关键词: 牙周炎, 桃红四物汤, 网络药理学, 分子对接技术, 抗炎机制

Abstract:

Objective To analyze the active ingredient-target-signaling pathway regulatory network of Taohong Siwu Decoction （THSWD） by network pharmacology and molecular docking technology， and to verify its role in the treatment of periodontitis through in vitro experiments， and to clarify its action targets and related regulatory mechanisms. Methods The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP） was used to obtain the active ingredients and potential targets of THSWD； the Online Mendelian Inheritance in Man （OMIM）， Human Gene Database （GeneCards）， and Therapeutic Target Database （TTD） were used to screen the periodontitis-related targets； the STRING platform was used to construct a protein-protein interaction （PPI） network of the intersecting targets of THSWD and periodontitis， and to screen the core components and targets； the Metascape database was used for Gene Ontology （GO） functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） signaling pathway enrichment analysis； molecular docking technology was used to verify the binding ability of the core components to the targets. In the in vitro experiments， the RAW264.7 cells were induced by Porphyromonas gingivalis lipopolysaccharide （P.g-LPS） to establish the in vitro model of periodontitis； cell counting kit-8 （CCK-8） method was used to detect the cytotoxicities of THSWD in various groups； real-time fluorescence quantitative PCR （RT-qPCR） was used to detect the mRNA expression levels of inflammatory factors in various groups； 2'，7'-dichlorodihydrofluorescein diacetate （DCFH-DA） probe method was used to detect the levels of reactive oxygen species （ROS） in the cells； Western blotting method was used to detect the expression levels of pathway-related proteins in various groups. Results Network pharmacology screening identified 45 potential active ingredients （quercetin， kaempferol， luteolin， etc.） of THSWD， and 81 intersecting genes with periodontitis， among which the core targets included prostaglandin-endoperoxide synthase 2 （PTGS2）， heat shock protein 90 alpha family class A member 1 （HSP90AA1）， B-cell lymphoma 2 （Bcl-2）， protein kinase B1 （AKT1）， mitogen-activated protein kinase 1 （MAPK1）， and v-rel reticuloendotheliosis viral oncogene homolog A （RELA）. The KEGG signaling pathway enrichment analysis results showed that THSWD might affect the inflammatory and immune processes of periodontitis by regulating signaling pathways such as the Janus kinase/signal transducer and activator of transcription （JAK/STAT） signaling pathway. The molecular docking technology results showed that the core components of THSWD had strong binding ability to the key targets of periodontitis. In the in vitro experiments， 0.2， 0.4， 0.8， and 1.6 g·L^-1 THSWD were selected as the administration concentrations， and the half-maximal inhibitory concentration （IC??） of the drug was 41.72 g·L^-1. The RT-qPCR results showed that compared with control group， the expression levels of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， interleukin-6 （IL-6）， and interleukin-10 （IL-10） mRNA in the cells in lipopolysaccharide （LPS） group were significantly increased （P<0.01）； compared with LPS group， the expression levels of TNF-α， IL-1β， and IL-6 mRNA in the cells in 0.2， 0.4， 0.8， and 1.6 g·L^-1 THSWD groups were significantly decreased （P<0.01）， and the expression level of IL-10 mRNA was significantly increased （P<0.05 or P<0.01）. The DCFH-DA probe method results showed that compared with control group， the ROS level in the cells in LPS group was significantly increased （P<0.01）； compared with LPS group， the ROS levels in the cells in 0.2， 0.4， and 0.8 g·L^-1 THSWD groups were significantly decreased （P<0.01）. The Western blotting method results showed that compared with control group， the expression levels of TNF-α， IL-1β， IL-6， and phosphorylated Janus kinase 1 （p-JAK1） proteins in the cells in LPS group were significantly increased （P<0.01）； compared with LPS group， the expression levels of TNF-α， IL-1β， IL-6， and p-JAK1 proteins in the cells in 0.2， 0.4， and 0.8 g·L^-1 THSWD group and ruxolitinib group were significantly decreased （P<0.05 or P<0.01）. Conclusion THSWD can treat periodontitis through multi-component， multi-target， and multi-pathway approaches.The in vitro experiments confirm that THSWD can inhibit the activation of JAK/STAT signaling pathway and has anti-inflammatory and antioxidant effects in vitro.

Key words: Periodontitis, Taohong Siwu Decoction, Network pharmacology, Molecular docking technology, Anti-inflammatory mechanism