Fig 2: CEP increased cell viability and inhibited lipid peroxidation of bEnd.3 at 24 h after RSL3 exposure, and systematic inhibition of AA metabolizing enzymes identified ALOX15 as a target for ferroptosis. (a) RSL3 decreased cell viability detected by CCK-8. *p < 0.05 vs. control, n = 6 per group. (b) The CCK-8 results depicted that CEP saves RSL3-induced cell death in a concentration-dependent manner. *p < 0.05 vs. control, #p < 0.05 vs. DMSO group, n = 6 per group. (c) Representative morphological images of bEnd.3 cells exposed to RSL3 with or without CEP. Scale bar = 20 µm. (d, e) Representative picture of BODIPY 581/591 C11 staining and quantitative fluorescence ratio analysis of green (representing lipid peroxidation)/red (conventional coloring). (f) The table clarified that inhibitors of 15-lipoxygenase and 5-lipoxygenase, but not other types of oxygenase which catalyze AA, suppressed RSL3-induced ferroptosis in bEnd.3 cells.

Fig 3: Oxylipin metabolite profile in melanoma brain metastasis mouse sera. (a) PLS-DA score plot and (b) loading plot of oxylipin profiles for Group 1: sham vs. tumor control; Group 2: tumor control vs. dLGG–10 vs. dLGG–25 vs. dLGG–10 + Avastin–5; Group 3: tumor control vs. Lipo-DOX–2 vs. Lipo-DOX–2 + Avastin–5 vs. dLGG–10 + Lipo-DOX–2. (c) Heat map showing the oxylipin metabolite intensity of sham and treated groups relative to tumor control arranged based on fatty acid substrates LA, AA, EPA, and DHA and the corresponding metabolic enzymes (n = 4 per treatment group and four technical repeats for each biological sample). (d) The summation of serum oxylipin concentration (ng/mL) derived from 5-LOX, 12/15-LOX, and CYP450 enzymes is shown. Data are mean ± SD, n = 4; Kruskal–Wallis test; * p = 0.05 compared with tumor control. (e) Representative immunofluorescence images of mouse brain tissues with positive staining of 15-LOX and CYP450-4A in melanoma cell (Mel-A) and microglia/macrophages (F4/80). Data are mean ± SD, n = 3; Kruskal–Wallis test; * p = 0.05 compared with the tumor control.

Fig 4: Schematic diagram of arachidonate metabolic cascade in liver inflammation and the intervention strategies. CCl4 activates AA metabolism and leads to increased M1-type macrophages and neutrophils, as well as elevated cytokines, chemokines and ROS levels in the liver. Oral administration of gigantol alleviates liver inflammation partly through inhibiting the JNK/cPLA2/12-LOX pathway. The image was created by using Adobe Illustrator CC 2019 (https://www.adobe.com/products/illustrator.html).

Fig 5: The sensitivity of different types of microglia to ferroptosis, and the effect of CEP on the expression of ALOX15 and GPx4 in hemin-induced BV2in vitro. (a, c) Representative images of iNOS and CD206 immunostaining in different polarization types of BV2 cells. (b, d) RSL3 decreased cell viability in M1- (b) and M2- (d) type microglia detected by CCK-8. *p < 0.05 vs. control, n = 6 per group. (e–g) Representative Western blot bands and densitometric quantification showed the protein level of ALOX15 and HO-1 in different polarization types of microglia. *p < 0.05 vs. M0 group, #p < 0.05 vs. M1 group, n = 6 per group. (h–j) Representative Western blot bands and densitometric quantification showed the effect of CEP on GPx4 and ALOX15 expression in hemin-treated M2-type microglia. *p < 0.05 vs. control, #p < 0.05 vs. hemin, n = 6 per group.