCR analyses. The values in panels B, C, E represent means ± SD. (n=6 WT, n=4 ALDH2^−/− or ALDH2^−/−STAT3^Hep−/−). *P < 0.05.

Fig. 5. ALDH2 deficiency accelerates liver inflammation after ethanol plus CCl₄ administration

WT and ALDH2^−/− mice were fed an ethanol diet or pair-fed, and these mice were co-administrated with CCl₄ for 8 weeks. The mice were euthanized 24 hours post the last CCl₄ injection. (A) Serum ALT levels. (B) Representative hematoxylin and eosin (H&E) staining (X100). (C) Representative immunohistochemical staining of F4/80 (X200). (D) The hepatic expression of cytokines was measured using real-time PCR analyses. The values represent means ± SD. (n=8 mice in each pair-fed or ethanol-fed WT or ALDH2^−/− group) *P < 0.05.

Fig. 6. ALDH2^−/− mice show higher degrees of hepatic fibrosis after 8-week ethanol plus CCl₄ treatment than do WT mice

(A-D) WT and ALDH2^−/− mice were treated with ethanol-fed plus CCl₄ or pair-fed plus CCl₄ for 8 weeks. (n=8 mice in each pair-fed or ethanol-fed WT or ALDH2^−/− group). (A, B) Representative photographs of Sirius red staining (A) and immunohistochemical analysis with an anti-α-SMA antibody (B). (C) The Sirius red⁺ and α-SMA⁺ areas were quantified from panels A and B, respectively. (D) Real-time PCR analyses of hepatic fibrosis-associated genes. The values represent means ± SD. *P < 0.05.

Fig. 7. A model depicting the mechanisms underlying increased inflammation and fibrosis but reduced fatty liver and hepatocellular damage after ethanol feeding in ALDH2^−/− mice

Alcohol consumption leads to MAA adduct accumulation in the livers of ALDH2^−/− mice. MAA then stimulates Kupffer cells to produce proinflammatory cytokines, such as IL-6, leading to inflammation and subsequently promoting liver fibrosis. IL-6 also activates STAT3 in hepatocytes, followed by the upregulated expression of antioxidative stress genes and the downregulated expression of fatty acid synthesis genes, subsequently ameliorating hepatocellular damage and steatosis. In addition, acetaldehyde, MAA and IL-6 activate ERK1 and STAT3 in HSCs, thereby accelerating liver fibrosis in ALDH2^−/− mice.