Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of biologically active lipids with anti-inflammatory and anti-diabetic properties. Despite the possible link between endogenous FAHFA levels and nuclear factor erythroid 2-related factor 2 (Nrf2), their possible function as antioxidants and the mechanisms involved in this are unknown. Here, we investigate FAHFAs’ plausible antioxidant potential with reference to their effect on the Nrf2 levels, oxidative stress, and lipid droplet oxidation in human hepatocytes (C3A). Six authentic FAHFAs were chemically synthesized and performed activity-based screening by reporter gene assay. Among them, eicosapentaenoic acid (EPA) esterified 12-hydroxy stearic acid (12-HSA) and 12-hydroxy oleic acid (12-HOA) FAHFAs showed less cytotoxicity compared to their free fatty acids and potent activators of Nrf2. To define their mode of action, relative levels of nuclear Nrf2 were determined, which found a higher amount of Nrf2 in nucleus of cells treated with 12-EPAHSA compared to the control. Furthermore, 12-EPAHSA increased the expression of Nrf2-dependent antioxidant enzyme genes (NQO1, GCLM, GCLC, SOD-1, and HO-1). Fluorescence imaging analysis of linoleic-acid-induced lipid droplets (LDs) in C3A cells treated with 12-EPAHSA revealed the strong inhibition of small-size LD oxidation. These results suggest that EPA-derived FAHFAs as a new class of lipids with less cytotoxicity, and strong Nrf2 activators with plausible antioxidant effects via the induction of cytoprotective proteins against oxidative stress, induced cellular damage.

Nonalcoholic steatohepatitis (NASH) is a prevalent disease related to lipid metabolism disorder and oxidative stress. Lipid hydroperoxidation is known to be a critical driving force of various disorders and diseases. However, the combination of both intact and hydroperoxidized lipids in NASH has not yet been studied. In this work, the liver and kidney samples from NASH-model mice were comprehensively investigated by using the LC/MS-based lipidomic analysis. As a result, triglycerides showed the amount accumulation and the profile alteration for the intact lipids in the NASH group, while phosphatidylethanolamines, lysophosphatidylethanolamines, plasmalogens, and cardiolipins largely depleted, suggesting biomembrane damage and mitochondria dysfunction. Notably, the lipid hydroperoxide species of triglyceride and phosphatidylcholine exhibited a significant elevation in both the liver and the kidney of the NASH group and showed considerable diagnostic ability. Furthermore, the relationship was revealed between the lipid metabolism disturbance and the lipid hydroperoxide accumulation, which played a key role in the vicious circle of NASH. The present study suggested that the omics approach to the lipid hydroperoxide profile might be the potential diagnostic marker of NASH and other oxidative stress-related diseases, as well as the evaluative treatment index of antioxidants.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a new class of endogenous lipids with interesting physiological functions in mammals. Despite their structural diversity and links with nuclear factor erythroid 2-related factor 2 (NRF2) biosynthesis, FAHFAs are less explored as NRF2 activators. Herein, we examined for the first time the synthetic docosahexaenoic acid esters of 12-hydroxy stearic acid (12-DHAHSA) or oleic acid (12-DHAHOA) against NRF2 activation in cultured human hepatoma-derived cells (C3A). The effect of DHA-derived FAHFAs on lipid metabolism was explored by the nontargeted lipidomic analysis using liquid chromatography-mass spectrometry. Furthermore, their action on lipid droplet (LD) oxidation was investigated by the fluorescence imaging technique. The DHA-derived FAHFAs showed less cytotoxicity compared to their native fatty acids and activated the NRF2 in a dose-dependent pattern. Treatment of 12-DHAHOA with C3A cells upregulated the cellular triacylglycerol levels by 17-fold compared to the untreated group. Fluorescence imaging analysis also revealed the suppression of the degree of LDs oxidation upon treatment with 12-DHAHSA. Overall, these results suggest that DHA-derived FAHFAs as novel and potent activators of NRF2 with plausible antioxidant function.

Lipid droplet accumulation (LDA) in hepatocytes is the initial stage of nonalcoholic fatty liver disease (NAFLD). In the search for natural compounds for the prevention of NAFLD, a series of β-carboline alkaloid derivatives, inspired by flazin and its derivative, newly identified in Crassostrea gigas Thunberg. extracts, were examined for LDA inhibition (LDAI) activity in oleic acid–loaded hepatocytes (HepG2). Eight compounds with a piperidine or pyridine C-ring were chemically synthesized (1–8). Among them, compounds 2 and 4 (flazin) with a carboxy group at C-3 and furfuryl alcohol moiety at C-1 showed low cytotoxicity and they exhibited significant LDAI activity. Compound 2 with piperidine C-ring was identified for the first time in C. gigas extract, and ameliorated the lipid accumulation with the LDAI value of 25.4%. Active compounds 2 and 4 significantly inhibited triacylglycerol species accumulation in cells. These compounds upregulated ATGL and downregulated SREBP1, FASN, and SCD1 genes, suggesting that they activated lipolysis and suppressed lipogenesis, respectively. These results suggest that β-carboline alkaloids, especially compounds 2 and 4, might be potentially useful for preventing NAFLD.

Targeting bioactive compounds to prevent lipid droplet accumulation in the liver, we explored an antioxidative extract from vanilla bean (Vainilla planifolia) after chemo-selective derivatization through heating and acid modification. The chemical analysis of vanilla bean extract through chemoselective derivatization resulted in the identification of sixteen compounds (34–50) using LC-MS/MS analysis. A β-carboline alkaloid with a piperidine C-ring and a vanillin moiety at C-1 (34) was identified by molecular networking and diagnostic fragmentation filtering approaches. β-carboline alkaloid 34 exhibited significant inhibitory activity of lipid droplet accumulation (LDAI) in oleic acid-loaded hepatocellular carcinoma HepG2 cells. The LDAI activity was associated with both activation of lipolysis and suppression of lipogenesis in the cells. The study indicates that crude plant extracts, following chemoselective derivatization, may contain bioactive compounds that could be beneficial in preventing hepatosteatosis and could serve as a source of lead compounds for drug development. This approach may be useful to investigate other mixtures of natural products and food resources.