背景:皂角(GSF)在传统医学中通常用于治疗支气管哮喘等呼吸系统疾病。然而,目前尚缺乏对GSF的化学组成及GSF治疗支气管哮喘的药理物质和作用机制的研究。
目的:采用超高效液相色谱-四极杆-Orbitrap高分辨质谱(UHPLC-Q-OrbitrapHRMS)对GSF的化学成分进行分析。在这项研究中,我们结合了网络药理学,分子对接技术,并进行实验验证,探讨GSF治疗支气管哮喘的疗效及潜在机制。
方法:使用UHPLC-Q-OrbitrapHRMS对GSF的化学成分进行表征。对鉴定的化学成分进行瑞士吸收中的活性成分筛选,Distribution,代谢,和排泄(ADME)数据库。相关数据库用于检索与支气管哮喘疾病相关的活性成分和靶标的靶蛋白。并选择了两者之间的共同目标。随后,使用String数据库和Cytoscape软件构建蛋白质-蛋白质相互作用(PPI)网络,以识别关键靶标.使用Metascape数据库进行基因本体论(GO)和京都基因和基因组百科全书(KEGG)富集分析。使用Cytoscape构建了“组件共同目标”网络,以识别主要活性成分。使用AutoDock软件进行分子对接验证。采用卵清蛋白(OVA)建立支气管哮喘小鼠模型,并测量小鼠的肺器官指数。用苏木精和伊红(HE)观察肺组织病理变化,高碘酸希夫(PAS),和Masson染色。使用肺功能测试仪器评估小鼠的呼吸阻力(Penh)。酶联免疫吸附试验(ELISA)用于确定IgE的水平,小鼠血清中的IL-4、IL-5和IL-13。免疫荧光染色检测肺组织中AKT和PI3K的蛋白表达水平。进行体外实验以观察棘突胞酸(EA)对IL-4刺激的人ASMC(hASMC)的作用。使用CCK-8测定法测量细胞活力以计算EA的IC50值。进行伤口愈合试验以观察EA对愈合程度的影响。RT-qPCR检测EA对ALBmRNA表达水平的影响,SRC,TNF-α,细胞中的AKT1和IL6。
结果:从GSF中鉴定出总共95种化学成分。其中,37被鉴定为活性成分。活性成分和疾病靶标之间有169个重叠的靶标。蛋白质-蛋白质相互作用(PPI)网络的拓扑分析确定了核心靶标为IL6,TNF,ALB,AKT1和SRC。富集分析显示GSF治疗支气管哮喘主要涉及AGE-RAGE信号通路和PI3K-Akt信号通路,在其他人中。主要活性成分包括13(s)-HOTRE,亚麻酸,还有acacetin.分子对接结果表明,GSF的关键成分与核心靶标之间具有良好的结合活性。动物实验研究表明GSF能有效改善症状,肺功能,OVA诱导的哮喘小鼠的肺组织病理变化,同时减轻炎症反应。GSF降低了AKT和PI3K蛋白的荧光强度。EA的IC50值为30.02μg/ml。EA(30)显著促进IL4刺激的hASMCs细胞的增殖。EA(30)显著增加ALB和SRCmRNA的表达,降低TNF-α的表达,AKT,和IL6mRNA。
结论:在GSF中发现的多种活性成分通过多种靶标和途径发挥其抗炎作用。这项初步研究揭示了其治疗支气管哮喘的核心靶标和作用机制。这些发现为进一步研究GSF的药理物质和质量控制提供了有价值的见解。
BACKGROUND: Gleditsiae Sinensis Fructus (GSF) is commonly used in traditional medicine to treat respiratory diseases such as bronchial asthma. However, there is a lack of research on the chemical composition of GSF and the pharmacological substance and mechanism of action for GSF in treating bronchial asthma.
OBJECTIVE: The chemical constituents of GSF were analyzed using ultrahigh-performance liquid chromatography-quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS). In this study, we combined network pharmacology, molecular docking techniques, and experimental validation to explore the therapeutic efficacy and underlying mechanism of GSF in the treatment of bronchial asthma.
METHODS: Characterization of the chemical constituents of GSF was conducted using UHPLC-Q-Orbitrap HRMS. The identified chemical components were subjected to screening for active ingredients in the Swiss Absorption, Distribution, Metabolism, and Excretion (ADME) database. Relevant databases were utilized to retrieve target proteins for the active ingredients and targets associated with bronchial asthma disease, and the common targets between the two were selected. Subsequently, the protein-protein interaction (PPI) network was constructed using the String database and Cytoscape software to identify key targets. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using the Metascape database. The \"component-common target\" network was constructed using Cytoscape to identify the primary active ingredients. Molecular docking validation was conducted using AutoDock software. The bronchial asthma mouse model was established using ovalbumin (OVA), and the lung organ index of the mice was measured. Lung tissue pathological changes were observed using hematoxylin and eosin (HE), Periodic Acid-Schiff (PAS), and Masson staining. The respiratory resistance (Penh) of the mice was assessed using a pulmonary function test instrument. An enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of IgE, IL-4, IL-5, and IL-13 in the mouse serum. Immunofluorescence staining was performed to detect the protein expression levels of AKT and PI3K in the lung tissues. An in vitro experiment was performed to observe the effects of echinocystic acid (EA) on IL-4 stimulated Human ASMCs (hASMCs). Cell viability was measured using a CCK-8 assay to calculate the IC50 value of the EA. A wound healing test was conducted to observe the effect of EA on degree of healing. RT-qPCR was performed to detect the influence of EA on the mRNA expression levels of ALB, SRC, TNF-α, AKT1, and IL6 in the cells.
RESULTS: A total of 95 chemical constituents were identified from the GSF. Of these, 37 were identified as active ingredients. There were 169 overlapping targets between the active ingredients and the disease targets. A topological analysis of the protein-protein interaction (PPI) network identified the core targets as IL6, TNF, ALB, AKT1, and SRC. An enrichment analysis revealed that the treatment of bronchial asthma with GSF primarily involved the AGE-RAGE signaling pathway and the PI3K-Akt signaling pathway, among others. The primary active ingredients included 13(s)-HOTRE, linolenic acid, and acacetin. The molecular docking results demonstrated a favorable binding activity between the critical components of GSF and the core targets. Animal experimental studies indicated that GSF effectively improved symptoms, lung function, and lung tissue pathological changes in the OVA-induced asthmatic mice, while alleviating inflammatory responses. GSF decreased the fluorescent intensity of the AKT and PI3K proteins. The IC50 value of EA was 30.02μg/ml. EA (30) significantly promoted the proliferation of IL4-stimulated hASMCs cells. EA (30) significantly increased the expression of ALB and SRC mRNA and decreased the expressions of TNF-α, AKT, and IL6 mRNA.
CONCLUSIONS: The multiple active ingredients found in GSF exerted their anti-inflammatory effects through multiple targets and pathways. This preliminary study revealed the core target and the mechanism of action underlying its treatment of bronchial asthma. These findings provided valuable insights for further research on the pharmacological substances and quality control of GSF.