Oxidative Stress and the ER Stress Response in a Murine Model for Early-Stage Alcoholic Liver Disease

Abstract

Alcoholic liver disease (ALD) is a primary cause of morbidity and mortality in the United States and constitutes a significant socioeconomic burden. Previous work has implicated oxidative stress and endoplasmic reticulum (ER) stress in the etiology of ALD; however, the complex and interrelated nature of these cellular responses presently confounds our understanding of ethanol-induced hepatopathy. In this paper, we assessed the pathological contribution of oxidative stress and ER stress in a time-course mouse model of early-stage ALD. Ethanol-treated mice exhibited significant hepatic panlobular steatosis and elevated plasma ALT values compared to isocaloric controls. Oxidative stress was observed in the ethanol-treated animals through a significant increase in hepatic TBARS and immunohistochemical staining of 4-HNE-modified proteins. Hepatic glutathione (GSH) levels were significantly decreased as a consequence of decreased CBS activity, increased GSH utilization, and increased protein glutathionylation. At the same time, immunoblot analysis of the PERK, IRE1α, ATF6, and SREBP pathways reveals no significant role for these UPR pathways in the etiology of hepatic steatosis associated with early-stage ALD. Collectively, our results indicate a primary pathogenic role for oxidative stress in the early initiating stages of ALD that precedes the involvement of the ER stress response.

Figure 1

Chronic ethanol ingestion leads to increased hepatic lipid accumulation and lipid peroxidation. (a) H&E staining reveals marked pan-lobular lipid accumulation following 6 weeks of ethanol consumption. (b) Liver triglycerides are significantly increased following 6 weeks of ethanol ingestion. (c) Immunohistochemical analysis with 4-HNE antibodies reveals increased staining in ethanol-fed mice throughout zones 1 and 2 as early as week 3. (d) Hepatic TBARSs are significantly elevated following 6 weeks of ethanol ingestion. Magnification: 400x. Scale bar represents approximately 50 μm. PT: portal triad; CV: central vein (n = 12, *P < 0.05, **P < 0.01).

Figure 2

Ethanol ingestion results in increased protein-SSG. (a) Immunohistochemical staining with antibodies directed against protein-SSG reveals a marked increase in the zone 3 staining in the week 6 ethanol-fed mice. (b) pan-GST activity and (c) GRx activity significantly elevated at week 6. Magnification: 400x. Scale bar represents approximately 50 μm. PT: portal triad: CV: central vein (n = 6, **P < 0.01, ***P < 0.001).

Figure 3

Early-Stage ALD leads to a significant increase in protein ubiquitination. (a) Immunohistochemical analysis reveals marked staining of ubiquitinated proteins in zone 3 and zone 2 of ethanol-fed mice following 6-week ingestion. PT: portal triad; CV: central vein. (n = 4 pairs; **P < 0.01). Magnification, 400x; Scale bar represents approximately 50 μm. (b) Hepatic proteasome activity revealed no change at the week 6 time point (n = 6 pairs; P = n.s.).

Figure 4

Western blotting reveals a disconnect between the UPR and oxidative stress in the pathogenesis of ALD. (a) Standard western blotting shows a lack of activation in classical UPR signaling cascades at any time point. (b) No induction of the Ero1 proteins or Nox4 was observed, confirming a lack of UPR-induced oxidative stress. β-actin was utilized as a loading control for total liver extracts while Lamin B1 was used as a nuclear loading control.