β-Caryophyllene reverts free fatty acids-induced steatosis 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. The accumulation of lipids causes oxidative stress and inflammation, that may lead to the progression of NASH (nonalcoholic steatohepatitis). The scientific interest for natural compounds has increased exponentially in the last years, along with the number of studies on nutraceuticals, aimed to test their effects on many disorders, including obesity, NAFLD and also cancer. The sesquiterpene (E)-β-caryophyllene (BCP), widely distributed in the plant kingdom1 , is one of the most promising natural compounds in chronic inflammation studies2 , with significant effects on reduction of lipid accumulation3 . In our study we demonstrate its ability to revert free fatty acid (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, and lipid content was quantified by fluorescent 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. 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 protective4. To reveal the potential change in HepG2 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 cotreatment with BCP induces a reduction of palmitic and stearic acids, oleic acid, elaidic 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. 2 Scandiffio et al. Nutrients, 2020, 12, 3273. 3 Geddo et al. Nutrients, 2019, 11, 2788. 4 Oteng et al. Adv. Nutr., 2020, 11(3):697-708.