基于网络药理学和分子对接技术探讨异钩藤碱治疗脑卒中的作用机制

摘要(中文译文)

本研究旨在阐明异钩藤碱（IRN）在脑卒中中的神经保护机制。利用网络药理学方法，通过SwissTargetPrediction、HERB和TCMIP 2.0数据库预测IRN的作用靶点。我们确定了IRN与脑卒中共同的65个靶点。使用R软件进行GO和KEGG富集分析，并构建了化合物-靶点-通路-疾病网络。此外，进行分子对接实验以研究IRN与其主要靶点之间的结合相互作用。随后，在缺氧条件（1% O₂）下使用人神经母细胞瘤细胞系SH-SY5Y建立氧糖剥夺/复氧（OGD/R）模型。通过CCK-8法测定细胞活力以确定IRN的有效工作浓度。然后，进行ELISA实验检测炎症因子IL-1β和IL-10的表达。采用双荧光检测法测定活性氧（ROS）的活性。采用TUNEL染色评估细胞凋亡。最后，使用Western blot检测IRN对细胞中EGFR和AKT表达的影响。网络药理学分析结果表明，IRN治疗脑卒中的主要靶点为EGFR、mTOR、JAK2、AKT和JUN。富集分析显示IRN与神经递质受体功能相关，并参与EGFR信号通路。分子对接结果显示其与AKT1具有特别强的结合亲和力（结合能≈-8.7 kcal/mol）。体外实验表明，IRN处理显著减轻了OGD/R诱导的损伤。具体而言，IL-1β和ROS水平降低，IL-10水平升高，凋亡率下降。Western blot结果显示，与OGD/R组相比，IRN显著上调了p-EGFR和p-AKT的表达。我们通过计算预测和实验验证证明，IRN采用多靶点途径对抗脑卒中病理。网络药理学、分子对接和体外实验的汇聚使得关键靶点的鉴定成为可能，包括EGFR、AKT1和JAK2。这些方法还表明IRN可同时调控炎症、氧化应激和凋亡过程。与单靶点药物相比，IRN展现出更优越的多靶点治疗潜力。这些来自数据库分析和基于细胞的实验的发现，全面概述了IRN的药理作用，与其已知的神经保护益处相一致。尽管这些结果来自基于细胞的模型和预测数据库，但它们提供了连贯的机制依据，与IRN所具有的神经保护特性相一致。仍需在动物模型和临床环境中开展进一步研究，以将这些见解应用于治疗目的。IRN可通过调控包括EGFR在内的关键靶点发挥对抗脑卒中的神经保护作用。其具有抗炎、抗氧化和抗凋亡活性，为将IRN开发为治疗脑卒中的药物提供了合理依据。

Abstract

This study aimed to elucidate the neuroprotective mechanism of Isorhynchophylline (IRN) in stroke. Using network pharmacology, the action targets of IRN were predicted using the SwissTargetPrediction, HERB, and TCMIP 2.0 databases. We identified sixty-five targets common to IRN and stroke. We conducted GO and KEGG enrichment analyses using R software and constructed a compound- target- pathway- disease network. Additionally, molecular docking experiments were performed to investigate the binding interactions between IRN and its main targets. Subsequently, an oxygen-glucose deprivation/reoxygenation (OGD/R) model was established using the human neuroblastoma cell line SH-SY5Y under hypoxic conditions (1% O2). The effective working concentration of IRN was determined by measuring cell viability using the CCK-8 assay. Then, an ELISA experiment was conducted to detect the expression of inflammatory factors IL-1β and IL-10. The activity of reactive oxygen species (ROS) was measured using the dual fluorescence detection method. TUNEL staining was employed to assess the apoptosis of cells. Finally, a Western blot was used to examine the effect of IRN on EGFR and AKT expression in cells. The results of network pharmacology analysis indicated that the main targets of IRN in treating stroke are EGFR, mTOR, JAK2, AKT, and JUN. Enrichment analysis revealed that IRN is associated with neurotransmitter receptor function and participates in the EGFR signaling pathway. Molecular docking results showed that it has a particularly strong binding affinity for AKT1 (binding energy ≈ -8.7 kcal/mol). The in vitro experiments showed that IRN treatment significantly alleviated the damage induced by OGD/R. Specifically, the levels of IL-1β and ROS decreased, the level of IL-10 increased, and the apoptosis rate decreased. Western blot results showed that, compared with the OGD/R group, IRN significantly upregulated the expression of p-EGFR and p-AKT. We demonstrated that IRN employs a multi-target approach to counteract stroke pathology, as revealed by computational predictions and experimental validation. The convergence of network pharmacology, molecular docking, and in vitro assays enabled the identification of key targets, including EGFR, AKT1, and JAK2. These methods also indicate that IRN can simultaneously regulate the inflammatory, oxidative stress, and apoptotic processes. Compared to singletarget agents, IRN exhibits superior multi-target therapeutic potential. These findings, derived from database analyses and cell-based assays, provide a comprehensive overview of IRN's pharmacological effects, which are consistent with its known neuroprotective benefits. Although these results are obtained from cell-based models and predictive databases, they provide a cohesive mechanistic rationale that is consistent with the neuroprotective properties ascribed to IRN. Further studies in animal models and in clinical settings must be conducted to apply these insights for therapeutic purposes. IRN can exert neuroprotective effects against stroke by regulating key targets, including EGFR. It exhibits anti-inflammatory, anti-oxidative, and anti-apoptotic activities, which provide a reasonable basis for developing IRN as a drug for treating stroke.

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