S-nitroso-N-acetylcysteine attenuates liver fibrosis in experimental nonalcoholic steatohepatitis

Abstract

S-Nitroso-N-acetylcysteine (SNAC) is a water soluble primary S-nitrosothiol capable of transferring and releasing nitric oxide and inducing several biochemical activities, including modulation of hepatic stellate cell activation. In this study, we evaluated the antifibrotic activity of SNAC in an animal model of nonalcoholic steatohepatitis (NASH) induced in Sprague-Dawley rats fed with a choline-deficient, high trans fat diet and exposed to diethylnitrosamine for 8 weeks. The rats were divided into three groups: SNAC, which received oral SNAC solution daily; NASH, which received the vehicle; and control, which received standard diet and vehicle. Genes related to fibrosis (matrix metalloproteinases [MMP]-13, -9, and -2), transforming growth factor β-1 [TGFβ-1], collagen-1α, and tissue inhibitors of metalloproteinase [TIMP-1 and -2] and oxidative stress (heat-shock proteins [HSP]-60 and -90) were evaluated. SNAC led to a 34.4% reduction in the collagen occupied area associated with upregulation of MMP-13 and -9 and downregulation of HSP-60, TIMP-2, TGFβ-1, and collagen-1α. These results indicate that oral SNAC administration may represent a potential antifibrotic treatment for NASH.

Keywords: NASH; S-nitroso-N-acetylcysteine; diethylnitrosamine; fibrogenesis; nitric oxide.

Figure 1

Scheme of the study design describing the three groups of animals treated over eight weeks with: standard diet and oral vehicle (control group); choline-deficient high fat diet plus diethylnitrosamine (CDHFD + DEN) and oral vehicle (nonalcoholic steatohepatitis group); and CDHFD + DEN and oral S-nitroso-N-acetylcysteine (SNAC) solution (1.4 mg/kg) (SNAC group). Abbreviations: CDHFD, choline-deficient high fat diet; DEN, diethylnitrosamine; NASH, nonalcoholic steatohepatitis; SNAC, S-nitroso-N-acetylcysteine.

Figure 2

(A and B) Ultraviolet-visible spectral changes associated with the formation of S-nitroso-N-acetylcysteine (SNAC) through the S-nitrosation of N-acetylcysteine (NAC) with nitrous acid showing the two absorption bands characteristic of primary S-nitrosothiols with maxima at 336 and 545 nm. The concentration of the SNAC solution is 1 and 40 mM, respectively. (C) Kinetic curve of SNAC formation during the S-nitrosation of NAC based on the absorption band of SNAC at 545 nm. Inset: SNAC structure. (D) Kinetic curve of SNAC decomposition in simulated gastric fluid at a concentration of 1 mM, pH 1.5 and 37°C over 4 hours based on the absorption band of SNAC at 336 nm. Note: *Exited state of a molecular orbital.

Figure 3

Representative picrosirius red staining images of hepatic histology of (A) control, (B) nonalcoholic steatohepatitis (NASH), and (C) S-nitroso-N-acetylcysteine (SNAC) treated animals. Magnification is × 200. (D) Collagen quantification of the control, SNAC, and NASH groups.

Figure 4

Representative nitrotyrosine immunostaining of liver sections. Notes: Absent nitrotyrosine staining or hepatocyte injury is seen in the control group (A). Intense positivity is seen in both nonalcoholic steatohepatitis (NASH) (B) and S-nitroso-N-acetylcysteine (SNAC) (C) groups. (D) Nitrotyrosine immunostaining quantification of the NASH and SNAC groups.

Figure 5

Fold change in matrix metalloproteinase (MMP)-13 and -9, and tissue inhibitor of metalloproteinases (TIMP)-1 and -2 mRNA expression in the nonalcoholic steatohepatitis groups against S-nitroso-N-acetylcysteine groups. Abbreviations: NASH, nonalcoholic steatohepatitis; SNAC, S-nitroso-N-acetylcysteine.

Figure 6

Fold change in heat-shock protein (HSP)-60 and -90, transforming growth factor β-1, and collagen 1α mRNA expression in the nonalcoholic steatohepatitis groups against S-nitroso-N-acetylcysteine groups. Abbreviations: NASH, nonalcoholic steatohepatitis; SNAC, S-nitroso-N-acetylcysteine.