基于体外试验和网络药理学研究金银花抗炎抗菌活性及分子机制

Molecular Mechanism of Anti-inflammatory and Antibacterial Activity of Lonicerae Japonicae Flos Based on in Vitro Experiments and Network Pharmacology

关键词:  金银花  网络药理  分子对接  RAW264.7  抗炎

Key words:  Lonicerae Japonicae Flos  network pharmacology  molecular docking  RAW264.7  anti-inflammation

摘要:

目的 利用网络药理学和体外试验研究，对金银花的抗炎抗菌作用机制进行挖掘，并为其深入研究提供参考依据。方法 通过网络药理学构建了金银花抗炎抗菌蛋白质相互作用（protein-protein interaction，PPI）网络，并通过分子模拟技术进行了金银花活性成分的筛选。体外细胞试验采用LPS诱导的RAW264.7炎症细胞模型，通过细胞活性检测法检测不同浓度金银花提取物细胞的毒性反应；采用Griess法检测金银花提取物对细胞中一氧化氮（nitric oxide，NO）的影响；荧光定量PCR法和Western blotting分别测定金银花提取物对RAW264.7细胞中关键靶标的mRNA表达量和蛋白含量的影响；免疫荧光分析检测金银花提取物对NF-kB p65的影响。结果 网络药理学研究显示，金银花抗炎抗菌的关键靶点为IL-1β、IL-6、COX-2等。分子对接结果显示，金银花中ZINC03978781、beta-sitosterol、Stigmasterol成分具有潜在的抗炎抗菌活性。体外试验结果显示，与LPS组相比，0.19，0.38，0.75，1.50 mg·mL^–1的金银花提取物组RAW264.7细胞中NO的生成量均显著降低（P<0.001）；金银花提取物在0.19，0.38，0.75，1.50 mg·mL^–1的浓度下能够抑制IL-1β，IL-6，COX-2 mRNA的表达（P<0.05或P<0.01或P<0.001）；在1.50 mg·mL^-1的浓度下能够抑制IL-1β、IL-6蛋白的表达（P<0.01 或P<0.001），并且抑制NF-kB p65向细胞核内的转运。结论 该结果为深入研究金银花抗炎抗菌作用及进一步合理开发利用金银花提供了实验依据。

Abstract:

OBJECTIVE To excavate the anti-inflammatory and antibacterial mechanism of Lonicerae Japonicae Flos by using network pharmacology and in vitro experimental research, and to provide the reference basis for its in-depth research. METHODS The Lonicerae Japonicae Flos anti-inflammatory and anti-bacterial protein-protein interaction(PPI) network was constructed through network pharmacology research. Afterwards, the active ingredients of Lonicerae Japonicae Flos were screened by molecular docking technology. The in vitro cell experiment used the RAW264.7 inflammatory cell model induced by LPS. The cell viability test(MTT) method was used to detect the toxicity of Lonicerae Japonicae Flos at different concentrations to cells. The Griess method was used to detect the effect of Lonicerae Japonicae Flos on nitric oxide(NO) in cells. Fluorescence quantitative PCR and Western blotting were used to determine the effects of Lonicerae Japonicae Flos on the mRNA expression and protein content of key targets in RAW264.7 cells. Immunofluorescence analysis was used to detect the effect of Lonicerae Japonicae Flos on NF-κB p65. RESULTS Network pharmacology studies had shown that the key targets of Lonicerae Japonicae Flos anti-inflammatory and antibacterial were IL-1β, IL-6 and COX-2. The molecular docking results showed that ingredients such as ZINC03978781, beta-sitosterol and Stigmasterol in Lonicerae Japonicae Flos had potential anti-inflammatory and antibacterial activities. The results of in vitro experiments showed that compared with the LPS group, the production of NO in the RAW264.7 cells of the Lonicerae Japonicae Flos group at 0.19, 0.38, 0.75, and 1.50 mg·mL^-1 was significantly reduced(P<0.001); at the concentration of 0.19, 0.38, 0.75, 1.50 mg·mL^-1, the Lonicerae Japonicae Flos could inhibit the expression of IL-1β, IL-6, COX-2 mRNA(P<0.05 or P<0.01 or P<0.001); at the concentration of 1.50 mg·mL^-1, it could inhibit the expression of IL-1β, IL-6 protein(P<0.01 or P<0.001), and inhibit the transport of NF-κB p65 to the nucleus. CONCLUSION This result provides an experimental basis for an in-depth study of the anti-inflammatory and antibacterial effects of Lonicerae Japonicae Flos and further rational development and utilization.