两地汤治疗绝经综合征机制研究

Mechanism study of Liangdi Decoction in treating menopausal syndrome

关键词:  两地汤  绝经综合征  炎症性老化  网络药理学  分子对接

Key words:  Liangdi Decoction  menopausal syndrome  inflammaging  network pharmacology  molecular docking

摘要:

[目的] 基于网络药理学分析及体内实验探讨两地汤治疗绝经综合征（MPS）的作用机制。[方法] 利用整合传统中医临床和实验证据的更新数据库（HERB）、有机小分子生物活性数据库（Pubchem）、药物分类和靶点预测数据库（superpred）获得两地汤的主要活性成分及靶点基因。通过人类基因的综合数据库（GeneCards）、人类在线孟德尔遗传搜索数据库（OMIM）、药物数据与全面药物靶点信息数据库（DrugBank）获得MPS的相关靶点。选取药物和疾病交集靶点，并将共同靶点录入STRING数据库，制作蛋白质-蛋白质相互作用网络图（PPI网络图），通过Cytoscape3.10.3软件获得“药物-活性成分-靶点”网络图。基因功能注释及通路富集分析通过DAVID在线数据库予以实施，具体包括基因本体（GO）功能注释与京都基因与基因组百科全书（KEGG）信号通路分析。基于AutoDock Tools 1.5.7软件和CB-Dock2平台，文章对网络药理学所预测的关键信号通路中的蛋白因子与筛选出的核心活性成分进行了分子对接，从而在分子层面验证其相互作用潜能。通过体内实验检测白细胞介素-6（IL-6）、信号转导和转录激活因子3（STAT3）、磷酸化STAT3（P-STAT3）、半胱氨酸天冬氨酸蛋白酶3（Caspase-3）、B淋巴细胞瘤-2（Bcl-2）、缺氧诱导因子-1（HIF-1）表达，验证其改善MPS卵巢衰老的作用机制。[结果] 筛选得到5个核心蛋白（IL-6、STAT3、HIF-1、Caspase-3、Bcl-2和8个关键活性成分（松柏苷、柳杉酚、儿茶素、异前胡醚、马美辛、白当归醇、金合欢素、醋酸橙黄胺），对接结合能均<-5 kcal/mol。实验表明，两地汤可以降低IL-6、STAT3、HIF-1、Caspase-3蛋白表达，提高Bcl-2蛋白表达。[结论] 两地汤可能通过抑制HIF-1信号通路，调控IL-6、STAT3、Caspase-3、Bcl-2等蛋白表达，降低缺氧诱导因子水平和炎症水平，增强抗凋亡表达，达到延缓卵巢衰老的作用。

Abstract:

[Objective] To explore the mechanism of Liangdi Decoction（LDD） in treating menopausal syndrome（MPS） based on network pharmacology and in vivo experiments. [Methods] The main active ingredients and target genes of LDD were obtained from databases including HERB（a high-throughput experiment- and reference-guided database of traditional Chinese medicine），PubChem，and SuperPred. Disease targets for MPS were retrieved from GeneCards，OMIM，and DrugBank. The common targets between LDD and MPS were selected and imported into the STRING database to construct a protein-protein interaction（PPI） network. The “drug-active ingredient-target” network was visualized using Cytoscape 3.10.3. Gene Ontology（GO） functional annotation and Kyoto Encyclopedia of Genes and Genomes（KEGG） pathway enrichment analysis were performed using the DAVID database. Molecular docking of core active ingredients with key protein factors in predicted signaling pathways was conducted using AutoDock Tools 1.5.7 and CB-Dock2 to validate potential interactions. In vivo experiments were performed to measure the expression levels of interleukin-6（IL-6），signal transducer and activator of transcription 3（STAT3），phosphorylated STAT3（p-STAT3），Caspase-3，B-cell lymphoma-2（Bcl-2），and hypoxia-inducible factor-1α（HIF-1α），verifying the mechanism of LDD in ameliorating ovarian aging in MPS. [Results] Six core proteins（IL-6，STAT3，HIF-1α，Caspase-3，Bcl-2） and eight key active ingredients（coniferin，sugiol，catechin，isoimperatorin，marmesin，byakangelicol，acacetin，poncimarin） were screened. The molecular docking binding energies were all< -5 kcal/mol. Experiments showed that LDD could down-regulate the protein expression of IL-6，STAT3，HIF-1α，and caspase-3，and up-regulate the expression of Bcl-2. [Conclusion] LDD may delay ovarian aging by inhibiting the HIF-1 signaling pathway，regulating the expression of proteins such as IL-6，STAT3，Caspase-3，and Bcl-2，thereby reducing hypoxia-inducible factor levels and inflammation，and enhancing anti-apoptotic expression.