system: [SYSTEM]

user: 3. 结果 3.1. 扶正抑瘤方活性化合物与靶标 经TCMSP及ETCM数据库筛选汇总得到扶正抑瘤方中各味药物活性成分195个(见表1)，通过SwissTargetPrediction数据库及STITCH数据库预测，去重后得到对应靶点676个。 3.2. 靶点映射与网络构建 将筛选得到的扶正抑瘤方中所含化合物作用靶点经过Uniprot数据库归一转换为Gene Symbol格式后，与MalaCards、DisGeNET等数据库搜集得到的免疫检查点相关靶标进行对应映射，绘制花瓣图(见图1)，得免疫检查点相关核心靶标44个，其中16个靶点与扶正抑瘤方作用靶点重合。 通过STRING数据库对上步所得的免疫检查点相关核心靶标–扶正抑瘤方作用靶点交集基因进行分析。选择分析模式为Multiple Proteins，设定物种类别为Homo Sapiens，互作分数阈值设为高度置信(0.700)，得到16个节点，边数58条，平均节点度值7.25，平均局部聚类系数0.752的PPI网络图(见图2)。 图1. 药物靶点与免疫检查点相关靶点花瓣图 Table 1 Active compounds contained in Fuzheng Yiliu decoctio Mol Herb Mol Herb Mol Herb (−)-ABA Dioscoreae Rhizoma DTY Dioscoreae Rhizoma lucidenic acid L Ganoderma (−)-alpha-Pinene Ganoderma EIC Ganoderma lucidenolactone Ganoderma Ligustri Lucidi Fructus Astragali Radix lucidone A Ganoderma ()-Borneol Ligustri Lucidi Fructus ergosta-4，6，8(14)， 22-tetraene-3-one Ganoderma lucidone B Ganoderma ()-Cuparene Ganoderma Ergosta-7，22-dien-3-one Ganoderma lucidone C Ganoderma (−)-Drimenol Ganoderma ergosta-7，22E-dien-3beta-ol Ganoderma Lucidumoside D Ligustri Lucidi Fructus (−)-nopinene Ligustri Lucidi Fructus ergosta-7，9(11)， 22-trien-3β，5α，6α-triol Ganoderma Lucidumoside D_qt Ligustri Lucidi Fructus (−)-Olivir Ligustri Lucidi Fructus ergosterol Ganoderma lupeol Astragali Radix (−)-taxifolin Dioscoreae Rhizoma Dioscoreae Rhizoma luteolin Ligustri Lucidi Fructus ()-Terpinen-4-ol Ligustri Lucidi Fructus eriodictyol Ligustri Lucidi Fructus luteolin-7-o-glucoside Ligustri Lucidi Fructus (+)-alpha-Curcumene Ganoderma eugenol Ligustri Lucidi Fructus L-Valin Dioscoreae Rhizoma (+)-Syringaresinol Astragali Radix FA Astragali Radix L-Xyl Dioscoreae Rhizoma (R)-linalool Ganoderma FERULIC ACID (CIS) Astragali Radix Mairin Astragali Radix Ligustri Lucidi Fructus Flavaxin Astragali Radix methly lucidenate L Ganoderma (R)-p-Menth-1-en-4-ol Ganoderma formononetin Astragali Radix Methose Dioscoreae Rhizoma (S)-Allantoin Dioscoreae Rhizoma Fucopyranose， L- Astragali Radix Methylcinnamate Ligustri Lucidi Fructus (s)-carvone Ganoderma FUM Ganoderma Mnk Ganoderma 1，8-Cineole Ganoderma GABA Ganoderma MTL Ganoderma 3，9-di-O-methylnissolin Astragali Radix Dioscoreae Rhizoma Ligustri Lucidi Fructus 3-Hydroxy-2-picoline Astragali Radix Astragali Radix Mucronulatol Astragali Radix 7-O-methylisomucronulatol Astragali Radix ganoderal B Ganoderma myristic acid Ganoderma ABK Dioscoreae Rhizoma ganoderenic acid H Ganoderma Nerol Ligustri Lucidi Fructus acteoside Ligustri Lucidi Fructus ganoderic acid E Ganoderma nicotinic acid Astragali Radix AIDS180907 Dioscoreae Rhizoma Ganoderic acid T Ganoderma Nonanal Ganoderma alexandrin Astragali Radix ganoderiol I Ganoderma nonane Ganoderma alpha-humulene Ligustri Lucidi Fructus ganolucidic acid E Ganoderma nonanoic acid Ganoderma AMG Ligustri Lucidi Fructus GCS Ganoderma Nonanol Ganoderma anethole Ganoderma geraniol Ligustri Lucidi Fructus Nouracid CS 80 Ganoderma apigenin Ligustri Lucidi Fructus GLB Dioscoreae Rhizoma OCTENAL Ganoderma Arabinose，d Astragali Radix GLY Dioscoreae Rhizoma o-Cymol Ganoderma ASI Dioscoreae Rhizoma Gulutamine Dioscoreae Rhizoma oleanolic acid Ligustri Lucidi Fructus Astraisoflavanin Astragali Radix GUP Dioscoreae Rhizoma Oleoside dimethyl ester_qt Ligustri Lucidi Fructus beta-Bazzanene Ganoderma hancinone C Dioscoreae Rhizoma Olitoriside_qt Ligustri Lucidi Fructus betaine Ganoderma hederagenin Astragali Radix Ononin Astragali Radix Astragali Radix hexanoic acid Ganoderma Ostreasterol Dioscoreae Rhizoma beta-Irone Ganoderma Hirsutrin Astragali Radix palmitic acid Astragali Radix beta-sitosterol Ganoderma h-Met-h Dioscoreae Rhizoma PEL Ligustri Lucidi Fructus Ligustri Lucidi Fructus Hyacinthin Ganoderma Pellitorin Dioscoreae Rhizoma BGC Dioscoreae Rhizoma hydroxytyrosol Ligustri Lucidi Fructus PENTADECYLIC ACID Ganoderma Bifendate Astragali Radix isoferulic acid Astragali Radix PENTYLFURAN Ganoderma C09704 Ganoderma Isofucosterol Dioscoreae Rhizoma PHA Dioscoreae Rhizoma Caffeate Astragali Radix isomucronulatol-7， 2'-di-O-glucosiole Astragali Radix piperlonguminine Dioscoreae Rhizoma caffeic acid Ligustri Lucidi Fructus isorhamnetin Astragali Radix Prolinum Dioscoreae Rhizoma Calycosin Astragali Radix Istidina Dioscoreae Rhizoma Astragali Radix campesta-7，22E-dien-3beta-ol Ganoderma Jaranol Astragali Radix quercetin Ligustri Lucidi Fructus campesterol Dioscoreae Rhizoma Kadsurenone Dioscoreae Rhizoma Astragali Radix caprylic acid Ganoderma kaempferol Ligustri Lucidi Fructus QUOSP Ligustri Lucidi Fructus Cedrol Ganoderma Astragali Radix RAM Ligustri Lucidi Fructus Ligustri Lucidi Fructus ketologanin_qt Ligustri Lucidi Fructus Astragali Radix Cerevisterol Ganoderma kingiside_qt Ligustri Lucidi Fructus Rhamnocitrin Astragali Radix cis-p-Coumarate Astragali Radix L- Dioscoreae Rhizoma rhamnocitrin-3-O-glucoside Astragali Radix cis-Thujopsene Ligustri Lucidi Fructus Astragali Radix rutin Ligustri Lucidi Fructus citral Ganoderma Lariciresinol Astragali Radix Astragali Radix CLR Dioscoreae Rhizoma lauric acid Ganoderma salidroside Ligustri Lucidi Fructus Coniferol Ligustri Lucidi Fructus L-Bornyl acetate Ligustri Lucidi Fructus Sinapyl alcohol Ligustri Lucidi Fructus copaene Ganoderma l-carvone Ganoderma Sitogluside Ligustri Lucidi Fructus Cosmetin Ligustri Lucidi Fructus Leucinum Dioscoreae Rhizoma ST5330609 Ganoderma coumarin Astragali Radix lignoceric acid Ganoderma stearic acid Ganoderma Crystal VI Astragali Radix L-Ile Dioscoreae Rhizoma Stigmasterol Dioscoreae Rhizoma daidzein Ligustri Lucidi Fructus LINALOOL (D) Ganoderma sucrose Ligustri Lucidi Fructus Astragali Radix linolenic acid Astragali Radix taxifolin Ligustri Lucidi Fructus Damascenone Ligustri Lucidi Fructus L-Limonen Ligustri Lucidi Fructus Tetracosane Ganoderma Daucene Ganoderma L-Lysin Dioscoreae Rhizoma Threonin Dioscoreae Rhizoma DBP Ligustri Lucidi Fructus LPG Dioscoreae Rhizoma Tormentic acid Ligustri Lucidi Fructus dec-2-enal Ganoderma L-Serin Dioscoreae Rhizoma trans-2，4-decadienal Ganoderma Decanal Ganoderma Lucialdehyde B Ganoderma Tyrosol Ligustri Lucidi Fructus delta-amorphene Ganoderma lucidenic acid B Ganoderma undecanal Ganoderma denudatin，a Dioscoreae Rhizoma lucidenic acid D1 Ganoderma ursolic acid Ligustri Lucidi Fructus Dioscoreside C_qt Dioscoreae Rhizoma lucidenic acid E1 Ganoderma vanillic acid Astragali Radix diosgenin Dioscoreae Rhizoma lucidenic acid G Ganoderma Vomifoliol Ligustri Lucidi Fructus dl-Thujone Ganoderma Lucidenic acid K Ganoderma XLS Astragali Radix 表1. 扶正抑瘤方所含活性化合物 将扶正抑瘤方中各味中药、对应活性成分、作用基因靶点等各个节点及映射关系导入Cytoscape软件，进行可视化分析并对其各项属性进行设置，得到如图3所示“药物–成分–靶点–通路”网络图，图中左侧绿色菱形代表扶正抑瘤方中各味中药，外圈黄色椭圆表示各有效成分，内圈三角形代表相对应的基因靶点，右侧蓝色方形表示关联性较高的通路。 图2. 蛋白–蛋白互作网络图 图3. 药物–成分–靶点–通路网络图(绿色：单味中药，黄色：中药化学成分，橙色：蛋白靶标，兰色：作用途径) 3.3. 靶点生物学功能分析 于Rstudio软件中加载clusterProfiler扩展包，对PPI网络中筛选出的核心靶点进行KEGG通路富集和Gene Ontology (GO)功能富集分析，pvalueCutoff与qvalueCutoff值均设定为0.05，得到扶正抑瘤方抗肝癌过程中的生物过程(Biological Process， BP)、细胞组分(Cellular Component， CC)、分子功能(Molecular Function， MF)与KEGG通路富集情况，进一步利用ggplot2扩展包绘图使结果可视化，绘制条形图及气泡图。 图4. GO富集分析图 由图4可知，靶点参与免疫系统过程的正向调节、细胞分化的正调控、细胞内信号转导的调节、免疫系统过程的调节、细胞内信号转导等生物过程，主要分布在质膜有界细胞投影及肥大细胞颗粒，涉及磷酸转移酶活性、信号受体结合、细胞因子受体结合、酶结合等分子功能。 图5. KEGG富集分析图 由图5结果可以推断，扶正抑瘤方抗肿瘤作用机制可能和PD-L1表达与PD-1检查点通路(PDL1 expression and PD1 checkpoint pathway in cancer)及T细胞受体信号通路(T cell receptor signaling pathway)高度相关。 PD-1及其配体PD-L1是关键的调节性生理免疫检查点，其可调节T细胞和B细胞在其他免疫细胞类型中的活化程度，维持生物体的自我耐受 [ 5 ]。在实体瘤中，肿瘤细胞通过增加细胞表面PD-L1的表达以沉默免疫系统。PD-L1的调控受HIF-1，STAT1，STAT3影响 [ 6 ]，阻断PD-1/PD-L1途径的治疗性抗体纳武利尤单抗(Opdivo)在肿瘤的临床治疗上已被广泛使用，具有较好的疗效 [ 7 ]。 受CTLA4介导的T细胞受体以及共刺激分子(例如CD28)通过与CD28反受体B7-1/B7-2分别相关的外源抗原(CD80， CD86)的相互作用导致一系列信号级联反应 [ 8 ]，包括蛋白质–酪氨酸激酶，磷酸酶，GTP结合蛋白和衔接子蛋白等 [ 9 ]，从而对T细胞增殖，细胞因子产生和分化成效应细胞产生影响。