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    基于网络药理学和细胞验证探讨蒙药冬青叶对肺功能异常治疗机制

    Therapeutic Mechanism of Mongolian Medicine Rhododendron Micranthum Turcz. Leaf in Treating Abnormal Pulmonary Function Based on Network Pharmacology and Cellular Verification

      摘要
    • 摘要:
      目的 探讨蒙药冬青叶活性成分组合防治肺功能异常的作用机制。
      方法 通过中药系统药理学数据库与分析平台和PubChem数据库筛选蒙药冬青叶活性成分，运用GeneCards数据库检索“肺功能异常”相关疾病靶点，获取蒙药冬青叶防治肺功能异常的交集靶点；用STRING数据库构建蛋白互作网络，DAVID数据库进行GO及KEGG通路富集分析。以SD大鼠为研究对象，采用腹腔注射“野百合碱”＋“幽门结扎”术建立肺功能异常病症模型，HE染色法及Masson染色法检测大鼠肺组织病理学改变；肺功能检测仪检测肺功能变化；能量代谢仪检测CO2排出量。在此基础上，用肺功能异常模型大鼠血清诱导肺动脉平滑肌细胞(pulmonary artery smooth muscle cells，PASMCs)建立肺功能异常PASMCs模型，CCK-8法检测细胞活力，筛选造模浓度及蒙药冬青叶核心成分防治肺功能异常的安全有效浓度；EdU法检测蒙药冬青叶核心成分对PASMCs细胞增殖能力的影响；Western blotting 检测PASMCs中AKT、P-AKT、mTOR、P-mTOR、EGFR、PI3K蛋白的表达。
      结果 网络药理学共筛选出40个蒙药冬青叶活性成分对应靶点321个，肺功能异常靶点2 027个，交集靶点为116个。其中蒙药冬青叶主要核心成分是槲皮素、丁香酸、莨菪亭、东莨菪苷等，且AKT、P-AKT、mTOR、P-mTOR、EGFR、PI3K等靶点可能是蒙药冬青叶防治肺功能异常的核心靶点。细胞实验表明，蒙药冬青叶核心成分组合(槲皮素、丁香酸、莨菪亭、东莨菪苷=1∶1∶1∶1)通过抑制AKT、P-AKT、mTOR、P-mTOR、EGFR、PI3K蛋白表达而起到防治肺功能异常的作用。
      结论 蒙药冬青叶能够通过抑制P-AKT、P-mTOR、EGFR、PI3K蛋白的表达水平，抑制模型大鼠血清诱导的PASMCs增殖，从而治疗肺功能异常，其核心成分为槲皮素、丁香酸、莨菪亭、东莨菪苷。

       

      Abstract:
      OBJECTIVE To explore the mechanism of active component combinations from Mongolian medicine Rhododendron micranthum Turcz. Leaf in the prevention and treatment of abnormal pulmonary function.
      METHODS The active components of Mongolian medicine Rhododendron micranthum Turcz. leaf were screened using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform and the PubChem database. Disease targets related to abnormal pulmonary function were retrieved from the GeneCards database, and the intersecting targets of Rhododendron micranthum Turcz. leaf for preventing and treating abnormal pulmonary function were obtained. Protein-protein interaction network was constructed via the STRING database, and Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses were performed using the DAVID database. SD rats were used as research subjects, and a abnormal pulmonary function model was established by intraperitoneal injection of monocrotaline combined with pyloric ligation. Histopathological changes in rat lung tissues were examined using HE staining and Masson staining; pulmonary function changes were detected with a pulmonary function tester; CO2 excretion was measured using an energy metabolism analyzer. Subsequently, a abnormal pulmonary function model of pulmonary artery smooth muscle cells(PASMCs) was established by inducing PASMCs with serum from abnormal pulmonary function model rats. Cell viability was assessed via the CCK-8 assay to screen the optimal modeling concentration and the safe and effective concentration of the core components of Rhododendron micranthum Turcz. leaf for preventing and treating abnormal pulmonary function. The effect of the core components of Rhododendron micranthum Turcz. leaf on PASMC proliferation was detected using the EdU assay. The expression levels of AKT, P-AKT, mTOR, P-mTOR, EGFR, and PI3K proteins in PASMCs were determined by Western blotting.
      RESULTS A total of 40 active ingredients were screened from Mongolian medicine Rhododendron micranthum Turcz. leaf via network pharmacology, corresponding to 321 targets; there were 2 027 targets related to abnormal pulmonary function, among which 116 intersecting targets were identified. The major core active ingredients of Rhododendron micranthum Turcz. leaf include quercetin, syringic acid acid, scopoletin and scopolin. Targets such as AKT, P-AKT, mTOR, P-mTOR, EGFR and PI3K may serve as core targets for the prevention and treatment of abnormal pulmonary function with Rhododendron micranthum Turcz. leaf . Cellular experiments demonstrated that the combination of core ingredients from Rhododendron micranthum Turcz. leaf(quercetin∶syringic acid acid∶scopoletin∶scopolin=1∶1∶1∶1) exerts therapeutic effects against abnormal pulmonary function by downregulating the protein expression of AKT, P-AKT, mTOR, P-mTOR, EGFR and PI3K.
      CONCLUSION Mongolian medicine Rhododendron micranthum Turcz. leaf can suppress the proliferation of pulmonary artery smooth muscle cells(PASMCs) induced by serum from model rats via reducing the expression levels of P-AKT, P-mTOR, EGFR and PI3K proteins, thereby alleviating abnormal pulmonary function. The main active constituents are quercetin, syringic acid acid, scopoletin and scopolin.

       

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