基于网络药理学联合孟德尔随机化分析骨碎补总黄酮治疗骨质疏松症的作用机制

Analysis of Mechanism of Action of Total Flavonoids from Drynariae Rhizoma in the Treatment of Osteoporosis Based on Network Pharmacology Combined with Mendelian Randomization

摘要:

目的：本研究旨在利用网络药理学、孟德尔随机化等生物信息学方法明确骨碎补总黄酮（TFRD）治疗骨质疏松症（OP） 的作用机制。方法：通过TCMSP、HERB、BATMAN等数据库及相关文献，筛选TFRD活性成分及对应靶点。从TTD、OMIM、GeneCards数据库及GEO数据集（GSE56815） 获取OP疾病靶点与差异表达基因。利用Venn图获取“药物-疾病-差异基因”交集靶点，并通过STRING数据库和Cytoscape软件构建蛋白质相互作用（PPI） 网络，进行拓扑分析。借助DAVID数据库进行基因本体（GO） 功能与京都基因与基因组百科全书（KEGG） 通路富集分析。利用AutoDock-Vina对核心活性成分与关键靶点进行分子对接验证，使用Gromacs软件对结合能最强的受体-配体复合物进行分子动力学模拟。利用孟德尔随机化验证基因与疾病的因果关系。结果：共筛选到TFRD的活性成分有木犀草素、黄芩素、圣草素等10个，对应靶点271个，获得42 个TFRD 治疗OP 的潜在靶点。PPI网络筛选出核心靶点为蛋白激酶B1 （AKT1）、胰岛素样生长因子1 受体（IGF1R）、表皮生长因子受体（EGFR）、B细胞淋巴瘤2（BCL2）、血小板衍生生长因子受体β（PDGFRB）、原癌基因（SRC）、B细胞淋巴瘤2样蛋白1（BCL2L1）、FMS样酪氨酸激酶3（FLT3）。GO分析显示潜在靶点主要富集于细胞凋亡过程的负调控、磷脂酰肌醇3-激酶/蛋白激酶B（PI3K/AKT） 信号通路的正调控、炎症反应等生物学过程；KEGG富集分析主要集中在PI3K/AKT信号通路。分子对接结果表明核心活性成分与核心靶点的分子对接结合能均≤-5 kcal/mol，结合稳定性良好。分子动力学模拟显示柚皮素与核心靶点BCL2能够形成构象稳定、结合紧密的复合物。孟德尔随机化分析显示BCL2与OP呈现密切相关性。结论：本研究揭示了TFRD可能通过激活PI3K/AKT信号通路影响BCL2的表达水平调控凋亡程序，从而改善骨代谢失衡，发挥抗骨质疏松作用。

Abstract:

Abstract：Objective：To elucidate the mechanism of action of total flavonoids from Drynariae Rhizoma （TFRD） in the treatment of osteoporosis （OP） using bioinformatics approaches including network pharmacology and Mendelian randomization. Methods： The active components of TFRD and their corresponding targets were screened using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）， HERB， and the Bioinformatics Analysis Tool for Molecular mechANism （BATMAN） databases， as well as relevant literature. OP disease targets and differentially expressed genes were obtained from The Therapeutic Target Database （TTD），Online Mendelian Inheritance in Man （OMIM）， and The Human Gene Database （GeneCards） databases， and the GEO dataset （GSE56815）. Intersection targets among drug and disease differentially expressed genes were identified using a Venn diagram. A protein-protein interaction （PPI） network was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins （STRING） database and Cytoscape software， followed by topological analysis. Gene Ontology （GO） functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis were performed using the Database for Annotation， Visualization， and Integrated Discovery （DAVID）. Molecular docking validation between core active components and key targets was conducted using AutoDock-Vina，and molecular dynamics simulations of the receptor-ligand complex with the strongest binding affinity were performed using Gromacs software. Mendelian randomization was employed to validate the causal relationship between genes and disease. Results： A total of 10 active components of TFRD were screened， including luteolin， baicalein， and eriodictyol， corresponding to 271 targets， and 42 potential targets of TFRD for OP treatment were identified. PPI network screening identified the core targets as protein kinase B1 （AKT1），insulin-like growth factor 1 receptor （IGF1R），epidermal growth factor receptor （EGFR），B-cell lymphoma 2 protein （BCL2），platelet-derived growth factor receptor beta （PDGFRB），proto-oncogene tyrosine-protein kinase Src （SRC），B-cell lymphoma 2-like protein 1 （BCL2L1）， and FMS-like tyrosine kinase 3 （FLT3）. GO analysis revealed that the targets were mainly enriched in biological processes such as negative regulation of apoptotic process， positive regulation of phosphatidylinositol 3-kinase/protein kinase B （PI3K/AKT） signaling pathway， and inflammatory response. KEGG enrichment analysis primarily focused on the PI3K/AKT signaling pathway. Molecular docking results showed that the binding energies between core active components and core targets were all ≤ -5 kcal/mol， indicating good binding stability. Molecular dynamics simulations demonstrated that naringenin could form a conformationally stable and tightly bound complex with the core target BCL2. Mendelian randomization analysis revealed a strong correlation between BCL2 and OP. Conclusion： This study reveals that TFRD may regulate the apoptotic program by activating the PI3K/AKT signaling pathway to affect the expression of BCL2，thereby ameliorating bone metabolic imbalance and exerting antiosteoporotic effects.