Action Mechanism of Nelumbo nucifera Leaf Alkaloids in the Treatment of Hyperuricemia Based on Network Pharmacology and Molecular Docking
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摘要: 目的：运用网络药理学及分子对接分析荷叶生物碱类化合物治疗高尿酸血症的作用机制。方法：通过数据库检索获取荷叶生物碱类化合物作用靶点以及高尿酸血症靶点，通过Cytoscape 3.8.0建立荷叶生物碱类化合物的“成分-疾病-靶点”网络，利用微生信平台进行基因本体论（GO）分析和京都基因与基因组百科全书（KEGG）通路分析，并将核心靶点与关键成分进行分子对接验证。结果：通过文献检索获取27种荷叶生物碱类化合物信息，包括荷叶碱、衡州乌药碱、莲心碱等，预测获得对应靶点337个，高尿酸血症相关靶点926个，关键靶点57个。荷叶生物碱类化合物治疗高尿酸血症的作用机制主要涉及GAPDH、STAT3、JUN、CASP3、PTGS2、XDH、MAPK1等靶基因，这些基因主要通过AGE-RAGE信号通路、PD-1通路、TNF信号通路、IL-17信号通路以及p53信号通路等调控蛋白表达发挥作用。分子对接结果显示化合物与分子靶点对接结果良好，荷叶碱、衡州乌药碱、莲心碱均可与关键靶点自发结合，其中莲心碱与蛋白PTGS2、JUN可形成最稳定结构，结合能可达−9.3 kcal/mol。结论：本研究预测荷叶生物碱类化合物通过调控多靶点、多通路发挥改善高尿酸血症的作用，为荷叶生物碱类化合物治疗高尿酸血症的分子研究提供了科学依据。Abstract: Objective: The mechanism of action of Nelumbo nucifera leaf alkaloids in the treatment of hyperuricemia was analyzed based on network pharmacology and molecular docking. Methods: The target of Nelumbo nucifera leaf alkaloids and disease targets related to hyperuricemia were obtained through database search. The "component-disease-target" network of Nelumbo nucifera leaf alkaloids was established through Cytoscape 3.8.0, and the gene ontology (GO) analysis and Kyoto encyclopedia of genes and genome (KEGG) pathway analysis were carried out using the bioinformatics-based platform. The core target and key components were verified by molecular docking. Results: The information of 27 kinds of Nelumbo nucifera leaf alkaloids, including nuciferine, coclaurine, liensinine, was obtained by literature search, and 337 corresponding targets, 926 hyperuricemia-related targets and 57 key targets were piedicted. The mechanism of action of Nelumbo nucifera leaf alkaloids in the treatment of hyperuricemia mainly involved GAPDH, STAT3, JUN, CASP3, PTGS2, XDH, MAPK1 and other target genes. These genes mainly played a role in regulating protein expression through AGE-RAGE signaling pathway, PD-L1 expression and PD-1 checkpoint pathway, TNF signaling pathway, IL-17 signaling pathway and p53 signaling pathway. The results of molecular docking showed that the compound had good docking results with the molecular target. Nuciferine, coclaurine and liensinine bound to the key targets spontaneously, among which liensinine formed the most stable structure with PTGS2 and JUN, and the binding energy could reach −9.3 kcal/mol. Conclusion: This study predicted that Nelumbo nucifera leaf alkaloids could ameliorate hyperuricemia by regulating multiple targets and action multiple pathways, which could provide scientific basis for molecular research of Nelumbo nucifera leaf alkaloids in the treatment of hyperuricemia.
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高尿酸血症（Hyperuricemia，HUA）是一种由于生物嘌呤合成代谢增加，尿酸产生过多或排泄不良而导致血液中尿酸含量升高的病症，在我国发病率高达13.3%[1]。当体内尿酸浓度过高时，尿酸从血液中析出后以钠盐的形式结晶而沉积在关节、软骨组织或肾脏中，引起关节肿大、畸形、痛风性肾结石等疾病[2−3]。目前临床上多采用抑制尿酸吸收，促进尿酸排泄等方式治疗高尿酸血症，常见药物有别嘌醇、非布司他、苯溴马隆和丙磺舒等，临床研究发现部分药品存在不同程度的副作用[4−5]。如何从天然产物中寻找安全性高且具有降尿酸活性的有效成分成为高尿酸血症相关的研究热点。
荷叶为睡莲科植物莲（Nelumbo nucifera Gaertn）的叶，其中荷叶生物碱类为其主要功能成分，不同产地、气候与水土环境会导致生物碱种类和含量差异显著[6]。荷叶生物碱具有降脂减肥[7]、抑菌杀菌[8]、抗病毒[9]、抗肿瘤[10]、抗炎[11]、降血脂[12]和舒张血管[13]等作用。现已证明荷叶生物碱中的荷叶碱可以通过调节动物肾脏有机离子转运蛋白表达而发挥其降尿酸功能[14]，但其他生物碱降尿酸效果及其作用机制尚不明确。
网络药理学是一门基于系统生物学和网络生物学、结合计算机科学和药理学的新兴学科，可以通过生物网络模型分析“成分-基因-靶点-疾病”间的相互作用关系，解释疾病治疗的原理和作用机制，指导具有高靶向性、疗效显著的新药研发[15−16]。分子对接技术基于药物与蛋白质分子呈现锁钥关系，通过计算机模拟对配体与受体之间作用位点以及作用力强弱进行分析[17]。因此本研究采用网络药理学结合分子对接的方法，预测作用靶点及机制，探寻荷叶生物碱类化合物降低高尿酸作用的物质基础和作用机制，为天然产物治疗高尿酸血症提供参考依据。
1. 材料与方法
1.1 荷叶成分及相关靶点筛选
通过PubChem（https://pubchem.ncbi.nlm.nih.gov/）获取已知荷叶生物碱类化合物的结构（SDF格式），将化合物结构分别导入SwissTarget Prediction数据库（http://www.swisstargetprediction.ch/），转化为SMILES结构进行检索，获取每种化合物前15个重要靶点进行信息富集[18]。将各化合物得到的药物靶点合并、去重，通过UniProt数据库（https://www.uniprot.org/）转换为Gene Symbol。
1.2 高尿酸血症相关靶点筛选
以“Hyperuricemia”为关键词，在GeneCards（https://www.genecards.org/）、Drugbank（https://go.drugbank.com/）、Disgenet（https://www.disgenet.org/）及OMIM（http://www.ncbi.nlm.nih.gov/omim）数据库检索并获得与高尿酸血症相关基因，将所得结果合并、去重，整合有效疾病靶点得到高尿酸血症相关靶点数据库。
1.3 筛选核心靶点
将荷叶生物碱类化合物与Hyperuricemia获得的相关靶点输入微生信（http://www.bioinformatics.com.cn/）在线平台绘制Venn图，得到核心靶点。
1.4 构建蛋白-蛋白相互作用（PPI）网络
将上述得到的核心靶点导入STRING（https://string-db.org/）数据库构建PPI网络，限定物种为“Homo sapiens”，选择最低相互作用阈值，下载TSV文件，将数据导入Cytoscape 3.8.0绘制PPI网络并分析。将节点（node）大小和颜色用于反映度值（degree）的大小，边（edge）的粗细用于反映结合分数的大小。
1.5 GO富集和KEGG通路富集分析
将得到的核心靶点导入Metascape数据库，限定物种为“Homo sapiens”，设定阈值为P≤0.05，进行基因本体（Gene ontology，GO）的生物进程（Biological process，BP）、细胞组分（Cellular component，CC）和分子功能（Molecular function，MF）分析，以及京都基因和基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）通路富集分析。根据P值，选取排名前20的GO富集上传至微生信平台绘制柱状图；根据P值、enrichment、count值，选取排名前20的KEGG通路上传至微生信平台绘制富集气泡图。
1.6 分子对接
采用Autodock Vina对荷叶生物碱类化合物和治疗高尿酸血症中关键靶点进行分子对接验证。靶点蛋白CASP3、PTGS2、JUN、GAPDH、STAT3结构来源于RCSB PDB数据库（www.rcsb.org，ID CODE：1NMS、5F19、5T01、6IQ6、6QHD）。采用Autodock Tools1.5.7对蛋白晶体结构删除水分子、配体，进行加氢处理，按照标准程序制备PDBQT文件。三种结构代表性荷叶生物碱（荷叶碱、衡州乌药碱、莲心碱）的2D结构由PubChem网站获得并将其导入Autodock Tools1.5.7进行加氢处理，得到三种荷叶生物碱的PDBQT文件。设置对接口袋将靶点蛋白的潜在活性位点全部覆盖，寻找对接构象，并取结合能最低的构象作为分子对接结果。使用Pymol对分子对接结果进行分析，呈现配体在受体对接口袋中的相对位置和相互作用关系。
2. 结果与分析
2.1 荷叶生物碱类化合物对应靶点预测
根据荷叶生物碱母核结构及连接基团的差异，可以分为阿朴啡类，如N-去甲基荷叶碱、荷叶碱等；单苄基异喹啉类，如亚美罂粟碱、衡州乌药碱等；双苄基异喹啉类，如莲心碱、甲基莲心碱等；氧化阿朴啡类，如鹅掌楸碱、观音莲明碱等；去氢阿朴啡类，如去氢荷叶碱、去氢莲碱等；原阿朴啡类和其他类[19−20]。在中国知网、Web of Science和Science Direct搜索荷叶中生物碱成分，共获取27种生物碱类化合物信息，其网络靶点分析顺序见表1，利用SwissTarget Prediction数据库预测化合物对应靶点337个，通过Cytoscape软件构建“荷叶生物碱类化合物-靶点”网络进行化合物与靶点关系分析。如图1所示，观音莲明碱和鹅掌楸碱关联靶点成分高达113个，荷叶碱、衡州乌药碱、莲碱、去氢番荔枝碱、去氢荷叶碱、去氢莲碱、亚美罂粟碱、O-去甲基荷叶碱和原荷叶碱的关联靶点均超过30个，表明荷叶生物碱类化合物可能通过作用于多靶点而发挥其生物活性。
表 1 荷叶生物碱类化合物网络靶点分析顺序Table 1. Analysis sequence of network target of Nelumbo nucifera leaf alkaloids序号 生物碱名称 序号 生物碱名称 序号 生物碱名称 HY1 亚美罂粟碱 HY10 甲基莲心碱 HY19 N-去甲基荷叶碱 HY2 衡州乌药碱 HY11 莲心季铵碱 HY20 巴婆碱 HY3 异衡州乌药碱 HY12 荷叶碱 HY21 去氢荷叶碱 HY4 N-甲基异衡州乌药碱 HY13 莲碱 HY22 去氢莲碱 HY5 N-甲基衡州乌药碱 HY14 番荔枝碱 HY23 去氢番荔枝碱 HY6 N-去甲基亚美罂粟碱 HY15 山矾碱 HY24 观音莲明碱 HY7 O-methylarmepavine HY16 北美鹅掌楸尼定碱 HY25 鹅掌楸碱 HY8 莲心碱 HY17 N-methylasimilobine HY26 原荷叶碱 HY9 异莲心碱 HY18 O-去甲基荷叶碱 HY27 N-methylnuciferine ![]()
图 1 “荷叶生物碱类化合物-靶点”网络Figure 1. Network diagram of "alkaloids of Nelumbo nucifera leaf-target"2.2 高尿酸血症相关靶点检索
通过检索GeneCards、Drugbank、Disgenet及OMIM等数据库，整合、去重，共获得高尿酸血症相关靶点926个。将以上所得化合物成分靶点和疾病靶点取交集，通过微生信（http://www.bioinformatics.com.cn/）在线平台绘制Venn图（图2），得到荷叶生物碱类化合物治疗高尿酸血症的作用靶点共60个，见表2。
表 2 荷叶生物碱类化合物对应高尿酸血症靶点Table 2. Targets of alkaloids of Nelumbo nucifera leaf for hyperuricemia序号 共同靶点 序号 共同靶点 序号 共同靶点 1 KCNJ1 21 CCND2 41 RXRA 2 DRD5 22 CYP17A1 42 ABCG2 3 GCK 23 JUN 43 ADRA2A 4 CCR2 24 PIK3CG 44 ADRB2 5 PIK3C2A 25 CDK1 45 ADRB3 6 PARP1 26 CCND3 46 FBP1 7 MAPK1 27 CCNH 47 GSR 8 SELE 28 CYP11B2 48 HPRT1 9 ACE 29 HTR1A 49 HTR2A 10 CDK4 30 HTR3A 50 MAOA 11 STAT3 31 KCNJ11 51 NCOA2 12 JAK2 32 TLR9 52 P2RX7 13 HDAC1 33 AGPAT2 53 PTGS2 14 ICAM1 34 CCR1 54 SLC22A12 15 MAPK8 35 CDK7 55 SLC6A3 16 CASP3 36 PARP2 56 TGFB1 17 DRD2 37 COMT 57 XDH 18 HDAC3 38 VCAM1 58 SLC47A1 19 PLAT 39 GAPDH 59 MTOR 20 CASP6 40 PLA2G7 60 AR 2.3 蛋白-蛋白相互作用（PPI）网络
将所得交集靶点通过STRING数据库和Cytoscape软件进行分析，PPI网络如图3所示，共得到关键靶点57个和341条相互关系。网络中度值越大表明其发挥作用越大[16]，由此推断GAPDH、STAT3、JUN、CASP3、PTGS2等靶点可能在治疗高尿酸血症中发挥关键作用。其中，GAPDH在肿瘤组织和正常细胞组织表达不一致时可作为肿瘤发生的标志物[21]，JUN原癌基因涉及人类恶性肿瘤易位和缺失的染色体区域并参与肿瘤细胞的生长调控[22]，由于高尿酸血症的血清尿酸水平与癌症发病率和死亡率之间存在线性关系[23]，因此GAPDH和JUN表达异常时血清尿酸含量可能出现异常。CASP3在细胞凋亡中起关键作用[24]，当CASP3表达水平升高时，大鼠肾小管水肿扩张且上皮细胞萎缩，肾脏损伤明显，抑制其表达后肾小管扩张减弱，上皮细胞变性减少[25]，因此CASP3的表达与肾脏功能密切相关。STAT3编码的蛋白在细胞过程如细胞生长和凋亡中起关键作用[26]，是肾纤维化进展中的关键信号之一，其抑制剂S3I-201可以降低高尿酸血症小鼠的血清尿酸水平，抑制高尿酸血症小鼠肾脏TGF-β/Smads、JAK/STAT和NF-κB纤维化信号通路，延缓肾纤维化发展[27]。PTGS2基因在调控炎症反应中发挥重要作用[28]，当细胞内尿酸水平过高时，尿酸激活NADPH氧化酶，产生活性氧，继而提高血清中MDA、IL-6、TNF-α和IL-8水平[29−30]，诱导炎症反应造成细胞损伤[5]，因此PTGS2的表达异常与肾脏转运尿酸能力密切相关。综上所述，上述靶点与细胞的增殖、分化、迁移及凋亡，蛋白质磷酸化，组织修复及炎症反应等相关，说明荷叶生物碱类化合物可能是通过多靶点、多通路调节以上生物过程发挥治疗高尿酸血症的作用。
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图 3 荷叶生物碱类化合物治疗高尿酸血症的PPI网络Figure 3. PPI network of Nelumbo nucifera leaf alkaloids for treatment of hyperuricemia2.4 GO功能分析及KEGG通路富集分析
关键靶点的GO功能分析见图4。从生物过程（BP）分析，荷叶生物碱类化合物治疗高尿酸血症主要涉及细胞运动的正向调节（positive regulation of cell motility）、蛋白质磷酸化的正向调节（positive regulation of protein phosphorylation）以及细胞中单原子离子运输调控（regulation of monoatomic ion transport）等；细胞组分（CC）结果显示，荷叶生物碱类化合物发挥作用主要涉及周期蛋白依赖性蛋白激酶全酶复合物（cyclin-dependent protein kinase holoenzyme complex）、蛋白激酶复合物（protein kinase complex）和RNA聚合酶Ⅱ转录调控复合物（RNA polymerase Ⅱ transcription regulator complex）等；分子功能（MF）表明荷叶生物碱类化合物治疗高尿酸血症的过程集中于去甲肾上腺素结合（norepinephrine binding）、G蛋白偶联受体活性（G protein-coupled amine receptor activity）和细胞周期蛋白依赖性蛋白激酶活性（cyclin-dependent protein kinase activity）等。以上结果表明荷叶生物碱类化合物通过参与多种生物调控过程减轻高尿酸症状。
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图 4 荷叶生物碱类化合物治疗高尿酸血症作用靶点GO分析Figure 4. GO analysis of Nelumbo nucifera leaf alkaloids for treatment of hyperuricemia关键靶点的KEGG通路富集分析得到124条信号通路，取富集分数前20的通路作图，见图5。结果表明荷叶生物碱类化合物治疗高尿酸血症的信号通路主要涉及糖尿病并发症中的AGE-RAGE信号通路（AGE-RAGE signaling pathway in diabetic complications）、癌症中PD-L1的表达和PD-1通路（PD-L1 expression and PD-1 checkpoint pathway in cancer）、TNF信号通路（TNF signaling pathway）、IL-17信号通路（IL-17 signaling pathway）以及p53信号通路（p53 signaling pathway）等。尿酸产生过程中的关键限速酶黄嘌呤氧化还原酶（XOR）可催化生成尿酸和ROS，参与各种疾病如代谢性综合征、心血管疾病和癌症等发挥促炎或抗炎作用，表明荷叶生物碱类化合物治疗高尿酸血症的信号通路具有高可信度[31]。KEGG通路富集分析相关基因见表3，作用靶点与通路网络分析见图6。
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图 5 荷叶生物碱类化合物治疗高尿酸血症关键靶点KEGG通路富集分析Figure 5. KEGG pathway enrichment analysis of key targets of Nelumbo nucifera leaf alkaloids for hyperuricemia表 3 KEGG通路富集分析相关基因Table 3. Related genes for KEGG pathway enrichment analysis通路名称 相关基因 P 基因数 AGE-RAGE signaling pathway in diabetic complications CASP3、CDK4、ICAM1、JAK2、JUN、MAPK1、MAPK8、SELE、STAT3、TGFB1、VCAM1 8.98E-17 11 Type II diabetes mellitus MTOR、GCKK、CNJ11、MAPK1、MAPK8 3.37E-08 5 Prolactin signaling pathway CCND2、CYP17A1、GCK、JAK2、MAPK1、MAPK8、STAT3 9.29E-11 7 Pancreatic cancer CDK4、MTOR、MAPK1、MAPK8、STAT3、TGFB1 9.37E-09 6 Th17 cell differentiation MTOR、JAK2、JUN、MAPK1、MAPK8、RXRA、STAT3、TGFB1 4.53E-11 8 Adipocytokine signaling pathway MTOR、JAK2、MAPK8、RXRA、STAT3 2.67E-07 5 TNF signaling pathway CASP3、ICAM1、JUN、MAPK1、MAPK8、PTGS2、SELE、VCAM1 6.08E-11 8 Colorectal cancer CASP3、MTOR、JUN、MAPK1、MAPK8、TGFB1 1.98E-08 6 p53 signaling pathway CASP3、CCND2、CCND3、CDK1、CDK4 3.55E-07 5 PD-L1 expression and PD-1 checkpoint pathway in cancer MTOR、JAK2、JUN、MAPK1、STAT3、TLR9 2.44E-08 6 Cell cycle CCND2、CCND3、CCNH、CDK1、CDK4、CDK7、HDAC1、TGFB1 1.67E-10 8 Toxoplasmosis CASP3、JAK2、PIK3CG、MAPK1、MAPK8、STAT3、TGFB1 2.63E-09 7 Kaposi sarcoma-associated herpesvirus infection CASP3、CDK4、CCR1、MTOR、ICAM1、JAK2、JUN、PIK3CG、MAPK1、MAPK8、PTGS2、STAT3 3.77E-15 12 Serotonergic synapse CASP3、HTR1A、HTR2A、HTR3A、MAOA、MAPK1、PTGS2 3.17E-09 7 Measles CASP3、CCND2、CCND3、CDK4、JUN、MAPK8、STAT3、TLR9 3.45E-10 8 IL-17 signaling pathway CASP3、JUN、MAPK1、MAPK8、PTGS2 1.26E-06 5 Chronic myeloid leukemia CDK4、HDAC1、MAPK1、TGFB1 1.63E-05 4 Regulation of lipolysis in adipocytes ADRB2、ADRB3、PTGS2 2.13E-04 3 Apoptosis PARP1、CASP3、CASP6、JUN、MAPK1、MAPK8、PARP2 1.02E-08 7 Lipid and atherosclerosis CASP3、CASP6、ICAM1、JAK2、JUN、MAPK1、MAPK8、RXRA、SELE、STAT3、VCAM1 4.68E-13 11 ![]()
图 6 荷叶生物碱类化合物治疗高尿酸血症作用靶点与通路网络分析Figure 6. Network analysis of targets and pathways of Nelumbo nucifera leaf alkaloids in the treatment of hyperuricemia结合表3和图6可知，糖尿病并发症中的AGE-RAGE信号通路（AGE-RAGE signaling pathway in diabetic complications）主要涉及CASP3、CDK4、ICAM1、JAK2、JUN、MAPK1、MAPK8、SELE、STAT3、TGFB1、VCAM1共11个基因，其中CASP3、MAPK1、JAK2与细胞生长、分化有关，调控细胞功能发挥和细胞凋亡[24,32−33]。杨晨曦[34]分析了Ⅱ型糖尿病伴高尿酸血症中医证型分布特征及影响证型的危险因素，验证了现代学者“消渴合并尿酸浊”理论，临床表明Ⅱ型糖尿病伴高尿酸血症患者的糖代谢紊乱更明显，且增高其并发症概率[35]。TNF信号通路（TNF signaling pathway）主要涉及CASP3、ICAM1、JUN、MAPK1、MAPK8、PTGS2、SELE、VCAM1共8个基因，ICAM1可使白细胞聚集到炎症部位，参与细胞信号转导与活化、免疫应答、炎症反应等重要生理过程[36]；PTGS2可被促炎因子、促肿瘤因子等高度诱导，参加炎症反应、细胞凋亡等多种生理过程[28]；MAPK8可启动JUN激酶活性和蛋白丝氨酸、苏氨酸、酪氨酸激酶活性，作用于多个过程的上游[37]。大量的研究集中在通过降低炎症反应而减轻高尿酸血症，Chen等[38]通过小鼠实验证明姜黄素可以减少血清中炎性因子的产生，抑制肾脏炎症小体表达而表现出治疗高尿酸血症的作用；青钱柳、荷叶碱、柚皮素、狼尾草醇提物均被证明可以通过改善炎症反应来缓解高尿酸血症[39−41]。癌症中PD-L1的表达和PD-1通路（PD-L1 expression and PD-1 checkpoint pathway in cancer）主要涉及MTOR、JAK2、JUN、MAPK1、STAT3、TLR9共6个基因，其中TLR9是表达于免疫系统细胞的重要受体，如树突状细胞、巨噬细胞，其可与MyD88相互作用，导致促炎因子反应的信号级联反应，诱导产生IL-6、TNF、IFN-α、IL-12和I型干扰素等[42]；STAT3是信号转导和转录激活因子，可介导细胞对白介素、KITLG、SCF、LEP和其他生长因子的反应[26]。在高尿酸血症与癌症关系研究中发现，XOR作为生成尿酸的关键限速酶，其表达和活性与癌症的发生和预后密切相关，统计数据表明，XOR在肝癌、乳腺癌、结直肠癌中活性减弱，在肺癌和脑肿瘤中活性增强[31]，因此，对于高尿酸血症的治疗可以通过癌症中的信号通路来调控XOR的表达。综上所述，荷叶生物碱类化合物治疗高尿酸血症涉及多个靶点和多个通路，证实高尿酸血症在体内是一个复杂的代谢性疾病。
2.5 分子对接验证
为了验证荷叶生物碱类化合物预测靶点的准确性，采用AutoDock Vine软件对不同母核结构成分中代表性化合物荷叶碱、莲心碱、衡州乌药碱和前文筛选出的CASP3、PTGS2、JUN、GAPDH和STAT3关键靶点蛋白进行分子对接，以自由结合能（binding energy）作为筛选条件，筛选出多构象中结合能最低的对接构象，结合能越低，说明受体与配体之间的结合作用越强，越有可能发生结合[43]。靶点与成分间的分子对接结合能见表4，配体在对接口袋中的相对位置和与受体的相互作用关系见图7。对接结果表明荷叶碱、莲心碱、衡州乌药碱均能与靶点形成氢键，其中莲心碱与PTGS2、JUN的结合能可达−9.3 kcal/mol，与GAPDH、CASP3的结合能分别达到−9.1、−9.0 kcal/mol，表明莲心碱与各个靶点氨基酸残基之间形成的相互作用最多，结合最稳定，这可能是由于莲心碱具有两个苄基异喹啉基团组成的骨架，与靶点的结合能力更强[44]。
表 4 关键靶点与关键成分的分子对接结合能（kcal/mol）Table 4. Binding energy of key component and key targets (kcal/mol)化合物 靶点 CASP3 PTGS2 JUN GAPDH STAT3 荷叶碱 −7.3 −7.0 −7.3 −7.4 −5.4 衡州乌药碱 −7.3 −8.5 −7.5 −7.1 −5.3 莲心碱 −9.0 −9.3 −9.3 −9.1 −5.6 ![]()
图 7 关键靶点与关键成分分子对接模式注：A.荷叶碱与CASP3最佳对接构象；B.荷叶碱与GAPDH最佳对接构象；C.衡州乌药碱与PTGS2最佳对接构象；D.衡州乌药碱与JUN最佳对接构象；E.莲心碱与PTGS2最佳对接构象；F.莲心碱与JUN最佳对接构象。Figure 7. Molecular docking pattern of key target and component3. 讨论与结论
本研究基于网络药理学的方法，借助相关数据库及软件对荷叶生物碱类化合物治疗高尿酸血症的作用靶点及作用机制进行了系统分析。研究结果显示，荷叶生物碱类化合物治疗高尿酸血症的关键靶点有57个，靶点与靶点之间的相互关系达341条。其作用机制主要涉及TNF信号通路、IL-17信号通路和糖尿病并发症中的AGE-RAGE信号通路等，这些通路与肥胖、糖尿病、心血管疾病等密切相关，证明高尿酸血症作为人体代谢性疾病，常与其他慢性病同时存在并相互影响[45−46]。在此网络主要涉及的靶点中，黄嘌呤脱氢酶（XDH）与高尿酸血症高度相关，研究表明，黄嘌呤氧化酶（XOD）是嘌呤代谢下游通路的关键靶点，当体内XOD活性异常增高时，会产生大量的尿酸，而XDH和XOD是同一酶类的两种不同存在形式，均由XDH mRNA表达，两者可相互转换，所以抑制XDH等同于抑制XOD活性，可达到抑制尿酸生成的效果[47−48]。同时，荷叶生物碱类化合物还能通过作用于MAPK1、PTGS2、STAT3等靶点起到抗炎作用，提示其可通过抑制炎症减轻肾损伤，从而保护肾脏[49]。
目前对于高尿酸血症的治疗停留在预防和控制阶段，随着网络药理学的发展，天然产物治疗高尿酸血症的研究逐步兴起。单味中药研究中，桑枝中关键成分可能通过MAPK1、IL6、TNF等靶点作用于TNF、TRP、Toll-like receptor等信号通路而发挥其改善高尿酸血症的作用[50]。尉雅洁等[51]通过网络药理学发现GAPDH、TP53、TNF、IL-6可能是荆防颗粒治疗高尿酸血症的关键靶点，动物实验代谢组学结果显示荆防颗粒能够通过改善高尿酸血症小鼠丙氨酸、天冬氨酸和谷氨酸的代谢、柠檬酸循环等途径改善代谢而发挥治疗高尿酸血症的作用。杨钊田[52]通过网络药理学分析中成药四神煎治疗痛风性关节炎的作用机制发现四神煎可以通过Toll-like receptor通路、Ⅱ型糖尿病通路、细胞因子-细胞因子受体相互作用通路等多种途径发挥抗炎作用，动物实验表明四神煎对痛风性关节炎大鼠具有抗炎作用且作用机制可能与调控TLR4/NF-κB通路有关。在基于肠道菌群和网络药理学探讨藏药十五味乳鹏丸抗高尿酸血症肾病的研究中发现木犀草素、山柰酚、表没食子酸儿茶素没食子酸酯等与关键靶点有较好的对接活性，大鼠实验验证藏药十五味乳鹏丸可以通过调节肠道菌群结构，调控AGE-RAGE、IL-17、TNF等信号通路治疗高尿酸血症[53]。以上研究从不同靶点、通路皆验证了高尿酸血症的调控是一个复杂的过程，与本篇网络药理学预测出的靶点、通路研究结果基本相符，验证了荷叶生物碱类化合物也可以作用于多靶点、多通路调控血尿酸含量[54−55]。
综上所述，本研究通过网络药理学方法预测了荷叶生物碱类化合物治疗高尿酸血症的作用靶点及作用通路，运用分子对接技术进行了初步验证，印证了高尿酸血症作为代谢性疾病与人体内多种代谢途径密切相关。但是网络药理学存在局限性，因其本身基于数据库和程序的应用无法证明其结论的正确性，因此今后需要结合功能评价实验进行验证。
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图 1 “荷叶生物碱类化合物-靶点”网络
Figure 1. Network diagram of "alkaloids of Nelumbo nucifera leaf-target"
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图 3 荷叶生物碱类化合物治疗高尿酸血症的PPI网络
Figure 3. PPI network of Nelumbo nucifera leaf alkaloids for treatment of hyperuricemia
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图 4 荷叶生物碱类化合物治疗高尿酸血症作用靶点GO分析
Figure 4. GO analysis of Nelumbo nucifera leaf alkaloids for treatment of hyperuricemia
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图 5 荷叶生物碱类化合物治疗高尿酸血症关键靶点KEGG通路富集分析
Figure 5. KEGG pathway enrichment analysis of key targets of Nelumbo nucifera leaf alkaloids for hyperuricemia
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图 6 荷叶生物碱类化合物治疗高尿酸血症作用靶点与通路网络分析
Figure 6. Network analysis of targets and pathways of Nelumbo nucifera leaf alkaloids in the treatment of hyperuricemia
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图 7 关键靶点与关键成分分子对接模式
注：A.荷叶碱与CASP3最佳对接构象；B.荷叶碱与GAPDH最佳对接构象；C.衡州乌药碱与PTGS2最佳对接构象；D.衡州乌药碱与JUN最佳对接构象；E.莲心碱与PTGS2最佳对接构象；F.莲心碱与JUN最佳对接构象。
Figure 7. Molecular docking pattern of key target and component
表 1 荷叶生物碱类化合物网络靶点分析顺序
Table 1 Analysis sequence of network target of Nelumbo nucifera leaf alkaloids
序号 生物碱名称 序号 生物碱名称 序号 生物碱名称 HY1 亚美罂粟碱 HY10 甲基莲心碱 HY19 N-去甲基荷叶碱 HY2 衡州乌药碱 HY11 莲心季铵碱 HY20 巴婆碱 HY3 异衡州乌药碱 HY12 荷叶碱 HY21 去氢荷叶碱 HY4 N-甲基异衡州乌药碱 HY13 莲碱 HY22 去氢莲碱 HY5 N-甲基衡州乌药碱 HY14 番荔枝碱 HY23 去氢番荔枝碱 HY6 N-去甲基亚美罂粟碱 HY15 山矾碱 HY24 观音莲明碱 HY7 O-methylarmepavine HY16 北美鹅掌楸尼定碱 HY25 鹅掌楸碱 HY8 莲心碱 HY17 N-methylasimilobine HY26 原荷叶碱 HY9 异莲心碱 HY18 O-去甲基荷叶碱 HY27 N-methylnuciferine 
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表 2 荷叶生物碱类化合物对应高尿酸血症靶点
Table 2 Targets of alkaloids of Nelumbo nucifera leaf for hyperuricemia
序号 共同靶点 序号 共同靶点 序号 共同靶点 1 KCNJ1 21 CCND2 41 RXRA 2 DRD5 22 CYP17A1 42 ABCG2 3 GCK 23 JUN 43 ADRA2A 4 CCR2 24 PIK3CG 44 ADRB2 5 PIK3C2A 25 CDK1 45 ADRB3 6 PARP1 26 CCND3 46 FBP1 7 MAPK1 27 CCNH 47 GSR 8 SELE 28 CYP11B2 48 HPRT1 9 ACE 29 HTR1A 49 HTR2A 10 CDK4 30 HTR3A 50 MAOA 11 STAT3 31 KCNJ11 51 NCOA2 12 JAK2 32 TLR9 52 P2RX7 13 HDAC1 33 AGPAT2 53 PTGS2 14 ICAM1 34 CCR1 54 SLC22A12 15 MAPK8 35 CDK7 55 SLC6A3 16 CASP3 36 PARP2 56 TGFB1 17 DRD2 37 COMT 57 XDH 18 HDAC3 38 VCAM1 58 SLC47A1 19 PLAT 39 GAPDH 59 MTOR 20 CASP6 40 PLA2G7 60 AR
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表 3 KEGG通路富集分析相关基因
Table 3 Related genes for KEGG pathway enrichment analysis
通路名称 相关基因 P 基因数 AGE-RAGE signaling pathway in diabetic complications CASP3、CDK4、ICAM1、JAK2、JUN、MAPK1、MAPK8、SELE、STAT3、TGFB1、VCAM1 8.98E-17 11 Type II diabetes mellitus MTOR、GCKK、CNJ11、MAPK1、MAPK8 3.37E-08 5 Prolactin signaling pathway CCND2、CYP17A1、GCK、JAK2、MAPK1、MAPK8、STAT3 9.29E-11 7 Pancreatic cancer CDK4、MTOR、MAPK1、MAPK8、STAT3、TGFB1 9.37E-09 6 Th17 cell differentiation MTOR、JAK2、JUN、MAPK1、MAPK8、RXRA、STAT3、TGFB1 4.53E-11 8 Adipocytokine signaling pathway MTOR、JAK2、MAPK8、RXRA、STAT3 2.67E-07 5 TNF signaling pathway CASP3、ICAM1、JUN、MAPK1、MAPK8、PTGS2、SELE、VCAM1 6.08E-11 8 Colorectal cancer CASP3、MTOR、JUN、MAPK1、MAPK8、TGFB1 1.98E-08 6 p53 signaling pathway CASP3、CCND2、CCND3、CDK1、CDK4 3.55E-07 5 PD-L1 expression and PD-1 checkpoint pathway in cancer MTOR、JAK2、JUN、MAPK1、STAT3、TLR9 2.44E-08 6 Cell cycle CCND2、CCND3、CCNH、CDK1、CDK4、CDK7、HDAC1、TGFB1 1.67E-10 8 Toxoplasmosis CASP3、JAK2、PIK3CG、MAPK1、MAPK8、STAT3、TGFB1 2.63E-09 7 Kaposi sarcoma-associated herpesvirus infection CASP3、CDK4、CCR1、MTOR、ICAM1、JAK2、JUN、PIK3CG、MAPK1、MAPK8、PTGS2、STAT3 3.77E-15 12 Serotonergic synapse CASP3、HTR1A、HTR2A、HTR3A、MAOA、MAPK1、PTGS2 3.17E-09 7 Measles CASP3、CCND2、CCND3、CDK4、JUN、MAPK8、STAT3、TLR9 3.45E-10 8 IL-17 signaling pathway CASP3、JUN、MAPK1、MAPK8、PTGS2 1.26E-06 5 Chronic myeloid leukemia CDK4、HDAC1、MAPK1、TGFB1 1.63E-05 4 Regulation of lipolysis in adipocytes ADRB2、ADRB3、PTGS2 2.13E-04 3 Apoptosis PARP1、CASP3、CASP6、JUN、MAPK1、MAPK8、PARP2 1.02E-08 7 Lipid and atherosclerosis CASP3、CASP6、ICAM1、JAK2、JUN、MAPK1、MAPK8、RXRA、SELE、STAT3、VCAM1 4.68E-13 11
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表 4 关键靶点与关键成分的分子对接结合能（kcal/mol）
Table 4 Binding energy of key component and key targets (kcal/mol)
化合物 靶点 CASP3 PTGS2 JUN GAPDH STAT3 荷叶碱 −7.3 −7.0 −7.3 −7.4 −5.4 衡州乌药碱 −7.3 −8.5 −7.5 −7.1 −5.3 莲心碱 −9.0 −9.3 −9.3 −9.1 −5.6
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