显齿蛇葡萄通过调控VEGFA/KDR/FGFR1介导的血管生成和GSK3β/Bcl-2/Bax介导的线粒体凋亡减轻BCAS小鼠的血管性认知障碍

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中文摘要

民族药理学相关性： 显齿蛇葡萄（Ampelopsis grossedentata (Hand.-Mazz.) W.T. Wang，藤茶）是中国南方土家族和苗族使用历史悠久的民族药物。传统上用于“清热解毒”、“活血通络”以及“平肝阳、化痰祛浊”。其水煎剂作为民间验方用于缓解眩晕、头晕及高血压相关的心脑血管症状。这些传统应用提示其可能与慢性脑低灌注所致血管性认知障碍的治疗相关。
研究目的： 本研究旨在探讨藤茶水提物对慢性脑低灌注所致血管性认知障碍的保护作用及其潜在机制，重点聚焦其对血管生成信号和线粒体凋亡相关通路的调控。
材料与方法： 采用UPLC-Q-TOF-MS对藤茶水提物的化学特征进行表征，并对选定成分进行定量。利用GSE122063数据集进行网络药理学和生物信息学分析，以识别活性成分和候选治疗靶点。通过分子对接和200 ns分子动力学模拟评估其结合稳定性。将雄性C57BL/6小鼠随机分为五组（每组n = 20）：假手术组、模型组、阳性对照组（尼莫地平，11.7 mg/kg）、VTL组（100 mg/kg）和VTH组（200 mg/kg）。通过微弹簧圈诱导双侧颈总动脉狭窄（BCAS）建立血管性认知障碍模型。恢复1周后，小鼠每日灌胃给药，持续30天。采用旷场实验、新物体识别实验和Morris水迷宫评估行为学表现。评估脑灌注、CD31阳性微血管密度、髓鞘完整性、神经元形态、神经元凋亡和线粒体超微结构。生化检测测定氧化应激、ATP和乳酸水平，并通过RT-PCR和Western blotting检测相关基因和蛋白。
结果： UPLC-Q-TOF-MS鉴定出31种化合物，其中对二氢杨梅素、槲皮素、花旗松素、香橙素、儿茶素、表没食子儿茶素和柚皮素（预测OB ≥ 20%，DL ≥ 0.18）进行了定量测定。网络分析识别出七个枢纽靶点，包括促血管生成模块（VEGFA、KDR、FGFR1）和凋亡相关模块（GSK3β、RAF1、BCL2、BAX）。分子对接和动力学模拟支持主要成分与枢纽靶点之间稳定的相互作用。在BCAS小鼠中，藤茶显著改善认知行为，恢复局部脑血流量（P < 0.01）并增加CD31阳性微血管密度（P < 0.05），同时保护髓鞘完整性（P < 0.01）和海马神经元存活（P < 0.05）。这些获益伴随线粒体超微结构损伤减轻，ATP、SOD和GSH水平升高，乳酸和MDA含量降低（P < 0.05），以及cleaved caspase-3/NeuN双阳性凋亡神经元减少（P < 0.05）。在机制上，VT恢复VEGFA/KDR/FGFR1表达，增加Bcl-2/Bax比值和GSK3β磷酸化，并使RAF1过度磷酸化正常化，提示其对血管生成信号和线粒体凋亡相关通路具有协同调控作用。
结论： 藤茶通过恢复VEGFA/KDR/FGFR1介导的血管生成信号并重新平衡GSK3β/RAF1和Bcl-2/Bax相关的线粒体凋亡通路，改善BCAS小鼠的认知缺陷和海马神经元损伤。这些发现提示藤茶作为血管性认知障碍的多靶点民族药物候选物具有前景，尽管这两个过程之间的因果联系仍需进一步的机制验证。

英文摘要

ETHNOPHARMACOLOGICAL RELEVANCE: Ampelopsis grossedentata (Hand.-Mazz.) W.T. Wang (vine tea) is a time-honored ethnomedicine used by the Tujia and Miao minorities in Southern China. It has traditionally been used for "clearing heat and detoxifying", "activating blood and unblocking collaterals", and "subduing liver-yang to resolve phlegm and eliminate turbidity". Its aqueous infusion is consumed as a folk remedy to alleviate vertigo, dizziness, and hypertension-related cardio-cerebrovascular symptoms. These traditional applications suggest its potential therapeutic relevance to chronic cerebral hypoperfusion-induced vascular cognitive impairment.
AIM OF THE STUDY: This study aimed to investigate the protective effects and underlying mechanisms of vine tea aqueous extract against chronic cerebral hypoperfusion-induced vascular cognitive impairment, focusing on its regulation of angiogenic signaling and mitochondrial apoptosis-related pathways.
MATERIALS AND METHODS: The chemical profile of vine tea aqueous extract was characterized by UPLC-Q-TOF-MS, and selected constituents were quantified. Network pharmacology and bioinformatics analysis using the GSE122063 dataset were performed to identify active constituents and candidate therapeutic targets. Molecular docking and 200-ns molecular dynamics simulations evaluated their binding stability. Male C57BL/6 mice were randomly divided into five groups (n = 20/group): Sham, Model, positive control (nimodipine, 11.7 mg/kg), VTL (100 mg/kg), and VTH (200 mg/kg). Vascular cognitive impairment was induced by microcoil-induced bilateral common carotid artery stenosis (BCAS). After a 1-week recovery period, mice received daily oral gavage for 30 days. Behavioral performance was assessed using the open field test, novel object recognition test, and Morris water maze. Cerebral perfusion, CD31-positive microvascular density, myelin integrity, neuronal morphology, neuronal apoptosis, and mitochondrial ultrastructure were assessed. Biochemical assays determined oxidative stress, ATP and lactic acid levels, while related genes and proteins were examined by RT-PCR and Western blotting.
RESULTS: UPLC-Q-TOF-MS identified 31 compounds, among which dihydromyricetin, quercetin, taxifolin, aromadendrin, catechin, epigallocatechin and naringenin (predicted OB ≥ 20%, DL ≥ 0.18) were quantitatively determined. Network analysis identified seven hub targets, including a pro-angiogenic module (VEGFA, KDR, FGFR1) and an apoptosis-related module (GSK3β, RAF1, BCL2, BAX). Molecular docking and dynamics simulations supported stable interactions between the major constituents and hub targets. In BCAS mice, vine tea significantly improved cognitive behavior, restored regional cerebral blood flow (P < 0.01) and increased CD31-positive microvascular density (P < 0.05), while preserving myelin integrity (P < 0.01) and hippocampal neuron survival (P < 0.05). These benefits were accompanied by attenuated mitochondrial ultrastructural damage, increased ATP, SOD, and GSH levels, and reduced lactate and MDA content (P < 0.05), as well as decreased cleaved caspase-3/NeuN double-positive apoptotic neurons (P < 0.05). Mechanistically, VT restored VEGFA/KDR/FGFR1 expression, increased Bcl-2/Bax ratio and GSK3β phosphorylation, and normalized RAF1 hyperphosphorylation, suggesting coordinated regulation of angiogenic signaling and mitochondrial apoptosis-related pathways.
CONCLUSIONS: Vine tea ameliorated cognitive deficits and hippocampal neuronal injury in BCAS mice by restoring VEGFA/KDR/FGFR1-mediated angiogenic signaling and rebalancing GSK3β/RAF1 and Bcl-2/Bax-related mitochondrial apoptotic pathways. These findings suggest vine tea as a promising multi-target ethnomedicinal candidate for vascular cognitive impairment, although the causal link between these two processes warrants further mechanistic validation.