基于网络药理学和实验验证探讨二十五味大汤丸治疗胃溃疡的作用机制

Investigation of the mechanism of Twenty-Five Flavour Soup Pills in treating Gastric Ulcers based on network pharmacology and experimental validation

关键词:  二十五味大汤丸  网络药理学  胃溃疡  分子对接

Key words:  Twenty-Five Flavour Soup Pills  network pharmacology  gastric ulcer  molecular docking

摘要:

目的 基于网络药理学结合体内动物实验探究二十五味大汤丸治疗胃溃疡的作用机制。方法 首先使用超高效液相色谱-线性离子阱-静电场轨道阱高分辨质谱技术(UPLC-LTQ-Orbitrap-MS)检测二十五味大汤丸的入血成分。接着采用网络药理学技术预测二十五味大汤丸治疗胃溃疡的潜在靶点和通路,通过分子对接对结果进行验证。最后,采用苦寒泻下、饥饱失常法结合无水乙醇、阿司匹林溶液建立脾胃虚寒胃溃疡大鼠模型,采用苏木素-伊红(HE)染色法对胃组织进行形态病理分析,应用酶联免疫吸附法(ELISA)法检测血清中TNF-α、IL-6、GAS的含量,运用实时荧光定量聚合酶链反应(qRT-PCR)和蛋白质印迹法(Western blot)测定胃组织中IL-6、caspase-3、PI3K、EGFR表达量。结果 共鉴定出二十五味大汤丸入血成分10个。网络药理学分析获得了503个化合物靶点和5856个疾病相关靶点,PPI分析筛选出TP53、SRC、PIK3CA等7个核心靶点,KEGG分析表明PI3K、EGFR酪氨酸激酶抑制剂耐药等信号通路在二十五味大汤丸抗胃溃疡中发挥关键作用,分子对接结果显示大波斯菊苷、姜油酮及阿福豆苷与核心靶点结合力较强。动物实验结果显示,相较于模型组,各给药组血清中的TNF-α、IL-6含量显著降低(P<0.01),GAS含量升高；胃组织IL-6、caspase-3、PI3K表达降低,EGFR表达增加。结论 二十五味大汤丸通过控制PI3K-AkT信号通路,缓解炎症反应并减少细胞凋亡,从而发挥对胃溃疡的治疗作用。

Abstract:

OBJECTIVE An investigation into the mechanism of action of the Twenty-Five Flavor Soup Pills in the treatment of gastric ulcers, based on network pharmacology combined with in vivo animal experiments. METHODS Initially, ultra-high-performance liquid chromatography–linear ion trap–Orbitrap mass spectrometry (UPLC-LTQ-Orbitrap-MS) was employed to identify the absorbable constituents of the Twenty-Five Flavor Soup Pills. Subsequently, network pharmacology techniques were employed to predict potential targets and pathways for the treatment of gastric ulcers by the Twenty-Five Flavor Soup Pills, and the results were validated through molecular docking. Finally, a rat model of gastric ulcer complicated by spleen-stomach deficiency cold syndrome was established using a combination of purgative and cold-inducing herbs, irregular feeding schedules, and administration of absolute ethanol and aspirin solution. Histopathological changes in gastric tissues were assessed via hematoxylin and eosin (HE) staining. Serum levels of TNF-α, IL-6, and GAS were measured using enzyme-linked immunosorbent assay (ELISA). Moreover, the expression levels of IL-6, caspase-3, PI3K, and EGFR in gastric tissues were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. RESULTS From serum samples after administration of Twenty-Five Flavor Soup Pillsl, ten ingredients that enter the bloodstream were characterized. A network pharmacological approach identified 503 potential targets associated with the formula''s components and 5856 genes linked to gastric ulcer pathology. Subsequent PPI network topology analysis enabled the selection of seven hub targets, namely TP53, SRC, and PIK3CA among others. KEGG analysis indicated that the PI3K and EGFR tyrosine kinase inhibitor resistance signaling pathways were critically involved in mediating the anti-ulcer activity of this Tibetan medicine. Furthermore, molecular docking simulations suggested that Cosmosiin, Zingerone, and Afzelin bound strongly to the identified core targets. The results of animal experiments showed that, compared with the model group, serum levels of TNF-α and IL-6 were significantly reduced (P < 0.01) in all treatment groups, whilst GAS levels were elevated; in gastric tissue, the expression of IL-6, caspase-3 and PI3K was reduced, whilst EGFR expression was increased. CONCLUSION The Twenty-Five Flavor Soup Pills exert their therapeutic effect on gastric ulcer by modulating the PI3K-Akt signalling pathway, thereby alleviating inflammatory responses and reducing cell apoptosis.