The protective effect of ACL can be inhibited by hmox1a knockout in zebrafish. (A) Zebrafish hmox1a mRNA level in control, FAC, FAC + ACL treated wildtype or hmox1a−/− larvae were analyzed by qPCR, * p < 0.05. (B,C) ROS production of embryos (5dpf) from two zebrafish lines, wildtype and hmox1a−/− larvae treated with FAC, FAC + ACL and FAC + ACL + Fer1 were detected and quantified by staining with DCF-DA, * means p < 0.05 between two indicated groups (n = 10). (D,E) Determination of mRNA expression level of ferroptosis-related genes in 5 dpf zebrafish larvae or hmox1a−/− larvae treated with 200 µg/mL FAC with or without 0.5 mg/L ACL. (F) Calcein staining images of wildtype and hmox1a−/− zebrafish larvae (7 dpf) treated with FAC, FAC + ACL. (G,H) The fluorescence intensity of vertebrae of zebrafish larvae in different groups were quantified and compared. * means p < 0.05 between two indicated groups (n = 15). (I–K) The relative mRNA expression of zebrafish osteogenesis related genes, spp1, bglap and runx2a, in wildtype and hmox1a−/− zebrafish larvae treated with FAC, FAC + ACL were determined by qPCR. The mean value of FAC treated wildtype group was set to 1.0. The concentrations used in the experiments were as follows: FAC (200 μg/mL), ACL (0.5 mg/L), and Fer1 (1 μM), * means p < 0.05 between two indicated groups (n = 6). Data are displayed as mean ± SD.
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