Electroacupuncture pretreatment alleviates cerebral ischemia-reperfusion injury in rats by inhibiting ferroptosis through the gut-brain axis and the Nrf2/HO-1 signaling pathway

doi:10.12122/j.issn.1673-4254.2025.05.03

Abstract

Abstract:

Objective To investigate the neuroprotective effects of electroacupuncture (EA) preconditioning against cerebral ischemia-reperfusion injury (CIRI) mediated by gut microbiota modulation, Nrf2/HO-1 pathway activation, and ferroptosis suppression. Methods Adult male SD rats were divided into sham operation group, CIRI model group, and EA preconditioning group. In the latter two groups, rat models of CIRI were established by middle cerebral artery occlusion (MCAO), and in EA preconditioning group, EA was applied at Baihui (DU20) and Zusanli (ST36) for 3 days before modeling. Neurological deficits, cerebral infarction, and hippocampal pathology of the rats were evaluated using behavioral tests, TTC staining, and Nissl and HE staining, and the oxidative stress markers (MDA, ROS, and SOD), apoptosis/ferroptosis-related proteins (Bax, Bcl-2, GPX4, and SLC7A11), and changes in gut microbiota were analyzed. Results EA preconditioning significantly reduced neurological deficits, decreased infarct volume, promoted hippocampal neuronal survival, and improved structural integrity of the hippocampal neurons in MCAO rats. EA preconditioning also significantly lowered MDA and ROS and increased SOD levels, upregulated Bcl-2, GPX4, and SLC7A11 expressions, and downregulated Bax expression in the hippocampal tissue of the rats, causing also activation of Nrf2/HO-1 signaling and improvement of gut microbiota composition. Conclusion EA preconditioning alleviates CIRI in rats by suppressing ferroptosis and apoptosis, enhancing antioxidant defenses via activating Nrf2/HO-1 signaling, and regulating the gut-brain axis.

Key words: electroacupuncture, cerebral ischemia-reperfusion injury, oxidative stress, gut microbiota, ferroptosis

Anbang ZHANG, Xiuqi SUN, Bo PANG, Yuanhua WU, Jingyu SHI, Ning ZHANG, Tao YE. Electroacupuncture pretreatment alleviates cerebral ischemia-reperfusion injury in rats by inhibiting ferroptosis through the gut-brain axis and the Nrf2/HO-1 signaling pathway[J]. Journal of Southern Medical University, 2025, 45(5): 911-920.

Figures/Tables 8

Tab.1 PCR primers for target genes

Target gene	Forward primer	Reverse primer
Nrf2	CTTCTGGGGATACAGTACAGCC	CTCCTTGGACATCATTTCATTG
HO-1	AGGTCCTGAAGAAGATTGCG	GGCGAAGAAACTCTGTCTGTGA
Bax	GCGATGAACTGGACAACAAC	GCAAAGTAGAAAAGGGCAACC
Bcl-2	GCGTCAACAGGGAGATGTCA	TTCCACAAAGGCATCCCAGC
β-actin	GCCATGTACGTAGCCATCCA	GAACCGCTCATTGCCGATAG

Fig.1 Effect of electroacupuncture (EA) preconditioning on neurological deficit scores and cerebral infarct volume in rats with cerebral ischemia-reperfusion injury (CIRI). A: Neurological deficit scores of the rats. B: Locomotor activity counts of the rats. C: Locomotor activity duration. D, E: TTC staining for determining cerebral infarct volume percentage in the rats. Data are presented as Mean±SE (n=10). *P<0.05 vs sham group; #P<0.05 vs I/R group.

Fig.2 Histopathological examination of rat brain tissue with Nissl and HE staining (Original magnification: ×200).

Fig.3 Serum levels of MDA, SOD and ROS in the rats detected by ELISA. *P<0.05 vs sham group; #P<0.05 vs I/R group.

Fig.4 Protein expressions of Bax, Bcl-2, GPX4 and SLC7A11 in rat brain tissue. A-C: Western blotting for Bax and Bcl-2 protein expressions in the brain tissue. D, E: Immunofluorescence staining of Bax and Bcl-2 expression in the brain tissue (×400). F: Western blotting of GPX4 and SLC7A11 protein expressions in the brain tissue. Data are presented as Mean±SE (n=10). *P<0.05 vs sham group; #P<0.05 vs I/R group.

Fig.5 Relative protein and mRNA expressions of Nrf2 and HO-1 in rat brain tissues. A-C: Western blotting for Nrf2 and HO-1 protein expressions. D, E: mRNA expression levels of Nrf2 and HO-1. Data are presented as Mean±SE (n=10). *P<0.05 vs sham group; #P<0.05 vs I/R group.

Fig.6 Expression levels of Nrf2, HO-1 and GPX4 in each group. A, B: Immunofluorescence staining of Nrf2, HO-1 and GPX4 expression in rat brain tissue (×50). *P<0.05 vs sham group; #P<0.05 vs I/R group.

Fig.7 EA pretreatment improves gut microbiota in rats with cerebral ischemia-reperfusion injury. A: Principal component analysis (PCA) of OTU level. B: Relative abundance of gut microbiota. C: Heatmap of gut microbiota abundance. D: Percentage composition of gut microbiota (%). *P<0.05 vs sham group.

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