基于网络药理学及实验验证探讨地骨皮治疗牙周炎的作用机制

Mechanism of the treatment of periodontitis by Lycii Cortex based on network pharmacology and experimental validation

目的基于网络药理学及实验验证探讨地骨皮治疗牙周炎的作用机制。方法查询中药系统药理学数据库与分析平台(TCMSP)筛选地骨皮的药物成分, 使用SwissTargetPrediction预测药物成分的作用靶点;运用GeneCards获取牙周炎的疾病靶点, 利用Venny 2.1获取交集靶点。使用STRING进行蛋白质-蛋白质相互作用(PPI)分析并使用Cytoscape构建网络图, 利用Metascape进行基因本体(GO)功能注释及京都基因和基因组百科全书(KEGG)信号通路分析。使用Cytoscape软件构建"药物-靶点-通路"网络图。将大鼠随机分为模型组和治疗组, 每组5只。建模后8周, 治疗组局部注射1 ml地骨皮药液(将150 mg地骨皮颗粒溶于1 ml水中), 模型组组局部注射等量生理盐水, 治疗4周。结果地骨皮的10个有效成分通过多条通路直接作用于55个疾病靶点治疗牙周炎, 其中β-谷甾醇、豆甾醇、莨菪碱、金合欢素、阿托品、常春藤素等是核心成分, V-Rel网状内皮增生病毒癌基因同源物A(RELA)、B淋巴细胞瘤-2(Bcl-2)、前列腺素内过氧化物合酶2(PTGS2)、JUN、含半胱氨酸的天冬氨酸蛋白水解酶-3(CASP3)、肿瘤蛋白53(TP53)、核受体共激活因子2(NCOA2)是重要的靶点。GO分析显示交集基因最可能相关的生物过程(BP)主要涉及对类固醇激素的反应、细胞对有机环化合物的反应、程序性细胞死亡的正调控、对激素的反应、凋亡信号通路、白细胞凋亡过程、正调控神经元凋亡过程、对氧气水平的反应、凋亡过程的正向调节等, 细胞组分(CC)主要涉及细胞器外膜、外膜、转录调控复合体、线粒体外膜、RNA聚合酶Ⅱ转录调控复合物、线粒体膜、膜筏、膜微区、细胞质核周区等, 分子功能(MF)主要涉及蛋白质结构域特异性结合、转录因子结合、半胱氨酸型内肽酶活性参与凋亡信号通路、DNA结合转录因子结合、RNA聚合酶Ⅱ特异性DNA结合转录因子结合、半胱氨酸型内肽酶活性参与细胞凋亡过程、一般转录起始因子结合、泛素蛋白连接酶结合、泛素样蛋白连接酶结合等。KEGG分析结果提示地骨皮主要通过p53、细胞凋亡、晚期糖基化终产物及其受体(AGE-RAGE)、磷脂酰肌醇3-激酶-蛋白激酶B(PI3K-Akt)、白细胞介素-17(IL-17)、缺氧诱导因子-1(HIF-1)、肿瘤坏死因子(TNF)、丝裂原活化蛋白激酶(MAPK)、核因子-κB(NF-κB)等信号通路治疗牙周炎。动物实验显示, 地骨皮能够明显改善牙周炎, 同时也能改善RELA、Bcl-2、PTGS2、JUN、CASP3、TP53、NCOA2表达水平。结论地骨皮主要通过调节p53、细胞凋亡、AGE-RAGE、PI3K-Akt、IL-17、HIF-1、TNF、MAPK、NF-κB等信号通路的RELA、Bcl-2、PTGS2、JUN、CASP3、TP53、NCOA2等疾病靶点, 调节酶类活性、抗炎等生物过程治疗牙周炎。

Objective To explore the mechanism of action of Lycii Cortex in the treatment of periodontitis based on network pharmacology and experimental validation.Methods Traditional Chinese medicine systems pharmacology database and analysis platform(TCMSP)was used to screen the drug components of Lycii Cortex.Swiss Target Prediction was used to predict the action targets of the drug components.GeneCards was used to obtain the disease targets of periodontitis. Venny 2.1 was used to obtain the intersecting targets. Protein-protein interaction (PPI) network analysis was performed using the STRING, and network diagrams were constructed using Cytoscape. Gene ontology (GO) functional annotation and Kyoto encyclopedia of genes and genomes (KEGG) signaling pathway analysis were performed using Metascape. The Cytoscape software was used to construct “drug-target-pathway” network diagrams. The rats were randomly divided into model group and treatment group, with 5 rats in each group. After modeling for 8 weeks, the treatment group was injected with 1 ml of Lycii Cortex (150 mg Lycii Cortex Granules were dissolved in 1 ml water), and the model group was injected with the same amount of normal saline locally for 4 weeks. Results A total of 10 active components of Lycii Cortex were founded. These components acted directly on 55 disease targets through multiple pathways to treat periodontitis. The results showed that β-sitosterol, stigmasterol, scopoletin, chrysin, atropine, and ivytin were the core components, and V-Rel reticuloendotheliosis hyperplasia viral oncogene homolog A (RELA), B-cell lymphoma-2 (Bcl-2), prostaglandin-endoperoxide synthase 2 (PTGS2), JUN, cysteinyl aspartate specific proteinase-3 (CASP3), tumor protein 53 (TP53), and nuclear receptor coactivator 2 (NCOA2) were important targets. GO analysis revealed that the most likely biological process (BP) associated with the intersecting genes was mainly involved in the response to steroid hormones, cellular response to organic ring compounds, programmed cell death positive regulation, response to hormones, apoptosis signaling pathway, leukocyte apoptotic process, positive regulation of neuronal apoptotic process, response to oxygen levels, positive regulation of apoptotic process, etc. The cellular component (CC) of the Lycii Cortex was mainly involved in the outer membrane of organelles, outer membrane, transcriptional regulatory complex, outer membrane of mitochondria, RNA polymerase Ⅱ transcriptional regulatory complex, mitochondrial membrane, membrane rafts, membrane microregion, perinuclear region of cytoplasm, etc. The molecular function (MF) of the Lycii Cortex was mainly involved in protein structural domain-specific binding, transcription factor binding, cysteine-type endopeptidase activity involved in apoptotic signaling pathway, DNA-binding transcription factor binding, RNA polymerase Ⅱ-specific DNA-binding transcription factor binding, cysteine-type endopeptidase activity involved in apoptotic process, general transcription initiation factor binding, ubiquitin protein ligase binding, ubiquitin-like protein ligase, etc. The results of KEGG analysis suggested that dermatophytes were mainly involved in the apoptotic process through p53, apoptosis, advanced genetically engineered end products and their receptors (AGE-RAGE), phosphatidylinositol 3-kinase-protein kinase B (PI3K-Akt), interleukin-17 (IL-17), hypoxia-inducible factor-1 (HIF-1), tumor necrosis factor (TNF), mitogen-activated protein kinase (MAPK), and nuclear factor-κB (NF-κB) signaling pathways for the treatment of periodontitis. Animal experiments showed that diclofenac could significantly improve periodontitis, as well as improve the expression levels of RELA, Bcl-2, PTGS2, JUN, CASP3, TP53, and NCOA2. Conclusions Lycii Cortex mainly regulates enzymatic activity, anti-inflammatory, and other biological processes such as RELA, BCL2, PTGS2, JUN, CASP3, TP53, NCOA2, and other disease targets of the signaling pathways such as p53, apoptosis, AGE-RAGE, PI3K-Akt, IL-17, HIF-1, TNF, MAPK, and NF-κB, etc., to treat periodontitis.