痹宁汤基于网络药理学–转录组学分析通过PPAR信号通路抑制痛风肾的作用机制

Bining Decoction Based on Network Pharmacology-Transcriptome Analysis of the Mechanism of Inhibition of Gout Kidney through PPAR Signal Pathway

摘要: 目的：痛风肾是一种因血尿酸产生过多或排泄减少而导致大量血尿酸沉积于肾小管而引起的常见肾小球疾病。痹宁汤是治疗痛风肾的有效方剂。然而，其作用机制尚不明确。本研究运用网络药理学、分子对接技术以及转录组学分析，探讨痹宁汤治疗痛风肾的潜在分子机制、方法。方法：首先，使用网络药理学方法找到痹宁汤对痛风肾的潜在靶点：从公共数据库TCMSP获取痹宁汤相关9味中药有效化合物靶点，从OMIM、GenBank、GeneCards、Disgenet数据库中获取痛风肾相关基因靶点，进一步筛选和聚类。然后，将网络药理学预测的痹宁汤靶点与筛选的痛风肾相关基因重叠，利用Venny2.1.0获取相交靶标，找到交集靶点，然后利用Cytoscape软件构建活性成分——靶标网络。通过String数据库和Cytoscape软件绘制蛋白–蛋白相互作用(PPI)网络。然后，通过DAVID数据库进行《京都基因与基因组百科全书》(KEGG)的基因本体(GO)和途径富集分析。最后，使用Discovery Studio 2019 Client进行分子对接。另外，以动物实验的方法进行痛风肾建模，以痹宁汤对痛风肾小鼠进行治疗，并采集样本进行分析。将其结果以转录组学方法进行测序实验分析，并作出表达量差异分析。结果：根据筛选标准，共筛选出痹宁汤的413个活性化合物和1085个潜在靶点。发现了118个基因。在活性成分–靶点网络中，β-谷甾醇beta-sitosterol、谷甾醇sitosterol、甘露醇Mandenol、豆甾醇Stigmasterol是重要的活性成分。在PPI网络中，MAPK8、JAK2、PTPN11、ESR1、HSP90AA1、MAPK14是核心靶点。将其进行分子对接，发现MAPK8与化合物豆甾醇Stigmasterol有比较好的结合亲和力，且豆甾醇Stigmasterol化和物广泛存在于痹宁汤中黄柏、山慈菇、薏苡仁中药成分中。因此在分子对接方面，痹宁汤活性成分与核心靶点具有良好的亲和力。将转录组学数据与118个靶基因相交，推测PPAR信号通路可能是痹宁汤的有效机制。体内实验中痹宁汤治疗后的痛风肾小鼠肾功、尿蛋白有显著改善。这些结果证实痹宁汤主要通过PPAR信号通路在痛风肾疾病中发挥有效作用。结论：本研究初步预测了痹宁汤治疗痛风肾的主要有效成分、潜在靶点、信号通路，为进一步研究痹宁汤对痛风肾的保护机制及临床应用提供新的思路。

Abstract: Objective: Gouty nephropathy is a common glomerular disease caused by excessive uric acid pro-duction or decreased uric acid excretion, which leads to a large amount of uric acid deposition in renal tubules. Bining Decoction is an effective prescription for treating Gouty nephropathy. However, its mechanism of action is still unclear. In this study, network pharmacological molecular docking technology and transcriptomics analysis were used to explore the potential molecular mechanism of Bining Decoction in treating Gouty nephropathy. Method: Firstly, the potential targets of Bining Decoction on Gouty nephropathy were found by using network pharmacology methods: the effective compound targets of 9 traditional Chinese medicines related to Bining Decoction were obtained from public database TCMSP, and the Gouty nephropathy-related gene targets were obtained from OMIM, GenBank, GeneCards, Disgenet database for further screening and clustering. Then the net-work pharmacological prediction of Bining Decoction target and the Gouty nephropathy-related genes was overlapped using Venny2.1.0 to obtain the intersection target and then use Cytoscape software to construct the active ingredient-target network. Protein-protein interaction (PPI) net-work is plotted by String database and Cytoscape software. Then the gene ontology (GO) and path-way enrichment of Kyoto Encyclopedia of Genomics and Genomics (KEGG) were analyzed by DAVID database. Finally, Discovery Studio 2019 Client is used for molecular docking. In addition, the model of Gouty nephropathy was established by animal experiment, and the mice with Gouty nephropathy were treated with Bining Decoction and samples were collected for analysis. The results were se-quenced and analyzed by transcriptomics method, and the difference of expression quantity was analyzed. Results: According to the screening criteria, 413 active compounds and 1085 potential targets of Bining Decoction were screened out. 118 genes were discovered. Beta-sitosterol sitos-terolmannitol Mandenolstigmasterol is an important active component in the active compo-nent-target network. MAPK8, JAK2, PTPN11, ESR1, HSP90AA1, MAPK14 are the core target in PPI network. It was found that MAPK8 had good binding affinity with stigmasterol and stigmasterol widely existed in Chinese herbal medicine components of Phellodendron amurensis, mushroom and coix seed in Bining Decoction. Therefore, the active ingredients of Bining Decoction have good affin-ity with the core target in molecular docking. Combining the transcriptomics data with 118 target genes, it is speculated that PPAR signaling pathway may be an effective mechanism of Bining De-coction. In vivo experiment, urinary protein of kidney function in Gouty nephropathy mice treated with Bining Decoction was significantly improved. These results confirmed that BND plays an effec-tive role in Gouty nephropathy disease mainly through PPAR signaling pathway. Conclusion: This study predicted the potential target signal pathway of the main effective components of BND in treating Gouty nephropathy, and provided a new idea for further study on the protective mecha-nism of BND on Gouty nephropathy and its clinical application.