N-acetylcysteine attenuates progression of liver pathology in a rat model of nonalcoholic steatohepatitis

Abstract

A "2-hit" model for nonalcoholic steatohepatitis (NASH) has been proposed in which steatosis constitutes the "first hit" and sensitizes the liver to potential "second hits" resulting in NASH. Oxidative stress is considered a candidate for the second hit. N-acetylcysteine (NAC), an antioxidant, has been suggested as a dietary therapy for NASH. We examined the effects of NAC in a rat total enteral nutrition (TEN) model where NASH develops as the result of overfeeding dietary polyunsaturated fat. Male Sprague-Dawley rats consumed pelleted AIN-93G diets ad libitum or were overfed a 9200 kJ.kg(-0.75).d(-1) liquid diet containing 70% corn oil with or without 2 g.kg(-1).d(-1) NAC i.g. for 65 d. Hepatic steatosis was not influenced by dietary supplementation with NAC; however, the liver pathology score was lower (P </= 0.05) and NAC provided partial protection against alanine aminotransferase release (P </= 0.05). NAC attenuated increased hepatic oxidative stress (TBARS; P </= 0.05) and prevented increases in cytochrome P450 2E1 apoprotein and mRNA and in tumor necrosis factor-alpha (TNFalpha) mRNA. Titers of auto-antibodies against proteins adducted to lipid peroxidation products were lower in serum of the NAC group than in the 70% corn oil group (P </= 0.05). NAC also decreased Picosirius red staining of collagen, a marker of fibrosis. However, markers of hepatic stellate cell activation were unaffected. Using NAC in a TEN model of NASH, we have demonstrated that NAC prevents many aspects of NASH progression by decreasing development of oxidative stress and subsequent increases in TNFalpha but does not block development of steatosis.

Figure 1. Representative Oil Red O (A,B,C), H&E (D,E,F) and Picosirius Red (G,H,I) stained liver sections from male rats overfed high fat diets with or without NAC supplementation

Representative Oil Red O stained liver sections: panels (A) C; (B) HF; and (C) HF + NAC (×20 magnification). Representative H&E-stained liver sections: panels (D) C; (E) HF; and (F) HF + NAC (×10 magnification). Representative Picosirius Red stained liver sections: panels (G) C; (H) HF; and (I) HF + NAC (×10 magnification).

Figure 2. NAC prevents increases in hepatic TBARS (A) and partially restores hepatic GSH concentrations (B) reduced by overfeeding rats a high fat diet

Values are means ± SEM, C (n = 4); HF (n = 7) and HF + NAC (n = 8). Means without a common letter differ, p≤0.05.

Figure 3. NAC supplementation reverses induction of rat hepatic CYP2E1 apoprotein (A) and hepatic CYP2E1 mRNA (B) expression following the overfeeding of a high fat diet

Values are means ± SEM, C (n = 4); HF (n = 7) and HF + NAC (n = 8). Means without a common letter differ, p≤0.05. In the representative western blot each lane represents the liver microsomal protein from individual animals.

Figure 4. Effects of overfeeding high fat with or without NAC supplementation on rat hepatic CD14 mRNA (A) and CD68/CD45 mRNA ratio (B) an indicator of leukocyte activation and infiltration

Values are means ± SEM, C (n = 4); HF (n = 7) and HF + NAC (n = 8). Means without a common letter differ, p≤0.05.

Figure 5. NAC supplementation reduces immune responses to protein adducts of malondialdehyde (A) and lipid hydroperoxides (B) in rats overfed a high fat diet

Values are means ± SEM, C (n = 4); HF (n = 7) and HF + NAC (n = 8). Means without a common letter differ, p≤0.05.

Figure 6. Effects of overfeeding high fat with or without NAC supplementation on relative mRNA expression of early markers of hepatic stellate cell activation: TGF-β (A), αSMA (B) and PDGFr-β (C)

Values are means ± SEM, C (n = 4); HF (n = 7) and HF + NAC (n = 8). Means without a common letter differ, p≤0.05.