Department of Pharmaceutical analysis, School of Pharmacy, Key Laboratory of Hui Ethnic Medicine Modernization, Ministry of Education, Ningxia Medical University, 1160 Shenli Street, Yinchuan, 750004, China.
Department of Pharmaceutical analysis, School of Pharmacy, Key Laboratory of Hui Ethnic Medicine Modernization, Ministry of Education, Ningxia Medical University, 1160 Shenli Street, Yinchuan, 750004, China; School of Pharmacy, Lanzhou University, 222 Tianshui South Road, Lanzhou, 730000, China.
J Ethnopharmacol. 2022 Mar 1;285:114882. doi: 10.1016/j.jep.2021.114882. Epub 2021 Nov 27.
To explore the effective components, potential targets and neuroprotective related mechanisms of Mijianchangpu decoction (MJCPD), a well-known TCM used by the Chinese Hui minorities to treat stroke, on the prevention and treatment of ischemic stroke (IS) by using experimental models combined with network pharmacology.
The neuroprotective efficacy of MJCPD was estimated by applying the middle cerebral artery occlusion (MCAO) induced cerebral ischemia rats, and the neurological deficits score, TTC and HE staining as well as behavioral evaluation tests were employed to evaluate the beneficial effects. Meanwhile, the bioactive components of MJCPD responsible for the neuroprotective effects were identified by detecting the constituents in the brain of the MCAO rats with UHPLC-QTOF-MS/MS techniques, and these compounds were then underwent for network pharmacology analysis. Firstly, the targets of the bioactive compounds of MJCPD were predicted using Pharmmapper database, and simultaneously, the targets of IS disease were obtained from disease databases including DisGenet, OMIM, and GeneCards. Secondly, the protein-protein interaction (PPI) network between the targets and diseases were established to give the possible therapeutic targets for IS. Thirdly, the go function and KEGG pathway enrichment analysis were carried out and the compound-target-pathway network was constructed by Cytoscape software. Finally, the effective compounds, core targets and possible pathways were obtained by analyzing the connectivity of the network. More importantly, the core targets were verified by western blot experiments to validate the reliability of this study.
MJCPD exhibited significant neuroprotective effect on IS, and 16 bioactive components of MJCPD were identified in the brain of the MCAO rats. 59 and 1982 targets related with IS disease were explored from Pharmapper and disease databases, respectively, and 32 intersecting targets were obtained as hypothetical therapeutic targets. Based on the results of the compound-target-pathway and PPI network with the degree was greater than the median, 8 effective compounds (suberic acid, epishyobunone, crocetin monomethyl ester, sfaranal, (Z)-6-octadccenoic acid, nerolidol and gurjunene) and 5 hub targets (SRC, MAPK8, MAPK14, EGFR and MAPK1) as well as 12 pathways were predicted. Western blot results showed that EGFR, p38, ERK and SRC proteins were expressed significantly different after MJCPD treatment as compared with the model group.
The present study employed network pharmacology, pharmacodynamics and molecular biology techniques to predict and validate the core potential targets and signaling pathways as well as the bioactive components of MJCPD responsible for the treatment of IS. All of which are very helpful to clarify the neuroprotective mechanism of MJCPD, and obviously, the active compounds and targets in this study can also provide clues for the treatment of IS.
本研究旨在采用实验模型结合网络药理学方法，探讨回族常用中药密煎肠汤（MJCPD）防治缺血性中风（IS）的有效成分、潜在靶点和神经保护相关机制。
通过大脑中动脉闭塞（MCAO）诱导的脑缺血大鼠模型评估 MJCPD 的神经保护作用，采用神经功能缺损评分、TTC 和 HE 染色以及行为评估试验评价其疗效。同时，采用 UHPLC-QTOF-MS/MS 技术检测 MCAO 大鼠脑内的 MJCPD 成分，鉴定其具有神经保护作用的生物活性成分，并对这些化合物进行网络药理学分析。首先，利用 Pharmmapper 数据库预测 MJCPD 生物活性成分的靶点，同时从 DisGenet、OMIM 和 GeneCards 等疾病数据库中获取 IS 疾病的靶点。其次，建立靶点与疾病之间的蛋白质-蛋白质相互作用（PPI）网络，为 IS 提供可能的治疗靶点。然后，通过 Cytoscape 软件进行 GO 功能和 KEGG 通路富集分析，构建化合物-靶点-通路网络。最后，通过分析网络的连通性，获得有效化合物、核心靶点和可能的通路。更重要的是，通过 Western blot 实验验证核心靶点，验证本研究的可靠性。
MJCPD 对 IS 具有显著的神经保护作用，在 MCAO 大鼠脑内鉴定出 16 种 MJCPD 的生物活性成分。从 Pharmapper 和疾病数据库中分别获得与 IS 疾病相关的 59 个和 1982 个靶点，获得 32 个交集靶点作为假设治疗靶点。基于化合物-靶点-通路和 PPI 网络的结果，选择度大于中位数的 8 种有效化合物（琥珀酸、菖蒲酮、藏红花酸单甲酯、sfaranal、（Z）-6-辛二烯酸、香叶醇和古芸烯）和 5 个核心靶点（SRC、MAPK8、MAPK14、EGFR 和 MAPK1）以及 12 条通路进行预测。Western blot 结果显示，与模型组相比，MJCPD 治疗后 EGFR、p38、ERK 和 SRC 蛋白表达明显不同。
本研究采用网络药理学、药效学和分子生物学技术，预测和验证了 MJCPD 治疗 IS 的核心潜在靶点和信号通路以及生物活性成分。所有这些都有助于阐明 MJCPD 的神经保护机制，而且本研究中的活性化合物和靶点也为 IS 的治疗提供了线索。
