Effects of genistein on non-alcoholic steatohepatitis (NASH) induced by high-fat high-fructose diet in bilateral ovariectomized rats

Abstract

To investigate the effects of genistein on estrogen deficiency with high-fat high-fructose (HFHF) diet-induced NASH rats. Female Sprague-Dawley rats (n=48) were randomly divided into ovariectomized (OVX) and non-ovariectomized (non-OVX) group. Both rat groups, further allocated into 3 subgroups; controls, rats fed with HFHF diet (NASH), and rats fed with HFHF diet plus daily 16 mg/kg BW of genistein (NASH+Gen) for 8 weeks. Serum samples were collected for liver enzymes (AST and ALT) and TNF-α level analysis. Liver tissue samples were harvested for histopathology, hepatic lipid accumulation (oil red o stained, FFA and TG level), hepatic lipid peroxidation (MDA level), hepatic inflammation (TNF-α and NF-kB) and hepatocytes apoptosis (TUNELs) examination. Protein expression of PPARγ, adiponectin, and estrogen receptor subtypes were analyzed by western blot. Rats fed with HFHF showed typical histopathology of NASH in both non-OVX and OVX groups as demonstrated by significantly increased of all histological feature scores of Brunt’s criteria when compared with controls. The most severe histopathological damage was observed in OVX rats with NASH group. Hepatic lipid accumulations (ORO, TG, and FFA) were augmented in both non-OVX and OVX rats fed with HFHF. These augmentation were significantly higher in NASH rats with OVX than non-OVX. Additionally, inflammatory markers; serum TNF-α, hepatic MDA, and NF-kB, were enhanced in diet-induced NASH with both non-OVX and OVX groups. Moreover, hepatocytes apoptosis, and hepatic PPARγ expression were increased in both non-OVX and OVX rats with NASH. Interestingly, similarly levels of hepatic MDA, FFA, PPARγ, and adiponectin were observed between control and NASH with OVX groups. These might be the influence of estrogen deficiency and suggested the impact of estrogen deficiency in NASH pathogenesis. Estrogen receptors (ERα and ERβ) expression in liver displayed the opposite pattern after induced to NASH, since, ERα increased and ERβ decreased in NASH rats of both non-OVX and OVX. NASH rats that received daily 16 mg/kg BW of genistein improved the histopathological damage in both non-OVX and OVX as exhibited significantly lowered of all histological features scores. Furthermore, genistein significantly attenuated the inflammation, lipid accumulation, oxidative stress, and hepatocyte apoptosis in both non-OVX and OVX rats with NASH. Genistein displayed the properties as PPARγ antagonist and adiponectin agonist in NASH rats. In both non-OVX and OVX rats, the expression of ERα was declined with genistein administration as compared to the NASH groups, whereas, ERβ expression was not altered. In conclusion, HFHF diet is able to develop NASH pathogenesis and could be intensified by estrogen deficiency, suggesting the protective effect of estrogen on NASH. Genistein administration may protect NASH in both non-OVX and OVX rats.