当归补血汤调控Nrf2/GPX4通路改善血管性痴呆大鼠认知功能的机制

Mechanism of Danggui Buxue decoction regulating Nrf2/GPX4 pathway to improve cognitive function of vascular dementia rats

摘要

目的: 探究当归补血汤(Danggui Buxue decoction,DBD)对双侧颈总动脉结扎(bilateral common carotid artery ligation,2-VO)血管性痴呆(vascular dementia,VD)模型大鼠核因子E2相关因子(nuclear factor erythroid 2-related factor 2, Nrf2)/谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)通路、脂质过氧化及突触可塑性的影响。方法: 60只雄性SPF级SD大鼠随机分为假手术组、双侧颈总动脉结扎VD模型组、2-VO+当归补血汤低剂量给药组、2-VO+当归补血汤中剂量给药组、2-VO+当归补血汤高剂量给药组、2VO+多奈哌齐组,每组10只。造模24 h后,连续给药4周后取材。采用Morris水迷宫实验、避暗实验评价大鼠学习记忆能力,试剂盒检测脂质过氧化水平,尼氏染色观察海马神经元情况,免疫荧光染色观察海马神经元的表达,蛋白免疫印迹法检测通路及突触可塑性相关蛋白表达,透射电镜观察海马神经元线粒体及突触超微结构,高尔基染色观察大鼠海马神经元树突棘形态及密度。结果: Morris水迷宫实验和避暗实验表明DBD可改善大鼠的学习记忆能力。尼氏染色和免疫荧光结果显示,DBD给药后可提高VD模型组大鼠海马神经元数量,Nrf2表达率及改善神经元损伤。VD模型大鼠表现出谷胱甘肽水平下降,脂质过氧化物及4-羟基壬烯醛水平上升,DBD给药可减轻氧化应激。在VD模型大鼠中出现烟酰胺腺嘌呤二核苷酸(磷酸)醌氧化还原酶1、GPX4、Nrf2、突触后密度蛋白95、突触素1、脑源性神经营养因子蛋白的表达降低;DBD-H组给药后均显著升高。透射电镜与高尔基染色显示,DBD可改善神经元线粒体结构和突触超微结构,增加树突棘密度。结论: 当归补血汤可通过调控Nrf2/GPX4信号通路,抑制神经元脂质过氧化,改善突触可塑性,从而发挥神经元保护作用,改善VD模型大鼠的学习记忆能力。

Abstract

Objective: To investigate the effects of Danggui Buxue Decoction (DBD) on the nuclear factor erythroid 2-related factor 2/glutathione peroxidase 4 pathway, degree of lipid peroxidation, and synaptic plasticity of rats with vascular dementia (VD) induced by bilateral common carotid artery ligation (2-VO). Methods: Sixty male SPF grade SD rats were randomly divided into sham surgery group, bilateral common carotid artery ligation VD model group, low-dose 2-VO+DBD group, medium dose 2-VO+DBD group, high-dose 2-VO+DBD group, and 2-VO+donepezil group, with 10 rats in each group. After 24 hours of modeling,continuous administration was performed for 4 weeks and samples were collected. The Morris water maze experiment and the dark avoidance experiment were used to evaluate the influence on the learning and memory ability of rats. The lipid peroxidation level was detected using a kit,and the morphology of hippocampal neurons was observed using Nissl staining. The expression of hippocampal neurons was observed using immunofluorescence staining, and the expression of pathway and synaptic plasticity-related proteins was detected using Western blotting. The mitochondria and synaptic ultrastructures of hippocampal neurons were observed using transmission electron microscopy, and the morphology and density of dendritic spines of hippocampal neurons were observed using Golgi staining. Results: The Morris water maze experiment and the dark avoidance experiment demonstrated that DBD could enhance the learning and memory abilities of rats. The results of Nissl staining and immunofluorescence showed that after DBD administration, the number of hippocampal neurons in the VD model group of rats increased, the expression rate of Nrf2 increased, and the neuronal damage was improved. VD rats showed a decrease in GSH levels, an increase in lipid peroxide and 4-hydroxynonenal levels, and DBD administration could alleviate oxidative stress. In VD rats, the expression of NAD(P)H quinone dehydrogenase 1, GPX4, Nrf2, postsynaptic density protein 95, Synapsin1, and brain-derived neurotrophic factor proteins decreased, and all of these significantly increased after administration of high dose DBD. Transmission electron microscopy and Golgi staining showed that DBD could improve the mitochondrial structure and synaptic ultrastructure of neurons, and increase the density of dendritic spines. Conclusion: DBD can exert a neuroprotective effect and improve the learning and memory abilities of VD model rats by regulating the Nrf2/GPX4 signaling pathway, inhibiting neuronal lipid peroxidation, and enhancing synaptic plasticity.    

关键词

当归补血汤 / 血管性痴呆 / 脂质过氧化 / 核因子E2相关因子/谷胱甘肽过氧化物酶4 / 突触可塑性

Key words

Danggui Buxue decoction / vascular dementia / lipid peroxidation / nuclear factor-erythroid 2-related factor 2/glutathione peroxidase 4 / synaptic plasticity

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基金资助

国家自然科学基金资助项目(82104757);2023年广州中医药大学第一附属医院2023年中青年骨干培育项目青优人才资助项目