Effects of apocynin and natural antioxidant from spinach on inducible nitric oxide synthase and cyclooxygenase-2 induction in lipopolysaccharide-induced hepatic injury in rat

The immunoreactivity of inducible nitric oxide synthase, and cyclooxygenase-2 was compared among groups of male Wistar rats comprising those injected with lipopolysaccharide following pretreatment with either natural antioxidant from spinach or the antioxidant apocynin, with lipopolysaccharide without pretreatment with antioxidants, with each of the two antioxidants alone, and untreated controls. The grade of staining of both inducible nitric oxide synthase and cyclooxygenase-2 increased with the severity of the inflammatory reaction in the lipopolysaccharide-treated animals, compared to the antioxidant-treated groups. Interpretation of the results of the immunostained tissues indicated that pretreatment with either antioxidant significantly (P<0.05) attenuated the lipopolysaccharide-stimulated inducible nitric oxide synthase induction. Analysis of the cycloxygenase-2-stained liver samples indicated that the pretreatment with the natural antioxidant NAO significantly (P<0.05) attenuated lipopolysaccharide-stimulated cycloxygenase-2 induction; whereas, in animals pretreated with apocynin, there was a trend of reduction in the cyclooxygenase-2 expression, but not statistically significant (P>0.05). The negative nitrotyrosine immunoreactivity of the lipopolysaccharide-related hepatic lesions may indicate that there was relatively low interaction between superoxide anions and nitric oxide to form peroxynitrite or that the expression levels of the nitrotyrosine were below the limit of detection. In all treatment groups a positive correlation (P<0.05, r=0.86) found between the inducible nitric oxide synthase and cyclooxygenase-2 scores suggests a strong relationship between these two parameters. The results indicate the possible therapeutic efficacy of NAO and apocynin in the prevention of liver damage related to clinical endotoxemia known to be associated with oxidative stress.