基于网络药理学的黄连-升麻药对治疗复发性口腔溃疡中西医学机制探讨

Chinese and Western Medicine Mechanism of Coptidis Rhizoma-Cimicifugae Rhizoma Herbal Pair in the Treatment of Recurrent Aphthous Ulcer Based on Network Pharmacology

[目的]探讨黄连-升麻药对治疗复发性口腔溃疡(recurrent aphthous ulcer,RAU)的现代药理机制,分析可能存在的中医治法相关疗效因素,进一步指导中医临床辨治。[方法]使用中药系统药理学数据库与分析平台(Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform,TCMSP)检索药物成分及靶点,通过在线人类孟德尔遗传(Online Mendelian Inheritance in Man,OMIM)数据库、人类基因组注释数据库(Human Genome Annotation Database,GeneCards)等检索疾病相关靶点,取交集后采用STRING平台进行蛋白互作(protein-protein interaction,PPI)分析,以CytoScape 3.7.2软件绘制PPI网络图并筛选出核心靶点,上传至MetaScape数据库,进行基因本体(gene ontology,GO)富集分析与京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)信号通路富集分析并构建网络图,利用AutoDock 4.2.6软件进行分子对接,并采用PyMOL软件将对接结果可视化。采用脊柱两侧注射完全弗氏佐剂的方法构建RAU大鼠模型;采用实时定量聚合酶链式反应(Real-time quantitative polymerase chain reaction,Real-time qPCR)检测各组大鼠口腔组织中核心靶基因的表达情况。[结果]筛选得到黄连、升麻活性成分共19个,靶点191个,药物与疾病交集靶点127个,药对干预RAU核心靶点23个,并依据连接度筛选出7个最关键的靶点,核心通路为白细胞介素-17(interleukin-17,IL-17)与肿瘤坏死因子(tumor necrosis factor,TNF)相关的信号通路。分子对接结果显示核心有效成分豆甾醇与各关键靶点均有较高的结合活性。动物实验发现,该药对可有效减少大鼠口腔溃疡数目。与空白对照组比较,模型组、低剂量组、中剂量组和高剂量组关键靶点表达明显更高(P<0.05);高剂量组靶点的mRNA相对表达和口腔溃疡数明显低于模型组、低剂量组、中剂量组(P<0.05)。[结论]黄连-升麻药对治疗RAU的分子机制,与其抗炎、保护口腔黏膜、调节免疫等作用有关,而相关的中医疗效机制涉及清热解毒、化瘀通络、托毒敛疮生肌等。

[Objective]To explore the modern pharmacological mechanisms of Coptidis Rhizoma-Cimicifugae Rhizoma herbal pair in the treatment of recurrent aphthous ulcer(RAU)and analyze the possible traditional Chinese medicine(TCM)therapeutic factors to further guide TCM clinical diagnosis and treatment.[Methods]The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)was used to retrieve drug components and targets.Disease-related targets were obtained from databases such as Online Mendelian Inheritance in Man(OMIM)and Human Genome Annotation Database(GeneCards).The intersection of targets was analyzed using the STRING platform for protein-protein interaction(PPI)analysis.The PPI network was visualized using Cytoscape 3.7.2,and core targets were selected.Gene ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were performed using the MetaScape database,and a network diagram was constructed.Molecular docking was conducted using AutoDock 4.2.6,and the docking results were visualized using PyMOL software.A rat model of RAU was established by bilateral subcutaneous injection of complete Freund’s adjuvant.Real-time quantitative polymerase chain reaction(Real-time qPCR)was used to detect the expression of core target genes in the oral tissues of rats in each group.[Results]A total of 19 active components of Coptidis Rhizoma-Cimicifugae Rhizoma were screened,along with 191 targets.There were 127 intersecting targets between herbs and diseases,and 23 core targets were identified for RAU intervention.Based on Degree,seven key targets were selected,and the core pathway was the signaling pathway associated with interleukin-17(IL-17)and tumor necrosis factor(TNF).The docking results showed that the core active component stigmasterol exhibited high binding activity with each key target.Animal experiments showed that the herbal pair treatment significantly reduced the number of oral ulcers in rats.Compared with blank control group,the expression of core targets in model group,low-dose group,medium-dose group and high-dose group was significantly higher(P<0.05).The relative mRNA expression and number of oral ulcers in the high-dose group were significantly lower than those in model group,low-dose group and medium-dose group(P<0.05).[Conclusion]The molecular mechanisms of Coptidis Rhizoma-Cimicifugae Rhizoma herbal pair in the treatment of RAU are related to its anti-inflammatory,oral mucosal protection and immunomodulatory effects.The relevant TCM therapeutic mechanisms involve heat-clearing and detoxifying,promoting blood circulation and removing blood stasis,eliminating toxins,promoting wound healing and other effects.