基于网络药理学探究桑黄总黄酮体外抗炎降尿酸作用机制

Study on the regulation of anti-inflammatory and lowering uric acid effect by total flavonoids of Phellinus Igniarius in vitro Based on network pharmacology

关键词:  网络药理学  桑黄总黄酮  高尿酸血症  炎症  尿酸转运蛋白

Key words:  network pharmacology  total flavonoids of Phellinus Igniarius  hyperuricemia  inflammation  uric acid transporter

摘要:

摘要：目的 基于网络药理学和实验验证探究桑黄总黄酮(TFPI)抗炎降尿酸的作用机制。方法 通过TCMSP和TCMID数据库筛选桑黄活性成分和有效靶点，使用DisGeNET数据库进行高尿酸血症(HUA)疾病靶点筛选，取交集靶点使用STRING数据库构建蛋白相互作用(PPI)网络，采用Metascape数据库进行Gene Ontology(GO)及Kyoto Encyclopedia of Genes and Genomes(KEGG)富集分析，利用微生信进行可视化处理。构建MSU诱导的HK-2细胞损伤模型，给予TFPI干预，通过氧化敏感探针(DCFH-DA)检测细胞活性氧(ROS)水平、酶联免疫法(Elisa)检测炎症因子、实时荧光定量PCR(qRT-PCR)、Western blot对部分预测结果进行实验验证。结果 网络药理学结果表明，TFPI治疗HUA的关键靶点涉及白细胞介素6(IL-6)、肿瘤坏死因子(TNF-α)和Toll样受体(TLRs)、NLRP3及核因子κB(NF-κB)。实验结果显示，与模型组相比，TFPI处理可有效抑制MSU诱导的HK-2细胞凋亡，降低细胞内ROS荧光水平，降低细胞上清液中IL-6、IL-1β、IL-18和TNF-α含量；Western blot结果显示，TLR4、NLRP3、ASC、Caspase-1、NF-κB、YAP、TAZ、ABCG2和OAT1的蛋白相对表达水平均显著下降，β-TRCP和URAT1蛋白相对表达水平显著增加；qRT-PCR结果表明，ABCG2和OAT1的mRNA的表达显著增加，URAT1的mRNA的表达显著下降。结论 研究证实了网络药理学的部分预测结果，表明TFPI通过多成分、多靶点、多通路的途径发挥抗炎降尿酸作用，为临床应用提供了科学依据。

Abstract:

Abstract: OBJECTIVE To investigate the mechanism of anti-inflammatory and lowering uric acid effect by total flavonoids of Phellinus Igniarius (TFPI) based on network pharmacology and experimental validation. METHODS The active ingredients and effective targets of Phellinus Igniarius were obtained through TCMSP and TCMID. DisGeNET was used to predict and screen targets of hyperuricemia(HUA). The protein-protein interaction (PPI) network was constructed using STRING database. Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment were analyzed using Metascape. The visualization analysis was carried out using the bioinformatics platform. The inflammation model of HK-2 cells induced by MSU was established. TFPI intervention was given, validation of some predictions by oxidation sensitive probe (DCFH-DA), enzyme linked immunoassay (Elisa), real-time quantitative PCR (qRT-PCR) and Western blot. RESULTS Network pharmacology results showed that the key targets of the treatment of HUA involved interleukin-6 (IL-6), tumor necrosis factor (TNF-α), toll-like receptors (TLRs), NLRP3 and nuclear factor κB (NF-κB). Compared with the model group, TFPI treatment could effectively inhibit the apoptosis of HK-2 cells induced by MSU, reduce the intracellular ROS fluorescence level, and reduce the contents of IL-6, IL-1β, IL-18 and TNF-α in the cell supernatant. Western blot results showed that the relative protein expression of TLR4, NLRP3, ASC, Caspase-1, NF-κB, YAP, TAZ, ABCG2 and OAT1 significantly decreased, and the relative protein expression levels of β-TRCP and URAT1 effectively increased. The qRT-PCR results showed that the expression of mRNA of ABCG2 and OAT1 significantly increased, and the expression of mRNA of URAT1 decreased. CONCLUSION This study confirmed the partial prediction results of network pharmacology, indicating that TFPI play anti-inflammatory and lowering uric acid effects through multi-component, multi-target and multi-pathway, providing scientific basis for clinical application.