Non-alcoholic fatty liver disease (NAFLD) is a rising global burden, affecting one in four adults. Despite the increasing prevalence of NAFLD, the exact cellular and molecular mechanisms remain unclear, and effective therapeutic strategies are still limited. In vitro models of NAFLD are critical to understanding the pathogenesis and searching for effective therapies; thus, we evaluated the effects of free fatty acids (FFAs) on NAFLD hallmarks and their association with the modulation of Annexin A2 (ANXA2) and Keratin 17 (KRT17) in HepG2 cells. Our results show that oleic and palmitic acids can differentially induce intracellular lipid accumulation, cell death, and promote oxidative stress by increasing lipid peroxidation, protein carbonylation, and antioxidant defense depletion. Moreover, a markedly increased expression of inflammatory cytokines demonstrated the activation of inflammation pathways associated with lipotoxicity and oxidative stress. ANXA2 overexpression and KRT17 nuclear translocation were also observed, supporting the role of both molecules in the progression of liver disease. Taken together, these data provide insights into the interplay between ANXA2 and KRT17 in NAFLD, paving the way for understanding molecular mechanisms involved with the disease and developing new therapeutic strategies.

Hepatic steatosis is the hallmark of metabolic dysfunction-associated steatotic liver disease (MASLD), the most prevalent chronic liver disease worldwide. Lipotoxicity, resulting from excessive accumulation of fatty acids and bioactive lipid intermediates, is associated with increased oxidative stress, potentially leading to cell death. Thus, antioxidant bioactive compounds are considered promising therapeutic agents. This study characterized the phytochemical composition and evaluated the effects of an aqueous extract of Hancornia speciosa leaves (EH) on HepG2 cells with fatty acid-induced lipid accumulation. HPLC-ESI-MS/MS analysis annotated 17 compounds in EH, predominantly phenolic compounds. Exposure of HepG2 cells to oleic and palmitic acids increased lipid accumulation, cytotoxicity, and antioxidant enzyme activities. Treatment with EH at 50 (EH50) and 100 µg/mL reduced lipid accumulation and reactive oxygen species and restored catalase and glutathione S-transferase activities to control levels. Notably, only EH50 showed reduced cytotoxicity. Untargeted metabolomic profiling showed an increase in intracellular abundance of the feature annotated as C16 sphinganine following fatty acid exposure and lower abundance after EH50 treatment. These findings indicate that H. speciosa leaf extract attenuates fatty acid-induced lipid accumulation, lipotoxicity, and redox imbalance in HepG2 cells, and suggest that its protective effects may be associated with sphingolipid-related metabolism, thereby strengthening evidence for the species’ therapeutic potential and supporting further studies to elucidate the underlying mechanisms.