Gastrodin inhibits ferroptosis to alleviate hypoxic-ischemic brain damage in neonatal mice by activating GPX4/SLC7A11/FTH1 signaling

doi:10.12122/j.issn.1673-4254.2025.10.03

Abstract

Abstract:

Objective To evaluate the therapeutic effect of gastrodin against hypoxic-ischemic brain damage (HIBD) in neonatal mice and explore the role of GPX4/SLC7A11/FTH1 signaling in mediating its effect. Methods Twenty-four 9- to 11-day-old C57BL/6J mice were randomized equally into 4 groups for sham operation, HIBD modeling by right common carotid artery ligation and subsequent exposure to hypoxia for 1 h, or gastrodin treatment at 100 or 200 mg/kg before and at 1 and 2 days after modeling. The mice then underwent neurological assessment (Zea-Longa scores), and the cerebral cortical penumbra tissue were collected for HE and Nissl staining, detection of ferroptosis biomarkers and protein expressions of GPX4, SLC7A11, and FTH1 with Western blotting and immunofluorescence co-localization, and observation of mitochondrial ultrastructure with electron microscopy. In cultured HT22 neuronal cells with oxygen-glucose deprivation (OGD) for 2 h, the effects of pretreatments with 0.5 mmol/L gastrodin, 10 μmol/L RSL3 (a GPX4 inhibitor), alone or in combination, were analyzed on expressions of ferroptosis-related proteins, cellular Fe²⁺, ROS, lipid peroxidation, MDA, and GSH levels, mitochondrial membrane potential (JC-1), and cell viability. Results Gastrodin treatment at the two doses both significantly ameliorated HIBD and neurological deficits of the mice, reduced mitochondrial damage and Fe²⁺, MDA and ROS levels, increased GSH level, and upregulated GPX4, SLC7A11, and FTH1 protein expressions. In HT22 cells, gastrodin pretreatment obviously attenuated OGD-induced ferroptosis and improved cell viability and mitochondrial function. Co-treatment with RSL3 potently abrogated the inhibitory effects of gastrodin on Fe²⁺, ROS, BODIPY-C11, and MDA levels and attenuated its protective effects on GSH level, cell viability, and mitochondrial membrane potential. Conclusion Gastrodin provides neuroprotective effects in neonatal mice with HIBD by suppressing neuronal ferroptosis via upregulating the GPX4/SLC7A11/FTH1 signaling pathway.

Key words: gastrodin, hypoxic-ischemic brain damage, oxygen-glucose deprivation, ferroptosis, GPX4/SLC7A11/FTH1, neurons

Tao GUO, Bolin CHEN, Jinsha SHI, Xianfeng KUANG, Tengyue YU, Song WEI, Xiong LIU, Rong XIAO, Juanjuan LI. Gastrodin inhibits ferroptosis to alleviate hypoxic-ischemic brain damage in neonatal mice by activating GPX4/SLC7A11/FTH1 signaling[J]. Journal of Southern Medical University, 2025, 45(10): 2071-2081.

Figures/Tables 6

Fig.1 Gastrodin produces neuroprotective effects in neonatal mice with HIBD. A: Representative images of HE staining. B, C: Representative images and count statistics of Nissl staining. D: Zea-Longa method for evaluating nerve function injury score. Scale bar=50 μm. ***P<0.001 vs Sham group; #P<0.05, ##P<0.01, ###P<0.001 vs HIBD group (n=6).

Fig.2 Effects of gastrodin on Fe2+, MDA, GSH and DHE levels in neonatal mice after HIBD. A-C: Relative levels of ferrous iron, MDA and GSH in different groups. D, E: Quantitative analysis and representative images of fluorescence intensity of DHE (Scale bar=10 μm). F: Representative images of mitochondrial structure observed by electron microscopy (Scale bar=500 nm). **P<0.01, ***P<0.001 vs Sham group; #P<0.05, ##P<0.01 vs HIBD group (n=3).

Fig.3 Effects of gastrodin on expressions of ferroptosis-related proteins in the ischemic penumbra of the cerebral cortex after HIBD. A-D: Western blotting protein bands of GPX4, SLC7A11 and FTH1 and their relative expression levels. E, F: Quantification and representative images of immunofluorescence double labeling of GPX4 and NeuN (Scale bar=10 μm). **P<0.01, ***P<0.001 vs Sham group; #P<0.05, ##P<0.01, ###P<0.001 vs HIBD group (n=3).

Fig.4 Effects of gastrodin on expressions of ferroptosis-related proteins in HT22 neurons after oxygen-glucose deprivation (OGD). Western blotting (A-D) and immunofluorescence double labeling (E-F) were used to detect the expression levels of GPX4, SLC7A11 and FTH1 proteins. Scale bar=10 μm. **P<0.01, ***P<0.001 vs Control group; #P<0.05, ##P<0.01 vs OGD group; &&P<0.01, &&&P<0.001 vs OGD+GAS group (n=3).

Fig.5 Effects of gastrodin on Fe2+, DCFH-DA, BODIPY-C11, MDA, and GSH levels in HT22 neurons after OGD. A, D: Representative images and quantification of Fe2+ level detected using the FerroOrange fluorescent probe (Scale bar=50 μm). B, E: Representative images and quantification of ROS detected using DCFH-DA fluorescent probe (Scale bar=50 μm). C, F, G: Representative images and quantification of lipid peroxidation using BODIPY-C11 fluorescent probe and MDA kit (Scale bar=10 μm). H: Quantification of antioxidant products by GSH. **P<0.01, ***P<0.001 vs Control group; #P<0.05, ##P<0.01 vs OGD group; &P<0.05, &&P<0.01 vs OGD+GAS group (n=3).

Fig.6 Neuroprotective effects of gastrodin against OGD-induced cell damage. A: CCK-8 assay of HT22 cell viability. B, C: Quantitative analysis and representative images of the ratio of mitochondrial membrane potential aggregates (red) and monomers (green) detected by JC-1. Scale bar=50 μm. ***P<0.001 vs Control group; #P<0.05, ##P<0.01 vs OGD group; &&P<0.01, &&&P<0.001 vs OGD+GAS group (n=3).

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