Schisandrin A alleviates neuronal ferroptosis in mice with hypoxic-ischemic brain injury by inhibiting the ACSL4/TFRC/PTGS2 signaling pathway

doi:10.12122/j.issn.1673-4254.2026.10.09

Abstract

Abstract:

Objective To investigate the inhibitory effect of schisandrin A (Sch A) on neuronal ferroptosis following hypoxic ischemic brain damage (HIBD) and the underlying mechanism. Methods Thirty-six neonatal C57BL/6J mice (7-10 days old) underwent sham operation (n=6) or permanent left carotid ligation followed by 40 min of hypoxic exposure and daily treatment with Sch A at 10, 30, or 90 mg/kg or with Fer 1 (5 mg/kg) for 3 consecutive days (n=6). Western blotting was used to detect protein expressions of ACSL4, TFRC, and PTGS2 in the cortical ischemic penumbra of the mice; ACSL4/4 HNE co localization in the neurons was detected by immunofluorescence staining, and the levels of ROS, MDA, GSH, and Fe²⁺ were quantified. In cultured HT22 hippocampal neurons with oxygen glucose deprivation (OGD), the effects of pretreatment (1 h prior to OGD) with Sch A (10 μmol/L) and PRGL493 (10 μmol/L), alone or in combination, were assessed by detecting expressions of ferroptosis related proteins, ROS production, lipid peroxidation, cell viability, mitochondrial membrane potential, and the levels of MDA, GSH, and Fe²⁺. Results In the neonatal mice, HIBD significantly upregulated ACSL4/TFRC/PTGS2, increased ACSL4/4 HNE co-localization and ROS, MDA, and Fe²⁺ levels, and decreased GSH in the cortical ischemic penumbra. Sch A dose-dependently reversed these changes with effects comparable to that of Fer-1. In cultured HT22 cells, OGD similarly upregulated these proteins, enhanced ACSL4/NeuN co-localization, increased ROS, lipid peroxidation, MDA, and Fe²⁺ levels, and reduced GSH, cell viability, and mitochondrial potential. Both Sch A and PRGL493 alone significantly ameliorated these changes, and their combined treatment further downregulated ACSL4, TFRC and PTGS2 expressions, more effectively alleviated oxidative stress and lipid peroxidation, restored cell viability and mitochondrial function, and reduced Fe²⁺ accumulation. Conclusion Sch A protects against HIBD in mice by inhibiting the ACSL4/TFRC/PTGS2 pathway and alleviating neuronal ferroptosis and produces synergistic effects with PRGL493.

Key words: schisandrin A, hypoxic-ischemic brain damage, ferroptosis, neurons, ACSL4/TFRC/PTGS2

Tao GUO, Jiahao HE, Yuexin DONG, Zhao WANG, Hanjun ZUO, Yanli ZHAO, Jinsha SHI, Min ZHAO, Juanjuan LI. Schisandrin A alleviates neuronal ferroptosis in mice with hypoxic-ischemic brain injury by inhibiting the ACSL4/TFRC/PTGS2 signaling pathway[J]. Journal of Southern Medical University, 2026, 46(10): 2393-2404.

Figures/Tables 6

Fig.1 Effects of schisandrin A (Sch A) on expressions of ferroptosis-related proteins ACSL4, TFRC and PTGS2 in neonatal mice with hypoxic ischemic brain damage (HIBD). A: Western blotting of ACSL4, TFRC, and PTGS2 in the ischemic penumbra of the cerebral cortex of the neonatal mice in each group. B-D: Statistical analysis of the relative expression levels. ***P<0.001 vs Sham group; #P<0.05, ##P<0.01, ###P<0.001 vs HIBD group (n=3). L: Low; M: Medium; H: High; Fer-1: Ferrostatin-1.

Fig.2 Effects of Sch A on expressions of ACSL4 and 4-HNE in brain neurons in mice with HIBD. A, D: Co-localization of ACSL4 or 4-HNE (red) with the neuronal marker NeuN (green) in the ischemic penumbra of the cerebral cortex detected by immunofluorescence double staining. B, C: Quantitative analysis of fluorescence intensity of ACSL4-NeuN and 4-HNE-NeuN. Scale bar=10 μm. ***P<0.001 vs Sham group; ##P<0.01, ###P<0.001 vs HIBD group (n=3).

Fig.3 Effects of Sch A on oxidative stress and iron overload following HIBD. A, B: ROS levels in the ischemic penumbra of mouse cerebral cortex detected by DHE fluorescence staining and quantitative analysis (Scale bar=10 μm). C: Relative expression levels of malondialdehyde (MDA). D: Relative expression levels of glutathione (GSH). E: Levels of ferrous ions (Fe²⁺). F: Ultrastructure of the neurons in the ischemic penumbra in each group observed by transmission electron microscopy (Original magnification: ×30 000; scale bar=500 nm). Red arrows indicate mitochondria. ***P<0.001 vs Sham group; #P<0.05, ##P<0.01, ###P<0.001 vs HIBD group (n=3).

Fig.4 Effects of Sch A combined with the ACSL4 inhibitor PRGL493 on OGD‑induced neuronal ferroptosis. A: Western blotting of ACSL4, TFRC, and PTGS2 protein expression levels in the neurons of each group. B-D: Statistical analysis of the relative expression levels of ACSL4, TFRC, and PTGS2 proteins. E, F: Immunofluorescence double staining for detecting co-localization of ACSL4 (red) with the neuronal marker NeuN (green). Scale bar=50 μm. ***P<0.001 vs Control group; ##P<0.01, ###P<0.001 vs OGD group; &P<0.05, &&&P<0.001 vs OGD+Sch A group (n=3). OGD: Oxygen-Glucose Deprivation.

Fig.5 Effects of Sch A on OGD induced neuronal oxidative stress and lipid peroxidation. A, B: ROS levels in the neurons detected by DCFH DA fluorescence staining. C, D: Oxidized/reduced lipid peroxidation levels in the neurons detected by BODIPY C11 fluorescence staining. E: Relative expression levels of MDA. F: Relative expression levels of GSH. Scale bar=50 μm. ***P<0.001 vs Control group; #P<0.05, ##P<0.01, ###P<0.001 vs OGD group; &P<0.05, &&P<0.01 vs OGD+Sch A group (n=3). MDA: Malondialdehyde; GSH: Glutathione.

Fig.6 Effects of Sch A on OGD induced neuronal viability, mitochondrial function, and iron accumulation. A: Neuronal viability detected by CCK-8 assay. B, C: Fluorescence staining of mitochondrial membrane potential detected by JC-1 staining. D, E: Neuronal iron ion levels detected by Fe²⁺ fluorescence staining. Scale bar=50 μm. ***P<0.001 vs Control group; #P<0.05, ##P<0.01, ###P<0.001 vs OGD group; &P<0.05, &&P<0.01 vs OGD+Sch A group (n=3).

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