β-CARYOPHYLLENE REVERTS FREE FATTY ACIDS-INDUCED CELLULAR STRESS IN HEPG2 HEPATOCYTES THROUGH CB2 AND PPAR-α RECEPTORS

Nonalcoholic fatty liver disease (NAFLD) is one of the most common cause of liver disorder, defined by excessive accumulation of triglycerides in hepatocytes due to both increased ingestion of free fatty acids (FFAs) and de novo hepatic lipogenesis. The accumulation of lipids causes oxidative stress, anomalies in hepatocytes and inflammation, that may lead to the progression of NASH (nonalcoholic steatohepatitis) and liver cancer. The scientific interest for natural compounds as potential drugs has increased exponentially in the last years, along with the number of studies on nutraceuticals and herbal extracts, aimed to test their effects on many disorders, including obesity, NAFLD and also cancer. The sesquiterpene hydrocarbon (E)-β-caryophyllene (BCP), widely distributed in the plant kingdom [1], is one of the most investigated and promising natural compounds in chronic inflammation studies [2], with significant effects on reduction of lipid accumulation [3]. In our study we demonstrate its ability to revert FFA-induced steatosis and modify the lipid profile in HepG2 hepatocytes by in vitro biological assays and lipidomic analysis. To simulate the condition of steatosis, HepG2 cells were treated with palmitate and oleate (the most abundant fats in our diet), and lipid content was quantified by AdipoRed fluorescence staining. Our results demonstrate that the treatment with a 0.5 mM mixture of palmitate and oleate causes 80% increase in intracellular triglycerides, while the 24h co-treatment with 0.5 µM BCP, determines a significant reduction in triglyceride accumulation with respect to steatotic control cells, without altering the cell viability. Moreover, we show that the BCP-induced triglyceride reduction could be mediated by the cannabinoid receptor 2 (CB2) and peroxisome proliferator-activated receptor alpha (PPAR-α). It is known that trans fatty acids promote inflammation and endoplasmic reticulum stress, whereas cis-unsaturated fatty acids are protective [4]. To reveal the potential change in HepG2 cell lipid profile induced by BCP treatment, we used a lipidomic approach based on gas chromatography-mass spectrometry (GC-MS). Our GC-MS data show that co-treatment with BCP induces a reduction of palmitic and stearic acids (both saturated fatty acids), oleic acid (monounsaturated fatty acid), elaidic acid (trans-polyunsaturated fatty acid) and an increase in palmitoleic acid (monounsaturated fatty acid). Taken together these results reveal interesting and novel properties of BCP, suggesting potential applications in the reduction of trans-fatty acid accumulation and cellular damages caused by the accumulation of fats, typical condition of NAFLD. REFERENCES 1. Maffei, Int. J. Mol. Sci. 2020, 21, 6540; doi:10.3390/ijms21186540. 2. Scandiffio et al. Nutrients 2020, 12, 3273; doi:10.3390/nu12113273. 3. Geddo et al. Nutrients 2019, 11, 2788; doi:10.3390/nu11112788. 4. Oteng et al. Adv. Nutr. 2020, 11(3):697-708; doi: 10.1093/advances/nmz125.