Oleocanthalic acid improves MASH features via a NOX1-dependent mechanism.

Metabolic dysfunction-associated steatohepatitis (MASH) is a chronic liver disease with increasing incidence. This study aimed at assessing the beneficial effects of oleocanthalic acid (OcA), extracted from extra virgin olive oil, in cellular models of MASH. OcA effects on lipid accumulation, oxidative stress, inflammation, and fibrosis were evaluated on 2D HepG2 cells treated with a palmitic and oleic acid mixture (1:2 0.1 mM) to induce steatosis, HepG2 co-cultured with THP-1-derived M1-like macrophages, multicellular spheroids (MCS) composed of HepG2 and TGFβ1-activated LX-2 cells co-cultured with macrophages, and spheroids of 3T3-L1-derived adipocytes. OcA effect was assessed by evaluating lipid droplets after Bodipy stain, reactive oxygen species (ROS), and mRNA expression by qPCR. The expression of the fibrogenic marker α-smooth muscle actin (Acta2) was assessed by ICC. OcA interaction with the pro-oxidant NADPH oxidases NOX1 and NOX4 was investigated via molecular docking simulations. OcA reduced the size and number of lipid droplets and ROS in HepG2 cells. In the MCS model, besides reducing lipid accumulation, OcA promoted a switch in macrophages towards an anti-inflammatory phenotype. OcA decreased Acta2 expression in activated LX-2 cells and prevented the LX-2 cell activation induced by the secretome of 3T3-L1-derived adipocyte spheroids. Molecular docking simulations suggested that OcA could act as a selective NOX1 inhibitor, binding to NOX1 in close contact with the FAD molecule. In conclusion, OcA demonstrated promising anti-steatotic, anti-inflammatory, and anti-fibrotic effects in different in vitro models of MASH, thus representing a potential therapeutic option for preventing lipid accumulation and fibrogenesis in MASH, with a NOX1-dependend mechanism of action.