Oral administration of S-nitroso-N-acetylcysteine prevents the onset of non alcoholic fatty liver disease in rats

Abstract

Aim: To evaluate the potential of S-nitroso-N-acetylcysteine (SNAC) in inhibition of lipid peroxidation and the effect of oral SNAC administration in the prevention of nonalcoholic fatty liver disease (NAFLD) in an animal model.

Methods: NAFLD was induced in Wistar male rats by choline-deficient diet for 4 wk. SNAC-treated animals (n=6) (1.4 mg/kg per day of SNAC, orally) were compared to 2 control groups: one (n=6) received PBS solution and the other (n=6) received NAC solution (7 mg/kg per day). Histological variables were semiquantitated with respect to macro and microvacuolar fat changes, its zonal distribution, foci of necrosis, portal and perivenular fibrosis, and inflammatory infiltrate with zonal distribution. LOOHs from samples of liver homogenates were quantified by HPLC. Nitrate levels in plasma of portal vein were assessed by chemiluminescence. Aqueous low-density lipoprotein (LDL) suspensions (200 microg protein/mL) were incubated with CuCl(2) (300 micromol/L) in the absence and presence of SNAC (300 micromol/L) for 15 h at 37 degree Celsius. Extent of LDL oxidation was assessed by fluorimetry. Linoleic acid (LA) (18.8 micromol/L) oxidation was induced by soybean lipoxygenase (SLO) (0.056 micromol/L) at 37 degree Celsius in the presence and absence of N-acetylcysteine (NAC) and SNAC (56 and 560 micromol/L) and monitored at 234 nm.

Results: Animals in the control group developed moderate macro and microvesicular fatty changes in periportal area. SNAC-treated animals displayed only discrete histological alterations with absence of fatty changes and did not develop liver steatosis. The absence of NAFLD in the SNAC-treated group was positively correlated with a decrease in the concentration of LOOH in liver homogenate, compared to the control group (0.7+/-0.2 nmol/mg vs 3.2+/-0.4 nmol/mg protein, respectively, P<0.05), while serum levels of aminotransferases were unaltered. The ability of SNAC in preventing lipid peroxidation was confirmed in in vitro experiments using LA and LDL as model substrates.

Conclusion: Oral administration of SNAC prevents the onset of NAFLD in Wistar rats fed with choline-deficient diet. This effect is correlated with the ability of SNAC to block the propagation of lipid peroxidation in vitro and in vitro.

Figure 1

Histological features of liver tissue of rats fed with choline-deficient diet. A: Control group showing a moderate macro and microvacuolar steatosis in periportal zone; B: SNAC-treated animals showing normal liver in periportal zone (hematoxylin-eosin stain-HE); C: Control group showing positive Scharlach staining; D: SNAC-treated animals showing negative Scharlach staining.

Figure 2

Concentration of hydroperoxides (LOOH) in liver homogenates of the control group, NAC and SNAC-treated animals.

Figure 3

Emission spectra of human LDL (200 μg/mL) suspended in aerated PBS. A: Freshly prepared suspension; B: after incubation with CuCl₂ (300 μmol/L) for 15 h; C: after co-incubation with CuCl₂ (300 μmol/L) and SNAC (300 μmol/L). The excitation and emission wavelengths were 360 and 433 nm, respectively.

Figure 4

Kinetic curves of linoleic acid (18.76 μmol/L) peroxidation catalyzed by SLO (A) (0.056 μmol/L), SLO co-incubated with NAC (B) (560 μmol/L), SLO co-incubated with SNAC (C) (56 μmol/L) and SLO co-incubated with SNAC (D) (560 μmol/L). Absorbance changes were monitored at 234 nm at 37 °C.

Figure 5

Barr graph showing the extent (Ext) and initial rates (V₀) of the peroxidation reaction of linoleic acid (LA) by SLO. Data were extracted from the curves of Figure 4.