电针预处理通过调节肠道-大脑轴及Nrf2/HO-1信号通路抑制铁死亡减轻大鼠脑缺血再灌注损伤

doi:10.12122/j.issn.1673-4254.2025.05.03

南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (5): 911-920.doi: 10.12122/j.issn.1673-4254.2025.05.03

电针预处理通过调节肠道-大脑轴及Nrf2/HO-1信号通路抑制铁死亡减轻大鼠脑缺血再灌注损伤

张安邦¹(), 孙秀颀¹, 庞博¹, 吴远华¹, 时靖宇¹, 张宁², 叶涛³()

^1.贵州中医药大学第一附属医院，神经内科，贵州贵阳 550001
^2.贵州中医药大学第一附属医院，药学部，贵州贵阳 550001
^3.贵州中医药大学第一附属医院，康复科，贵州贵阳 550001

收稿日期:2024-12-26 出版日期:2025-05-20 发布日期:2025-05-23
通讯作者: 叶涛 E-mail:544820318@qq.com;yetao008@gzy.edu.cn
作者简介:张安邦，博士，副主任医师，E-mail: 544820318@qq.com
基金资助:
国家自然科学基金(82060896);贵州省基础研究项目（QianKeHeJiChu-ZK [2021] General 505）;贵州省教育厅青年科技人才成长项目（QianJiaoHe KY Zi [2021]210）;贵州省卫健委科技基金项目(gzwkj2021-072);贵州省中医药管理局中药与民族医药科学技术研究项目(QZYY-2021-015);贵州中医药大学第一附属医院博士启动基金[GYZYYFY-BS-2021(01),GYZYYFY-BS-2021(02)]

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

Anbang ZHANG¹(), Xiuqi SUN¹, Bo PANG¹, Yuanhua WU¹, Jingyu SHI¹, Ning ZHANG², Tao YE³()

^1.Department of Neurology, First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, Guiyang 550001, China
^2.Department of Pharmacy, First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, Guiyang 550001, China
^3.Department of Rehabilitation Medicine, First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, Guiyang 550001, China

Received:2024-12-26 Online:2025-05-20 Published:2025-05-23
Contact: Tao YE E-mail:544820318@qq.com;yetao008@gzy.edu.cn
Supported by:
National Natural Science Foundation of China(82060896)

摘要/Abstract

摘要：

目的探讨电针（EA）预处理通过调节肠道微生物群和Nrf2/HO-1信号通路以及抑制铁死亡来减轻脑缺血再灌注损伤（CIRI）的潜力。方法诱导CIRI的大鼠接受大脑中动脉堵塞（MCAO）手术，并分为假手术组、模型组和EA治疗组，10只/组。EA治疗在百会穴（DU20）和足三里穴（ST36）于MCAO前3 d进行。评估神经功能缺损和开放场行为，使用TTC、Nissl和HE染色检查脑梗死和海马病理。通过ELISA测定脑组织中丙二醛、活性氧和超氧化物歧化酶水平，利用qRT-PCR和Western blotting分析Bax、Bcl-2、Nrf2、HO-1、GPX4和SLC7A11的表达。免疫组化检测脑组织中GPX4、Nrf2和HO-1的水平，并收集粪便样本以分析肠道微生物群。结果 EA预处理降低了大鼠的神经功能缺损评分（P<0.05），改善了自愿运动的频率和持续时间（均P<0.05）。TTC染色显示EA组的缺血性梗死面积小于模型组（P<0.05）。Nissl染色结果显示，模型组海马神经元数量减少且Nissl物质丢失，而EA组神经元存活率提高，Nissl物质含量恢复（P<0.05）。HE染色结果显示，模型组海马神经元排列不规则且病理变化明显，而EA组的结构和活力有所改善（P<0.05）。ELISA检测显示，模型组的丙二醛和活性氧水平升高，超氧化物歧化酶水平下降（均P<0.05）；EA组则相反。EA治疗降低了Bax蛋白表达，增加了Bcl-2蛋白表达（均P<0.05），同时上调了铁死亡关键调控蛋白GPX4和SLC7A11的表达，免疫组化结果一致。EA上调Nrf2（P<0.05）和HO-1（P<0.05）的表达，激活Nrf2/HO-1信号通路，并改善肠道微生物群。结论电针预处理通过调节肠脑轴、激活Nrf2/HO-1信号通路、抑制细胞凋亡和铁死亡，有效缓解了大鼠脑缺血再灌注损伤，为其在缺血性脑血管疾病中的临床应用提供了科学依据。

关键词: 电针, 脑缺血再灌注损伤, 氧化应激, 肠道微生物, 铁死亡

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

张安邦, 孙秀颀, 庞博, 吴远华, 时靖宇, 张宁, 叶涛. 电针预处理通过调节肠道-大脑轴及Nrf2/HO-1信号通路抑制铁死亡减轻大鼠脑缺血再灌注损伤[J]. 南方医科大学学报, 2025, 45(5): 911-920.

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.

图/表 8

表1 目标基因的引物序列

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

图1 EA对神经功能缺损评分和梗死面积的影响

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.

图2 脑组织Nissl和HE染色代表图

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

图3 ELISA检测血清中MDA、SOD和ROS的水平

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.

图4 Western blotting检测脑组织中Bax和Bcl-2蛋白的表达

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.

图5 Nrf2和HO-1在脑组织中的相对表达

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.

图6 各组中Nrf2、HO-1、GPX4的表达水平

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.

图7 电针预处理改善脑缺血再灌注大鼠肠道微生物群

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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电针预处理通过调节肠道-大脑轴及Nrf2/HO-1信号通路抑制铁死亡减轻大鼠脑缺血再灌注损伤

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

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