基于UPLC-Q-TOF-MS和网络药理学探究健脑通络胶囊治疗脑梗死作用机制

Investigating the Mechanism of Tongluo Jiannao Capsule in Treating Cerebral Infarction Using UPLC-Q-TOF-MS and Network Pharmacology

关键词:  健脑通络胶囊  UPLC-Q-TOF-MS  分子网络  网络药理学  脑梗死

Key words:  Jiannao Tongluo capsules  UPLC-Q-TOF-MS  molecular networking  network pharmacology  cerebral infarction

摘要:

目的? 采用超高效液相色谱-四级杆-飞行时间质谱(ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry, UPLC-Q-TOF-MS)联用分子网络(molecular networking)技术快速解析健脑通络胶囊(Jiannao Tongluo Capsule, JNTL)的化学成分；并结合网络药理学和体外实验验证JNTL治疗脑梗死(cerebral Infarction, CI)的潜在作用机制。方法? 采用正负离子模式质谱扫描获取JNTL二级质谱数据,并基于MS/MS碎片模式相似性构建全球天然产物社会分子网络(global natural products social molecular networking, GNPS),通过该网络中结相似结构分子簇、一/二级质谱数据和相关参考文献快速鉴定JNTL成分；通过网络药理学分析JNTL治疗CI的核心靶点和潜在作用机制,并建立可视化网络；最后通过体外建立HT22小鼠海马神经元细胞氧糖剥夺/复氧模型(oxygen-glucose deprivation/reoxygenation model, OGD/R)进行验证。结果? 在JNTL中鉴定出51个化学成分,包括有机酸类化合物11个、黄酮及其苷类化合物7个、三萜皂苷类化合物6个、环烯醚萜苷类化合物2个及其他类化合物25个。经网络药理学分析成分靶点与CI疾病靶点共有105个交集靶点,基因本体论(gene ontology, GO)功能富集分析得到608种生物过程(biological process, BP)、85种细胞组分(cellular component, CC)、159种分子功能(molecular function, MF)；京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes, KEGG)通路富集分析得到146条通路(P＜0.05)；蛋白互作网络(protein-protein interaction, PPI)分析得到白细胞介素-6(interleukin-6, IL-6)、肿瘤坏死因子(tumor necrosis factor, TNF)、蛋白激酶Bα(protein kinase B alpha, AKT1)等核心靶点；体外实验结果显示当JNTL血清浓度不高于20%时,对正常HT22细胞未产生明显毒性作用(P＞0.05)。在OGD/R模型中,JNTL可浓度依赖性地提高细胞存活率,显著减轻神经元损伤(P＜0.01)。进一步通过ELISA检测发现,JNTL能显著抑制OGD/R诱导的炎症因子TNF-α、IL-6和IL-1β的释放(P＜0.05)。Western blotting分析显示,JNTL可激活PI3K-Akt信号通路,表现为p-PI3K/PI3K和p-Akt/Akt比值显著上升,并有效抑制HIF-1α蛋白的异常积累(P＜0.01)。上述结果提示,JNTL可能通过抑制炎症反应、激活PI3K-Akt信号通路及调控HIF-1α表达,发挥神经保护作用。结论 本研究首次从JNTL中鉴定出51个化学成分，明确了其化学物质基础；并首次揭示JNTL可能通过激活PI3K-Akt信号通路、抑制炎症因子（TNF-α, IL-6, IL-1β）释放及下调HIF-1α蛋白表达，从而在OGD/R模型中发挥神经保护作用，并为阐明JNTL治疗CI的药效物质基础及分子机制提供了关键科学依据。

Abstract:

ABSTRACT: OBJECTIVE The ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF-MS) combined with molecular networking technology was employed to rapidly identify the chemical constituents of Jiannao Tongluo Capsule (JNTL); integrated with network pharmacology and in vitro experiments, the potential mechanism of JNTL in treating cerebral infarction (CI) was further validated. METHODS MS/MS spectral data of JNTL were acquired using both positive and negative ion mode mass spectrometry. A Global Natural Products Social Molecular Networking (GNPS) was constructed based on MS/MS fragmentation pattern similarity. Components of JNTL were rapidly identified by analyzing clusters of structurally similar molecules within the network, along with MS/MS data and relevant literature. Network pharmacology was applied to predict the core targets and potential mechanisms of JNTL in the treatment of cerebral infarction (CI), and visualization networks were generated. Finally, an in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) model was established using HT22 mouse hippocampal neuronal cells for experimental validation. RESULTS A total of 51 chemical constituents were identified in JNTL, including 11 organic acids, 7 flavonoids and their glycosides, 6 triterpenoid saponins, 2 iridoid glycosides, and 25 other compounds. Network pharmacology analysis revealed 105 overlapping targets between the component targets of JNTL and the disease targets of cerebral infarction (CI). Gene Ontology (GO) functional enrichment analysis identified 608 biological processes (BP), 85 cellular components (CC), and 159 molecular functions (MF). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated 146 significant pathways (P < 0.05). Protein-protein interaction (PPI) network analysis identified core targets such as interleukin-6 (IL-6), tumor necrosis factor (TNF), and protein kinase B alpha (AKT1). In vitro experimental results showed that JNTL-containing serum at concentrations up to 20% did not exhibit significant toxicity toward normal HT22 cells (P > 0.05). In the OGD/R model, JNTL concentration-dependently increased cell viability and significantly alleviated neuronal injury (P < 0.01). Furthermore, ELISA results demonstrated that JNTL significantly inhibited the release of the inflammatory factors TNF-α, IL-6, and IL-1β induced by OGD/R (P < 0.05). Western blotting analysis indicated that JNTL activated the PI3K-Akt signaling pathway, as evidenced by significantly increased ratios of p-PI3K/PI3K and p-Akt/Akt, and effectively suppressed the abnormal accumulation of HIF-1α protein (P < 0.01). These findings suggest that JNTL may exert neuroprotective effects by inhibiting inflammatory responses, activating the PI3K-Akt signaling pathway, and regulating HIF-1α expression. CONCLUSION For the first time, this study identified 51 chemical constituents from JNTL, thereby elucidating its chemical foundation. Furthermore, it demonstrated that JNTL may exert neuroprotective effects in the OGD/R model by activating the PI3K-Akt signaling pathway, inhibiting the release of inflammatory factors (TNF-α, IL-6, IL-1β), and downregulating HIF-1α protein expression. Additionally, it provided key scientific evidence for elucidating the pharmacodynamic material basis and molecular mechanisms underlying JNTL's therapeutic effects on CI.