枳椇属三萜类成分治疗非小细胞肺癌作用机制的网络药理学分析

Exploration on the Potential Mechanism of Hovenia Triterpenoids in the Treatment of Non-small Cell Lung Cancer:A Network Pharmacological Analysis

目的基于网络药理学方法及分子对接技术探讨枳椇属三萜类成分治疗非小细胞肺癌的关键成分及作用机制。方法利用TCMSP数据库结合文献检索获得枳椇属三萜类化合物,根据Lipinski规则筛选出活性成分。使用Swiss Target Prediction数据库预测出枳椇属三萜类活性成分的作用靶点;使用GeneCards、OMIM、TTD和PharmGkb等疾病数据库收集非小细胞肺癌疾病相关靶点;取枳椇属三萜类活性成分的作用靶点与非小细胞肺癌疾病相关靶点二者的交集(共同靶点),作为枳椇属三萜类成分治疗非小细胞肺癌的潜在作用靶点。通过STRING数据库与Cytoscape(V3.8.2)软件构建潜在作用靶点的蛋白互作(PPI)网络,并筛选出核心靶点。使用R 4.0.3软件中的“clusterProfiler”程序包对核心靶点进行GO功能及KEGG通路富集分析。利用Ualcan数据库获得核心靶点在肺腺癌或肺鳞癌中的表达情况并进行相关分析;通过Kaplan-Meier Plotter在线数据库分析核心靶点对非小细胞肺癌患者5年生存率的影响。联合使用AutoDock Vina及R软件对4个与非小细胞肺癌关联性较高的核心靶点分别与潜在活性成分进行分子对接验证。结果共筛选出枳椇属三萜类活性成分4个(Ceanothetric acid、Hovenic acid、Jujubogenin、Saponin H)及其作用靶点285个;筛选得到5916个非小细胞肺癌的疾病相关靶点;通过韦恩分析得到共同靶点186个,即枳椇属三萜类成分治疗非小细胞肺癌的潜在作用靶点。通过PPI分析、筛选出12个核心靶点:STAT3、SRC、PIK3CA、GRB2、MAPK3、MAPK1、AKT1、JAK1、JAK2、EGFR、MAPK14、IL2。潜在作用靶点涉及1161个生物过程条目、39个细胞组分条目、72个分子功能条目,KEGG信号通路主要包括EGFR酪氨酸激酶抑制剂抗性、癌症中PD-L1表达及PD-1检查点途径、JAK-STAT信号通路等。与正常肺组织相比,SRC的表达水平在肺腺癌和肺鳞癌中均明显升高(P<0.05),STAT3及GRB2的表达水平则明显降低(P<0.05);而PIK3CA在肺腺癌中的表达水平相较于正常肺组织明显降低(P<0.05),在肺鳞癌中的表达水平则明显升高(P<0.05)。SRC低表达、PIK3CA及GRB2高表达的肺癌患者5年生存率更高。4个活性成分与核心靶点STAT3、SRC、PIK3CA、GRB2的结合能力良好,其中Jujubogenin、Saponin H与PIK3CA存在极强结合能力。结论枳椇属三萜类成分通过多成分、多靶点、多通路治疗非小细胞肺癌,其机制可能与抗EGFR-TIKs耐药性及调节免疫功能的相关信号通路有关。

Objective To explore the key components and mechanism of Hovenia triterpenoids in the treatment of non-small cell lung cancer(NSCLC)based on network pharmacology and molecular docking technology.Methods The TCMSP database was used in conjunction with literature searches for Hovenia triterpenoids compound,and the active components were screened according to Lipinski rules.Swiss Target Prediction database was used to predict the targets of active components from Hovenia triterpenoids;GeneCards,OMIM,TTD and PharmGkb disease databases were used to collect NSCLC-related targets;take the intersection of the active components from Hovenia triterpenoids and NSCLC-related targets(common targets).The STRING database and Cytoscape(V3.8.2)software were used to build a protein-protein interaction(PPI)network of potential targets and to screen the core targets.GO function and KEGG pathway enrichment analysis of the core targets were performed using the"clusterProfiler"package in R 4.0.3 software.The Ualcan database was used to obtain and correlate the expression of the core targets in lung adenocarcinoma(LUAD)or lung squamous carcinoma(LUSC),and the Kaplan-Meier Plotter online database was used to analyze the impact of the core targets on the 5-year survival rate of patients with NSCLC.The 4 core targets with high association with NSCLC were jointly validated with potential active components using AutoDock Vina and R software for molecular docking.Results A total of 4 Hovenia triterpenoids(Ceanothetric acid,Hovenic acid,Jujubogenin,Saponin H)and 285 of their targets were screened;5916 disease-related targets for NSCLC were screened;186 common targets(intersecting targets)were obtained through Wayne analysis,i.e.Hovenia triterpenoids are potential targets for the treatment of NSCLC.Twelve core targets were identified through PPI analysis,which included STAT3,SRC,PIK3CA,GRB2,MAPK3,MAPK1,AKT1,JAK1,JAK2,EGFR,MAPK14,IL2.The potential targets of action involved 1161 biological process entries,39 cellular component entries and 72 molecular function entries.The KEGG signaling pathway mainly included EGFR tyrosine kinase inhibitor resistance,PD-L1 expression and PD-1 checkpoint pathway in cancer,and JAK-STAT signaling pathway.Compared with normal lung tissue,the expression level of SRC was significantly increased in both LUAD and LUSC(P<0.05),while the expression levels of STAT3 and GRB2 are significantly decreased(P<0.05).The expression level of PIK3CA in LUAD was significantly decreased compared with that in normal lung tissue,while the expression level in LUSC was significantly increased compared with that in normal lung tissue(P<0.05).The 5-year survival rate was relatively high in lung cancer patients with low expression of SRC and high expressions of PIK3CA and GRB2.Four active components bound well to the core targets STAT3,SRC,PIK3CA and GRB2,among which Jujubogenin and Saponin H had extremely strong binding ability to PIK3CA.Conclusion Hovenia triterpenoids are used to treat NSCLC through multi-component,multi-target and multi-pathway,and the mechanism may be related to anti-EGFR-TIKs resistance and related signaling pathways that regulate immune function.