基于网络药理学、分子对接与分子动力学模拟探讨华蟾酥毒基对翼状胬肉成纤维细胞的影响作用

Exploring the Effects of Cinobufagin on Human Pterygium Fibroblasts Using Network Pharmacology, Molecular Docking, and Molecular Dynamics Simulations

摘要: 目的：运用网络药理学、分子对接、分子动力学模拟和细胞实验预测华蟾酥毒基(Cinobufagin)对人翼状胬肉成纤维(Human Pterygium Fibroblasts, HPF)细胞影响作用及其可能作用靶点。方法：运用Swiss Target Prediction、GeneCards和OMIM等数据库筛选翼状胬肉(Pterygium)与华蟾酥毒基的相关靶点，绘制交集靶点韦恩图，STRING数据库构建蛋白相互作用网络图，运用GO与KEGG富集分析药物－疾病关键靶点的功能与通路，分子对接验证华蟾酥毒基与关键靶点相互结合能力，分子动力学模拟再次分析华蟾酥毒基与关键靶点的结构稳定性和相互作用；通过细胞培养，运用CCK-8法、RT-qPCR实验研究华蟾酥毒基对HPF细胞增殖、凋亡因子作用及关键靶点的影响。结果：检索获取华蟾酥毒基与翼状胬肉共同靶点36个；核心靶点涉及mTOR和MDM2；富集分析显示华蟾酥毒基主要作用在PI3K/AKT通路；分子对接发现其与mTOR、MDM2结合强烈；分子动力学模拟进一步确认其结构稳定性及相互作用。CCK8实验表明华蟾酥毒基抑制HPF细胞活力，RT-qPCR实验结果显示，华蟾酥毒基可干预相关靶点的mRNA表达。结论：通过网络药理学、分子对接、分子动力学模拟和RT-qPCR共同验证了华蟾酥毒基可抑制HPF细胞增殖及凋亡相关因子的表达；其作用机制可能与mTOR、MDM2等靶点有关。

Abstract: Objective: This study aims to predict the effects of cinobufagin on human pterygium fibroblasts (HPF) and identify its potential targets using network pharmacology, molecular docking, molecular dynamics simulations, and cellular experiments. Methods: We utilized databases such as Swiss Target Prediction, GeneCards, and OMIM to screen for targets associated with pterygium and cinobufagin. A Venn diagram was created to illustrate the intersecting targets, and the STRING database was employed to construct a protein-protein interaction network. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to explore the functions and pathways of key drug-disease targets. Molecular docking was performed to assess the binding affinity of cinobufagin to these key targets, while molecular dynamics simulations were used to further analyze the structural stability and interactions of cinobufagin with the identified targets. Additionally, cell culture and reverse transcription quantitative polymerase chain reaction (RT-qPCR) experiments were carried out to investigate the effects of cinobufagin on HPF cell proliferation, apoptotic factors, and key targets. Results: A total of 36 common targets for cinobufagin and pterygium were identified, with core targets including mTOR and MDM2. Enrichment analysis indicated that cinobufagin primarily acts on the PI3K/AKT signaling pathway. Molecular docking studies demonstrated strong binding affinity of cinobufagin to mTOR and MDM2. Molecular dynamics simulations further confirmed the structural stability and interactions of cinobufagin with these targets. Results from the RT-qPCR experiments suggested that cinobufagin could modulate the mRNA expression of related targets. Conclusion: The combined approaches of network pharmacology, molecular docking, molecular dynamics simulations, and RT-qPCR provide evidence that cinobufagin can inhibit HPF cell proliferation and the expression of apoptosis-related factors. Its mechanism of action may be associated with targets such as mTOR and MDM2.