BETA-CARYOPHYLLENE AS A THERAPEUTIC AGENT IN 2D AND 3D IN VITRO MODELS OF HEPATIC STEATOSIS: POTENTIAL INVOLVEMENT OF EXTRACELLULAR VESICLES

Non-alcoholic fatty liver disease (NAFLD) is a prevalent liver disorder characterized by accumulation of triglycerides within hepatocytes, leading to oxidative stress and inflammation. Despite its high prevalence, there is currently no approved pharmacological therapy specifically targeting NAFLD.1 In this study, we explored the therapeutic potential of (E)-β- caryophyllene (BCP), a natural product derived from several plants. To investigate the effects of BCP, we developed 2D and 3D in vitro models of liver steatosis using HepG2 human hepatoma cells. Steatosis was induced by incubating both spheroids and monolayer cells with a pathophysiological concentration of a free fatty acid mixture (FFAm) in serum-free MEM for 24 h. Through a fluorescent-based lipid quantification assay and gas chromatography-mass spectrometry (GC-MS) analysis, we demonstrated that BCP effectively decreases lipid accumulation in steatotic conditions. Notably, BCP altered the typical steatotic lipid profile by specifically reducing saturated fatty acids. Pharmacological studies revealed that BCP action is mediated by multiple receptors: CB2 cannabinoid receptor, peroxisome proliferator-activated receptor α (PPARα), and γ (PPARγ). Moreover, we observed that BCP is internalized by HepG2 cells, with a peak uptake occurring at 2 h, suggesting direct interaction of BCP with intracellular receptors. Since stressed hepatocytes can modify the released Extracellular Vesicles (EVs), which play a crucial role in intercellular communication, we isolated HepG2- derived EVs from spheroid conditioned media using high-speed centrifugation. Interestingly, nanoparticle tracking analysis (NTA) showed that BCP treatment significantly reduced the size and the number of released EVs in steatotic conditions. In conclusion, this study highlights BCP as a promising candidate for NAFLD therapy, implicating its multifaceted effects on lipid metabolism and intercellular communication via EVs. I