健胃消食口服液的免疫调节作用及网络药理学机制研究

Study on the Immunomodulatory Effect and Network Pharmacological Mechanism of Jianwei Xiaoshi Oral Liquid

目的:通过小鼠免疫功能抑制模型探讨健胃消食口服液的免疫调节作用,同时整合网络药理学方法探讨健胃消食口服发挥液健胃消食作用的机制。方法:按体质量随机分为对照组,模型组,阳性药及健胃消食口服液低、中、高剂量(0.62、1.24、2.48 g·kg^(-1))组,腹腔注射环磷酰胺制备小鼠免疫功能抑制模型。观察小鼠表观状态、体质量、脾指数和胸腺指数变化;运用淋巴细胞转化实验和B细胞抗体生成实验检测小鼠细胞免疫和体液免疫能力;结合流式细胞术检测淋巴细胞中辅助性T细胞(CD4^(+)T)和细胞毒性T细胞(CD8^(+)T)细胞比例,观察T淋巴细胞亚群的变化;应用中医药整合药理学研究平台(TCMIP)V2.0预测健胃消食口服液的潜在活性成分与靶点;根据主治病症临床表现,以“Gastritis”“Anorexia”为关键词在GeneCards等数据库获取疾病靶点;利用Cytoscape软件构建蛋白质-蛋白质相互作用网络,筛选健胃消食口服液核心作用靶点;使用欧易云平台对核心靶点进行基因本体(GO)及京都基因与基因组百科全书(KEGG)通路富集分析;应用液相色谱-质谱法检测脾脏中三羧酸循环代谢产物以初步了解代谢与免疫之间的关系。结果:体外实验证明,与对照组比较,模型组小鼠体质量持续下降。与模型组比较,给药组小鼠体质量从给药后第4天开始相继出现不同程度恢复,饮食、反应状态、蜷缩弓背情况也有不同程度的改善;给药组小鼠脾脏体比、胸腺脏体比及小鼠脾淋巴细胞转化能力和B细胞抗体生成能力显著提高。通过筛选得到健胃消食口服液中95个成分、650个成分靶点及1490个疾病靶点。结合CytoNCA插件筛选到56个核心靶点。富集分析发现,健胃消食口服液健胃消食的相关机制与C型凝集素受体信号通路和辅助性T细胞17(Th17)细胞分化等免疫通路有关。三羧酸循环中琥珀酸、二磷酸腺苷、乌头酸、柠檬酸、丙酮酸的代谢产物含量均呈现上升趋势,但是无剂量相关性。结论:初步揭示了健胃消食口服液的免疫相关作用机制,为健胃消食口服液的临床应用提供依据。

Objective:In this study,the immunosuppression model of mice was established to discuss the regulatory effect of Jianwei Xiaoshi oral liquid(JWXS)on immune function.The stomach strengthening and digestion mechanism of JWXS was explored by network pharmacology.Methods:The mice were randomly divided into control,model,positive and low,medium and high dose(0.62,1.24,2.48 g·kg^(-1))groups of Jianwei Xiaoshi oral liquid according to body mass.A mouse immune suppression model was established by intraperitoneal injection of cyclophosphamide.The changes in the apparent state,body weight,spleen index and thymus index of mice were observed.The cell-mediated immunity and humoral immunity of mice were tested by lymphocyte transformation and B cell antibody production experiments.Then the T helper cells(CD4^(+)T)and cytotoxic T cells(CD8^(+)T)proportion in lymphocytes was detected by flow cytometry to observe the varies of T lymphocyte subsets.Afterwards,the Integrative Pharmacology based Research Platform of Traditional Chinese Medicine(TCMIP V2.0)was used to predict the potential active ingredients and targets of JWXS.According to the clinical manifestations,disease targets were obtained by the keywords"Gastritis"and"Anorexia"in databases such as GeneCards database,etc.The protein-protein interaction(PPI)network was constructed by cytoscape to screen the core targets of JWXS.Then,the core targets were analyzed using the OECloud tools for the Gene Ontology(GO)function and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.Liquid chromatography-mass spectrometry(LC-MS)was further used to detect Citric acid cycle metabolites in the spleen to gain a preliminary understanding of the relationship between metabolism and immunity.Results:In vitro experiments have shown that compared to the continuous weight loss of the model group mice,the weight of the treatment group gradually recovered to varying degrees from the 4th day after administration,and the diet,reaction state,and hunched back of the treatment group mice also improved.Additionally,the ratios of spleen to body and thymus to body were significantly improved in the JWXS groups,along with enhanced transformation ability of mouse spleen lymphocytes and B cell antibody generation ability.The screening identified 95 components,650 component targets,and 1490 disease targets.Combined with the CytoNCA plugin,56 core targets were screened for enrichment analysis.The mechanisms of JWXS against gastritis and anorexia were suggested to immune pathways such as the C-type lectin receptor signaling pathway and Th17 cell differentiation.The content of metabolites such as succinic acid,adenosine diphosphate(ADP),aconitic acid,citric acid,and pyruvate in the tricarboxylic acid cycle showed an upward trend,but there was no dose correlation.Conclusion:This study preliminarily revealed the immune-related mechanism of JWXS,providing a foundation for its clinical application.