Zingerone attenuates steatosis via downregulation of AQP9 in hepatocytes

Zingerone (ZO) found in Zingiber officinale, belonging to methoxyphenol family and its related derivatives, has been reported to ameliorate nonalcoholic fatty liver disease (NAFLD) by enhancing lipid metabolism and exerting hepatoprotective effects. However, the effect of ZO on hepatic AQP9 expression, along with the underlying mechanisms driving its hepatoprotective potential, remains largely unexplored. This study aims to investigate the modulatory effect of ZO on AQP9 expression in a steatosis cell model through experimental and in silico analyses, providing insights that could contribute to therapeutic interventions for NAFLD. Oleate-palmitate (OA-PA) and free fatty acids (FFA) induced hepatic steatosis was established in Hepa 1-6 cells as in vitro model of NAFLD. Cell cytotoxicity and lipid accumulation were evaluated by MTT assay and Oil-RedO staining, respectively. ZO pretreatment significantly attenuated intracellular lipid accumulation and altered the expression of aquaporins, resulting in downregulation of AQP9 and upregulation of AQP3 and AQP7, as demonstrated by qRT-PCR analysis. These findings suggest that ZO ameliorates OA-PA induced hepatic steatosis primarily by inhibiting lipid accumulation and selectively modulating aquaporin expression. Molecular docking analysis revealed that ZO forms a complex with AQP9 with the minimum binding energy of -6.2 kcal/mol. The molecular dynamics simulations showed that this complex stays stable across time with consistent RMSD and low RMSF values. In summary, pre-treatment with ZO has beneficial health effects by inhibiting lipid accumulation and selectively modulating aquaporin expression, highlighting its potential in preventing fatty liver disease.