Oleocanthalic acid extracted form extravirgin olive oil reduces fatty acid accumulation and fibrogenesis in experimental in vitro models of NAFLD and NASH

Introduction The extravirgin olive oil (EVOO) phenol oleocanthal was demonstrated to exert an antifibrotic effect in preclinical models, counteracting the upregulation of NADPH oxidases, proinflammatory cytokines and metalloproteinases. Its mono-oxidized derivative oleochantalic acid (OcA) has recently been identified in aged EVOO, and limited information is available about its biological activities. Aim to assess the effect of OcA against fibrogenesis and lipid accumulation in 2D and 3D cellular models. Materials and Methods The effect of 24-h OcA treatment on fatty acid (FA) uptake was assessed in a 2D model of NAFLD obtained treating HepG2 cells with palmitic acid/oleic acid (PA/OA1:1, 0.1 mM) and in a 3D spheroid coculture of HepG2 and LX-2 cells treated with PA/OA by means of Bodipy and Nile red stain, respectively. To assess the effect of OcA on FA uptake in an inflammatory NASH-like microenvironment, PA/OA-treated HepG2 cells were co-cultured with THP-1-derived M1 proinflammatory macrophages. The effect of 24-h OcA treatment on fibrogenesis was also evaluated in LX-2 cells activated with TGFb1 (2ng/mL) and in cocultures of HepG2 and THP-1 derived M0 macrophages. aSMA expression, marker of LX-2 activation, was assessed by ICC. Results OcA significantly reduced the uptake of FAs in PA/OA-treated HepG2 cells in a dose dependent manner. The same effect (p<0.0001) could be observed in HepG2 cells cocultured with proinflammatory M1 macrophages, mimicking a NASH microenvironment, as well as in 3D spheroid coculture of HepG2 and LX-2 cells treated with PA/OA (p<0.01). Moreover, OcA significantly reduced LX-2 cell activation induced by both TGFb1 treatment and by coculture with HepG2 and M0 macrophages (p<0.0001). Conclusions This preliminary in vitro evaluation of OcA demonstrated a significant improvement of FA accumulation and HSC activation, which are both pivotal mechanisms in the development of NASH.