毛滴虫对肺腺癌作用机制的网络药理学预测及实验验证
Summary
本研究基于网络药理学和实验验证，揭示了毛 滴虫治疗肺腺癌的机制 。该研究还表明，PI3K / AKT信号通路在 Trichosanthes-Fritillaria thunbergii 治疗肺腺癌中的作用中起着至关重要的作用。
Abstract
本文旨在基于网络药理学和实验验证，研究毛 滴虫 治疗肺腺癌（LUAD）的机制。采用中药高通量实验和参考引导（HERB）数据库和相似合体法（SEA）数据库收集毛 滴虫 和 枸杞 子的有效成分和潜在靶点，并通过GeneCards和在线孟德尔遗传（OMIM）数据库查询LUAD相关靶点。利用Cytoscape软件构建了药物-成分-疾病-靶点网络。通过蛋白质-蛋白质相互作用（PPI）网络、基因本体（GO）功能、京都基因和基因组百科全书（KEGG）通路富集分析，获得核心靶点和关键通路。毛滴虫 -毛滴虫 和A549细胞的水提取物用于随后的实验验证。通过HERB数据库和文献检索，筛选出毛滴 虫 的有效化合物和157个潜在靶基因，其中144个是毛 滴 虫治疗肺腺癌的调控靶点。GO功能富集分析表明， 毛滴虫 对肺腺癌的作用机制主要是蛋白质磷酸化。KEGG通路富集分析表明， 毛滴虫 对肺腺癌的治疗主要涉及PI3K/AKT信号通路。实验验证表明， 毛滴虫 水提取物可以抑制A549细胞的增殖和AKT的磷酸化。通过网络药理学和实验验证，验证了PI3K/AKT信号通路在毛 滴虫 治疗肺腺癌中的作用中起着至关重要的作用。
Introduction
肺癌是指起源于肺支气管黏膜的恶性肿瘤，包括鳞状细胞癌、腺癌、大细胞癌、小细胞癌1。肺腺癌（LUAD）是最常见的肺癌类型，约占肺癌病例总数的40%2。大多数患者被诊断为晚期或有远处转移，因此失去了手术的机会3。在目前的临床治疗中，同步放化疗是治疗LUAD最常用的策略，但由于严重的不良反应，其应用受到限制4。
中医可有效缓解LUAD患者的临床症状，减少放化疗引起的不良反应，成为研究热点5，6，7。在中医中，肺癌属于“肺蓄积”和“肺岩”的范畴。气虚和痰、瘀、毒的相互作用是肺癌发病的重要因素。因此，补气、化痰止血是临床上治疗肺癌的主要方法8 根据中医理论9.基里洛维特里科桑特斯Maxim（Gualou）和Fritillaria thunbergii Miq（Zhebeimu）代表了治疗肺癌的常见药物对，这种组合具有清热降痰的作用10，11，12。然而，其作用机制仍不清楚，需要进一步研究。
网络药理学是一种基于系统生物学和多向药理学理论的综合方法，旨在揭示多种药物和疾病之间的复杂网络关系13。中国传统处方具有多组分、多靶点的特点，非常适合网络药理学研究14，15。近年来，网络药理学已成为中药方剂研究的有力途径，成为研究热点16，17。
然而，据我们所知，所有关于网络药理学的研究都是以文本形式呈现的。通过视频展示这项技术将大大降低学习门槛，促进这项技术的推广，这是本文的优势之一。本研究以 毛滴虫抗 肺腺癌为例，开展网络药理学预测和实验验证。
Protocol
所有网络药理学程序均按照《网络药理学评价方法指南》18进行。所有实验程序均按照北京中医药大学实验室管理规定进行。 1.网络药理预测 有源组件的选择打开HERB数据库（http://herb.ac.cn）19，以“Gualou”（ Trichosanthes kirilowii Maxim的中文名称）和“Zhebeimu”（Bulbus Fritillariae thunbergii的中文名称）作为关…
Representative Results
共鉴定出31个毛滴虫-松柏相关活性成分，包括21个毛滴虫和10个毛滴虫组分，以及144个相应的靶点。总体而言，分别从GeneCards数据库和OMIM数据库中提取了9，049个和67个LUAD相关基因。删除重复基因后，鉴定出9，057个与LUAD相关的基因。将LUAD相关基因与毛滴虫活性成分相关靶点进行交集，以获得潜在的治疗靶点。毛滴虫-毛滴虫对LUAD的药物-成分-疾病-靶点相互作用网?…
Discussion
通常，一个完整的网络药理学研究包括从数据库中识别活性成分，获取活性成分和疾病对应的靶点，构建药物-成分-疾病-靶点网络，以及核心靶点和途径的预测。通过计算机技术初步预测活性成分与核心蛋白之间的关联（分子对接），并通过实验进行最终验证。
相关数据库的选择是网络药理学最关键的部分，因为这决定了研究的质量。HERB数据库整合了Syμmap、TCMID、TCMSP和TCM-I…
Divulgations
The authors have nothing to disclose.
Acknowledgements
本研究得到了北京中医药大学创新培训计划（编号：202110026036）的支持。
Materials
| 0.25% trypsin-EDTA | Gibco | R001100 | |
| A549 cell line | Procell | CL-0016 | |
| AKT antibody | CST | 4691S | |
| BCA Protein Assay Kit | Solarbio | PC0020 | |
| Chemiluminescence detection system | Shanghai Qinxiang Scientific Instrument Factory | ChemiScope 6100 | |
| Dulbecco's modified eagle medium (DMEM) | Solarbio | 11995 | |
| Enhanced chemiluminescence (ECL) kit | ABclonal | RM00021 | |
| Fetal bovine serum | ScienCell | 0025 | |
| HRP Goat Anti-Rabbit IgG (H+L) | ABclonal | AS014 | |
| MTS assay kit | Promega | G3580 | |
| p-AKT antibody | CST | 6040S | |
| Penicillin streptomycin | Gibco | C14-15070-063 | |
| Phenylmethanesulfonyl fluoride (PMSF) | Solarbio | P0100 | |
| Phosphatase inhibitor | Beyotime | P1081 | |
| Phosphate buffered saline (PBS) | Solarbio | P1020 | |
| Polyvinylidene difluoride (PVDF) membranes | Millipore | ISEQ00010 | |
| RIPA lysis solution | Solarbio | R0010 | |
| Rotary evaporator | Shanghai Yarong Biochemical Instrument Factory | RE52CS-1 | |
| Vacuum freeze-drying mechanism | Ningbo Scientz Biotechnology | SCIENTZ-10 | |
| β-Actin antibody | ABclonal | AC026 |
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Citer Cet Article
Zhao, X., Yang, Y., Feng, J., Feng, C. Network Pharmacology Prediction and Experimental Validation of Trichosanthes–Fritillaria thunbergii Action Mechanism Against Lung Adenocarcinoma. J. Vis. Exp. (193), e64847, doi:10.3791/64847 (2023).