基于网络药理学-分子对接及体内实验探讨参芪抑瘤方治疗肝癌的作用机制

Mechanism of Shenqi Yiliu Decoction(参芪抑瘤方)in the Treatment of Liver Cancer Based on Network Pharmacology,Molecular Docking,and In Vivo Experiments

目的:运用网络药理学和分子对接技术探究参芪抑瘤方治疗肝癌的潜在机制,并以H22肝癌小鼠模型对核心靶点进行验证。方法:在中药系统药理数据等数据库检索参芪抑瘤方有效成分及作用靶点,通过GeneCards、OMIM、TTD、PharmGKB和DrugBank数据库获得药物及疾病相关靶点,取其交集靶点导入String数据库进行蛋白质相互作用分析,通过Bioconductor在线软件进行基因组数据的高通量分析,进行基因本体GO功能富集分析和京都基因与基因组百科全书KEGG通路富集分析。运用AutoDock vina对活性成分与核心靶点进行分子对接和结合能力预测。构建H22肝癌小鼠模型,进行肿瘤组织形态学观察并验证参芪抑瘤方对肿瘤组织中核心靶点的影响。结果:网络药理学分析结果得到参芪抑瘤方中包括槲皮素、β-谷甾醇、豆甾醇等核心成分;GO富集分析发现靶蛋白分子功能主要集中于核受体活性、配体活化、类固醇激素受体的活性等方面,细胞组成涉及膜筏、膜微域、膜区域的组成成分等方面,生物过程涉及对药物的反应、细胞对化学应激的反应、氧化应激反应等方面;KEGG富集结果分析得知,参芪抑瘤方是通过人巨细胞病毒感染、PI3K-AKT信号通路、乙肝、卡波西肉瘤相关疱疹病毒感染、MAPK等信号通路治疗肝癌;关键靶点涉及蛋白激酶B(protein kinase B,AKT1)、促分裂原活化蛋白激酶3(mitogen-activated protein kinase 3,MAPK3)、肿瘤坏死因子(tumor necrosis factor,TNF)等。分子对接结果表明3个核心成分与靶蛋白AKT1、MAPK3、TNF均有强烈的结合能力。为验证网络药理学与分子对接结果,应用H22肝癌荷瘤小鼠模型验证参芪抑瘤方对肝癌的干预作用。动物实验结果表明,与模型对照组比较,顺铂2.5×10g/kg组和参芪抑瘤方54 g/kg组瘤质量明显降低(P<0.05),其抑瘤作用明显;HE染色示各组肿瘤组织出现不同程度的坏死,参芪抑瘤方54 g/kg组最为明显;参芪抑瘤方能明显降低小鼠肝癌组织AKT1、MAPK3的蛋白表达,上调TNF-α蛋白的表达(P<0.05)。结论:参芪抑瘤方可能通过抑制AKT1、MAPK3蛋白的表达,上调TNF-α蛋白的表达,通过磷脂酰肌醇3激酶/蛋白激酶B(PI3K/AKT)及其他信号通路来调节肿瘤细胞的增殖、凋亡、迁移和血管生成等生物学过程,从而在肝癌治疗中发挥作用。

Objective:To investigate the potential mechanism of Shenqi Yiliu Decoction(参芪抑瘤方)for the treatment of liver cancer based on network pharmacology and molecular docking and to validate the core targets in the mouse model bearing H22 hepatoma.Methods:The active components and targets of Shenqi Yiliu Decoction were searched against the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP).The targets of liver cancer were screen out from GeneCards,Online Mendelian Inheritance in Man(OMIM),Therapeutic Target Database(TTD),PharmGKB,and DrugBank,and the common targets shared by the decoction and liver cancer were imported into STRING for protein-protein interaction(PPI)analysis.Bioconductor online was employed to conduct the high-throughput analysis of the genomic data.Gene ontology(GO)annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis were carried out.Molecular docking was carried out for the active component of the decoction and core targets in AutoDock Vina,and the binding energy was predicted.After the mouse model of H22 hepatoma was established,the tumor histomorphology was observed and the effect of Shenqi Yiliu decoction on the core target proteins in the tumor tissue was verified.Results:The active components of Shenqi Yiliu Decoction against tumor included quercetin,β-sitosterol,stigmasterol and so on.The GO annotation showed that the core targets were mainly involved in the molecular functions of nuclear receptor activity,ligand activation,and steroid receptor activity,the cellular components of membrane craft,membrane microdomain,and membrane region,and the biological processes of response to drug,cellular response to chemical stress,and response to oxidative stress.The KEGG pathway enrichment predicted that Shenqi Yiliu Decoction mainly treated liver cancer via human cytomegalovirus infection,phosphoinositide 3-kinase(PI3 K)-protein kinase B(AKT)signaling pathway,hepatitis B virus and Kaposi’s sarcoma-associated herpesvirus infections,and mitogen-activated protein kinase(MAPK)signaling pathway.The core targets included AKT1,MAPK3,and tumor necrosis factor(TNF).The results of molecular docking showed that the three core components had strong binding affinity to AKT1,MAPK3,and TNF.To verify the results of network pharmacology and molecular docking,we employed the H22 hepatoma-bearing mouse model to verify the therapeutic effect of Shenqi Yiliu Decoction on liver cancer.Compared with the model group,cisplatin(2.5×10^(-3) g/kg)and Shenqi Yiliu Decoction(54.06 g/kg)decreased the tumor mass(P<0.05),demonstrating significant anti-tumor effect.Shenqi Yiliu Decoction(especially at the dose of 54.06 g/kg)led to the necrosis of the tumor tissue.Furthermore,the decoction down-regulated the expression of AKT1 and MAPK3 and up-regulated that of TNF-αin the tumor tissue(P<0.05).Conclusion:Shenqi Yiliu Decoction may down-regulate the expression of AKT1 and MAPK3,up-regulate the expression of TNF-α,and regulate the proliferation,apoptosis,migration,and angiogenesis of tumor cells through the PI3 K/AKT and other signaling pathways.This study provides a basis for the clinical application and mechanism study of Shenqi Yiliu Decoction.