目的 基于高通量基因表达数据库(GEO)、网络药理学和分子对接技术分析归芍地黄汤对新生血管性年龄相关性黄斑变性的可能作用靶点和相关机制,并通过体内实验进行验证。方法 通过查阅文献及中国传统医学公共数据库(TCMSP)确定归芍地黄汤的主要成分及靶标,绘制成分-靶点网络拓扑图。通过整合GEO数据库和公共数据库对新生血管性年龄相关性黄斑变性的疾病靶点进行预测。使用Venny在线平台确定药物-疾病的交集靶点,构建蛋白质相互作用(PPI)网络图,利用Cytoscape软件筛选关键基因(Hub),利用R语言对交集靶点进行基因本体(GO)和京都基因与基因组百科全书(KEGG)富集分析。将归芍地黄汤核心成分与靶基因之间进行分子对接,最后利用光学相干断层扫描和免疫组化显示组织病理学变化,荧光素眼底血管造影观察脉络膜新生血管膜渗漏情况,并验证其对靶基因表达水平的影响。结果 从GSE29801数据集中获得50个差异基因,筛选得到药物-疾病交集靶点134个。核心靶点包括白蛋白(ALB)、肿瘤蛋白p53(TP53)、蛋白激酶B1(Akt1)、白细胞介素-6(IL-6)、肿瘤坏死因子(TNF)、半胱氨酸天冬氨酸蛋白酶-3(CASP3)、血管内皮生长因子A(VEGFA)、过氧化氢酶(CAT)、表皮生长因子受体(EGFR)、缺氧诱导因子1A(HIF1A)。这些基因主要富集在对外来生物刺激的反应、对营养水平的反应和创口愈合等生物学过程中,参与衰老、氧化应激、免疫炎症反应和细胞凋亡等相关途径。分子对接预测显示,归芍地黄汤核心成分与丝裂原活化蛋白激酶8(MAPK8)、VEGFA、TNF-α、IL-6表现出稳定的结合。动物实验显示,归芍地黄汤减轻了激光造模诱导的眼底病变,下调了VEGF、TNF-α水平,且治疗前后BN大鼠p38 MAPK表达水平存在差异。结论 归芍地黄汤可以减轻脉络膜新生血管膜渗漏大鼠视网膜病变,揭示了归芍地黄汤治疗新生血管性年龄相关性黄斑变性可能与激活MAPK信号通路有关。

Objective To explore the potential targets and related mechanisms of Guishao Dihuang Decoction in treatment of neovascular age related macular degeneration based on the GEO gene microarray database, network pharmacology and molecular docking technology.Methods The main targets and main component of Guishao Dihuang Decoction were identified by reviewing the literature and public databases of Chinese traditional medicine(TCMSP). Disease targets of neovascular age related macular degeneration were predicted by integrating GEO database and public databases. Drug-disease intersection targets were identified using the Venn online platform and the intersection targets were used to construct protein interaction network maps. Hub genes were screened using Cytoscape software, GO and KEGG enrichment analysis of the intersection targets were performed using R language. Molecular docking was performed between the core components of Guishao Dihuang Decoction and the target genes. Finally, optical coherence tomography and immunohistochemistry were used to reveal histopathological changes, and fluorescein fundus angiography was used to observe the leakage of choroidal neovascularization membrane, and to verify its influence on the expression level of target genes.Results There were 50 differentially expressed genes from the GSE29801 dataset. A total of 134 intersection targets were screened.The core targets included ALB, TP53, AKT1, IL6, TNF, CASP3, VEGFA, CAT, EGFR, and HIF1A. These genes were mainly enriched in biological processes such as response to xenobiotic stimulation, response to nutrient level, and wound healing, and involved in related pathways such as aging, oxidative stress, immune inflammatory response, and apoptosis. The result of molecular docking showed that the core components of Guishao Dihuang Decoction had a stable binding with MAPK8, VEGFA, TNFα, and IL-6. Animal experiments revealed that Guishao Dihuang Decoction alleviated fundus lesions induced by laser modeling, down-regulated the expression levels of VEGF and TNFα, and there was a difference in the expression level of p38MAPK in BN rats before and after treatment.Conclusion Guishao Dihuang Decoction can attenuate retinal lesions in leakage of the choroidal neovascularization membrane rats and preliminarily revealed that the treatment of neovascular age related macular degeneration with Guishao Dihuang Decoction may be related to the activation of MAPK signaling pathway.

R285

福建省自然科学基金资助项目(2020J011046);福建中医药大学附属人民医院中药制剂研发项目(ZYZJ2019003)