Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu, 610041, China.
Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu, 610041, China.
Phytomedicine. 2022 Oct;105:154363. doi: 10.1016/j.phymed.2022.154363. Epub 2022 Jul 28.
Secondary brain injury (SBI) has been confirmed as a leading cause for the poor prognosis of patients suffering from intracerebral hemorrhage (ICH). SBI co-exists in ischemia and hemorrhagic stroke. Neuro-excitotoxicity is considered the initiating factor of ICH-induced SBI. Our previous research has revealed alpha-asarone (ASA)'s efficacy against cerebral ischemia-reperfusion stroke by mitigating neuro-excitotoxicity. It is not yet known if ASA exhibit neuroprotection against ICH.
This work aimed to investigate ASA's therapeutic effects and potential mechanisms of action against ICH in a classic rat model induced by collagenase Ⅶ injection.
An in vivo ICH model of Sprague-Dawley rats was established by collagenase Ⅶ injection. We administrated different ASA doses (10, 20, or 40 mg/kg, i.p.) at 2 h post-ICH. Then, rats' short- and long-term neurobehavioral function, bodyweight change, and learning and memory ability were blindly evaluated. Histological, Nissl, and flow cytometry were applied to assess the neuronal damage post-ICH. The wet/dry method and Evans blue extravasation estimated brain edema and blood-brain barrier function. Pathway-related proteins were investigated by immunofluorescence staining, enzyme-linked immunosorbent assay, and Western-blot analysis.
The results demonstrated that ASA ameliorated neurological deterioration, bodyweight loss, and learning and memory ability of ICH rats. Histological, Nissl, and flow cytometry analyses showed that ASA reduced neuronal damage and apoptosis post-ICH. Besides, ASA probably mitigated brain edema and blood-brain barrier dysfunction via inhibiting astrocyte activation and consequent pro-inflammatory response. The mechanism investigation attributed ASA's efficacy to the following aspects: 1) promoting sodium ion excretion, thus blocking excitatory signal transduction along the axon; 2) preventing glutamate-involved pathways, i.e., decrease of N-methyl-d-aspartic acid receptor subunit 2B, increase of glutamate transporter-1, and alleviation of calcium-related cascades, mitochondrion-associated apoptosis, and neuronal autophagy; 3) enhancing the expression of GABARs, thus abating neuronal excitotoxicity.
Our study first confirmed the effect of ASA on ameliorating the neurobehavioral deterioration of ICH rats, possibly via alleviation of glutamate-involved neuro-excitotoxicity, i.e., calcium cascades, mitochondrion-involved apoptosis, neuronal autophagy, and astrocyte-related inflammation. These findings not only provided a promising drug candidate for clinical treatment of ICH but also shed light on the future drug discovery against ICH.
继发性脑损伤（SBI）已被证实是导致脑出血（ICH）患者预后不良的主要原因。SBI 存在于缺血性和出血性中风中。神经兴奋毒性被认为是ICH 引起 SBI 的起始因素。我们之前的研究表明，α-细辛脑（ASA）通过减轻神经兴奋毒性对脑缺血再灌注中风具有治疗作用。目前尚不清楚 ASA 是否对 ICH 具有神经保护作用。
本研究旨在通过胶原酶 VII 注射建立经典的 Sprague-Dawley 大鼠 ICH 模型，探讨 ASA 对 ICH 的治疗作用及其作用机制。
通过胶原酶 VII 注射建立 Sprague-Dawley 大鼠 ICH 模型。ICH 后 2 h 给予不同剂量的 ASA（10、20 或 40 mg/kg，腹腔注射）。然后，对大鼠的短期和长期神经行为功能、体重变化和学习记忆能力进行盲法评估。采用组织学、尼氏染色和流式细胞术评估 ICH 后神经元损伤。干湿法和 Evans 蓝渗出法评估脑水肿和血脑屏障功能。通过免疫荧光染色、酶联免疫吸附试验和 Western blot 分析检测通路相关蛋白。
结果表明，ASA 改善了 ICH 大鼠的神经功能恶化、体重减轻和学习记忆能力。组织学、尼氏染色和流式细胞术分析显示，ASA 减轻了 ICH 后神经元损伤和细胞凋亡。此外，ASA 可能通过抑制星形胶质细胞激活和随之而来的促炎反应来减轻脑水肿和血脑屏障功能障碍。机制研究表明，ASA 的疗效归因于以下几个方面：1）促进钠离子排泄，从而阻断轴突中的兴奋性信号转导；2）阻止谷氨酸相关通路，即减少 N-甲基-D-天冬氨酸受体亚单位 2B，增加谷氨酸转运体-1，并减轻钙相关级联、线粒体相关凋亡和神经元自噬；3）增强 GABAAR 的表达，从而减轻神经元兴奋毒性。
本研究首次证实了 ASA 改善 ICH 大鼠神经行为恶化的作用，可能通过减轻谷氨酸相关的神经兴奋毒性，即钙级联、线粒体相关凋亡、神经元自噬和星形胶质细胞相关炎症。这些发现不仅为 ICH 的临床治疗提供了有前途的候选药物，也为未来针对 ICH 的药物发现提供了思路。
