Fig 1: EMB alleviates 5‐LOX‐overexpression‐induced ferroptosis in PC12 cells. (A–F) Western blot analysis and quantitative results of p‐5‐LOX (Ser664), p‐5‐LOX (Ser272), 5‐LOX, GPX4, and SLC7A11 expression in PC12 cells transfected with empty vector or pET‐5‐LOX (5‐LOX overexpression plasmid), then treated with LPS and/or EMB. The levels of MDA were subjected to analysis. (H–K) Immunofluorescence staining showing DHE (ROS, H), RhoNox‐1 (Fe2+, H), 5‐LOX (green, I), GPX4 (red, J), and SLC7A11 (green, K) in PC12 cells. Scale bar: 50 μm; nuclei stained with DAPI (blue). Data are mean ± SEM; n = 6/group. Statistical significance: ns, non‐significant, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 versus indicated groups. 5‐LOX, 5‐lipoxygenase; DHE, dihydroethidium; EMB, Embelin; LPS, lipopolysaccharide; MDA, malondialdehyde; pET, prokaryotic expression vector; ROS, reactive oxygen species.
Fig 2: Compared to the LPS group, EMB downregulated the expression of 5‐LOX, GPX4, and SLC7A11 in the cortical and hippocampal neurons of neonatal rats, further confirming its neuroprotective effects. (A–C) Comparison of the expression of 5‐LOX, GPX4, and SLC7A11 in neurons (NeuN+) between CA1 and the cerebral cortex in the Ctrl and LPS groups. Scale bar = 50 μm. **p < 0.01 and ****p < 0.0001 versus the indicated groups (n = 6/group). Data are presented as mean ± standard error of the mean. 5‐LOX, 5‐lipoxygenase; Ctrl, control; EMB, Embelin; LPS, lipopolysaccharide.
Fig 3: The mechanism of action of EMB involves the reduction of intracellular reactive oxygen species and ferroptosis through the inhibition of 5‐LOX expression. (A–F) Western blot analysis of p‐5‐LOX (Ser664), p‐5‐LOX (Ser272), 5‐LOX, GPX4, and SLC7A11 expression in PC12 cells among the groups (LPS, LPS + EMB, LPS + Zileuton, LPS + siRNA‐5‐LOX, LPS + siRNA‐NC). ns, no significance, *p < 0.05, **p < 0.01, and ***p < 0.001 versus the indicated groups (n = 6/group). (G) MDA analysis of PC12 cells among the groups (LPS, LPS + EMB, LPS + Zileuton, LPS + siRNA‐5‐LOX, LPS + siRNA‐NC) (n = 6/group). ns, no significance, *p < 0.05, **p < 0.01, and ***p < 0.001 versus the indicated groups (n = 6/group). (H–K) Comparison of the level of DHE, RhoNox‐1, 5‐LOX, GPX4, and SLC7A11 in PC12 cells. Scale bar = 50 μm. **p < 0.01 and ****p < 0.0001 versus the indicated groups (n = 6/group). All data are presented as mean ± standard error of the mean. 5‐LOX, 5‐lipoxygenase; DHE, dihydroethidium; EMB, Embelin; LPS, lipopolysaccharide; MDA, malondialdehyde; siRNA‐NC, non‐specific control siRNA.
Fig 4: Intracerebroventricular injection of LPS resulted in increased 5‐LOX activity, oxidative stress, and ferroptosis in the cortical and hippocampal neurons of neonatal rats, indicating an inflammatory response. (A) Transmission electron microscopy (TEM) images showing mitochondrial ultrastructure in hippocampal and cerebral cortical neurons of neonatal rats in Ctrl and LPS groups at P7. In the hippocampus (left panel) and cerebral cortex (right panel), green arrows indicate normal mitochondria in the Ctrl group, while red arrows point to abnormal mitochondria in the LPS group. Scale bar = 0.5 μm. (B–D) Comparison of the changes in ROS levels over time (P6, P7, P9) in the cerebral cortex and cortical subplate, as well as the hippocampal areas (CA1, CA2, CA3, DG) of newborn rats in the Ctrl and LPS groups. n = 6 slices per group. Scale bar = 100 μm. ****p < 0.0001 versus the indicated groups. (E–J) Comparison of the expression of 5‐LOX, GPX4, and SLC7A11 in neurons (NeuN+) between CA1 and the cerebral cortex at P6, P7, and P9. Scale bar = 50 μm. ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 versus the indicated groups (n = 6/group). Data are presented as mean ± standard error of the mean. 5‐LOX, 5‐lipoxygenase; Ctrl, control; LPS, lipopolysaccharide; ROS, reactive oxygen species.
Fig 5: Following inflammatory stimulation, EMB demonstrated the ability to suppress 5‐LOX, oxidative stress, and ferroptosis in neurons of neonatal rats, highlighting its neuroprotective potential. (A–F) Western blot analysis of p‐5‐LOX (Ser664), p‐5‐LOX (Ser272), 5‐LOX, GPX4, and SLC7A11 expression among the groups in newborn rat brain tissue (Ctrl, LPS [P6, P7, P9]). ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 versus the indicated groups (n = 6/group). (G) MDA analysis between groups (Ctrl, LPS [P6, P7, P9]) (n = 6/group). ***p < 0.001 and ****p < 0.0001 versus the indicated groups. (H–I) Comparison of the changes in ROS levels of rat (P7) brain tissue between the Ctrl group and LPS group in hippocampal areas (CA1, CA2, CA3, DG) and the cerebral cortex. Scale bar = 100 μm. ****p < 0.0001 (n = 6/group). (K–P) Western blot analysis of p‐5‐LOX (Ser664), p‐5‐LOX (Ser272), 5‐LOX, GPX4, and SLC7A11 expression between the Ctrl group and LPS group. *p < 0.05 and **p < 0.01 versus the indicated groups (n = 6/group). (Q) MDA analysis between the Ctrl and LPS groups (n = 6/group). **p < 0.01 (n = 6/group). All data are presented as mean ± standard error of the mean. 5‐LOX, 5‐lipoxygenase; Ctrl, control; EMB, Embelin; LPS, lipopolysaccharide; MDA, malondialdehyde; ROS, reactive oxygen species.
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