Electroacupuncture Ameliorates Cerebral Ischemic Injury by Inhibiting Ferroptosis

Abstract

Background: Our previous study found that electroacupuncture (EA) can promote the recovery of neurological functions, reduce the volume of cerebral infarction, and protect the neurovascular unit in middle cerebral artery occlusion (MCAO) rats. Some studies have shown that ferroptosis is closely related to ischemic stroke; however, whether EA plays a protective role by regulating ferroptosis is unknown. Objective: We aimed to investigate the inhibitory effects of EA on ferroptosis in MCAO rats. Methods: We used 36 adult male Sprague-Dawley rats in this study. MCAO rats were established according to the Zea method and treated with EA at a continuous wave of 2/100 Hz and ~2-4 V for 30 min for 7 consecutive days. We analyzed the coordinated motor deficit and volume of cerebral infarction in vivo through 9.4-tesla magnetic resonance imaging. Then, the ischemic brain tissue was isolated and the levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and iron were determined. Western blotting and real-time quantitative PCR were performed to evaluate the expression of glutathione peroxidase 4 (GPX4), transferrin (Tf), transferrin receptor 1 (TfR1), and ferritin heavy chain 1 (FTH1). To confirm the results, we used a transmission electron microscope to observe the mitochondrial morphology. Results: EA intervention significantly decreased the oxidative stress level and inhibited ferroptosis. EA significantly improved coordinated motor deficit (P < 0.01) and decreased cerebral infarct volume (P < 0.01) in the EA + MCAO group, compared with the MCAO group. EA downregulated the level of MDA (P < 0.01) and total iron (P < 0.01) and upregulated the level of SOD (P < 0.01) and GSH (P < 0.01) in the EA + MCAO group, compared with the MCAO group. EA increased the levels of GPX4 and GPX4 mRNA (P < 0.01) and FTH1 and FTH1 mRNA (P < 0.05, P < 0.01), whereas it decreased the levels of Tf and Tf mRNA (P < 0.05, P < 0.01) and TfR1 and TfR1 mRNA (P < 0.01) in the EA + MCAO group, compared with the MCAO group. EA also promoted the recovery of mitochondrial morphology according to the mitochondrial classification system for the ischemic cerebral tissue. Conclusion: Our results indicate that EA can inhibit ferroptosis by regulating oxidative stress and iron-related proteins, thus conferring protection against MCAO in a rat model.

Keywords: electroacupuncture; ferroptosis; iron homeostasis; ischemic stroke; mitochondrial.

Figure 1

Coordinated motion time and cerebral infarction volume of rats in each group. (A) The coordinated motion time of rats in each group on the 7th day. (B,C) Evaluation of cerebral infarction volume of rats in different groups by 9.4T MRI T2-weighted imaging and analysis. Results are expressed as means ± SEM (n = 12, n = 3). **P < 0.01, compared with the Control group; ^##P < 0.01, compared with the MCAO group.

Figure 2

Levels of MDA (A), total iron (B), SOD (C), and GSH (D) in the samples in each group on the 7th day. Results are expressed as means ± SEM (n = 6). **P < 0.01, compared with the Control group; ^##P < 0.01, compared with the MCAO group.

Figure 3

Protein bands (A) and levels of GPX4 (B), FTH1 (C), Tf (D), and TfR1 (E) in each group. Results are expressed as means ± SEM (n = 6). **P < 0.01, compared with the Control group; ^#P < 0.05 and ^##P < 0.01, compared with the MCAO group.

Figure 4

Levels of GPX4 mRNA (A), FTH1 mRNA (B), Tf mRNA (C), and TfR1 mRNA (D) in each group. Results are expressed as means ± SEM (n = 6). **P < 0.01, compared with the Control group; ^##P < 0.01, compared with the MCAO group.

Figure 5

Electroacupuncture protects neuronal mitochondria. (A) TEM images of cortical cerebral tissues from each group. Scare bars: left, 2 μm; right, 500 nm. (B) Quantification of mitochondria counted blind to intervention of condition of three independent experiments. Results are expressed as means ± SEM. **P < 0.01, compared with the Control group; ^#P < 0.05, and ^##P < 0.01, compared with the MCAO group.