Study on the Mechanism of Anti-inflammatory and Lowering Uric Acid Effect by Total Flavonoids of Phellinus Igniarius in Vitro Based on Network Pharmacology

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 screen disease targets of hyperuricemia(HUA). Using STRING database to construct protein interaction network by taking intersection targets. 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. Construct a monosodium urate(MSU) crystal-induced HK-2 cell injury model and administer TFPI intervention. The partial predictions from network pharmacology were experimentally validated by detecting cellular reactive oxygen species(ROS) levels using the oxidation-sensitive probe(DCFH-DA), measuring inflammatory factors via enzyme-linked immunosorbent assay(ELISA), and assessing gene and protein expression levels through real-time quantitative PCR(qRT-PCR) and Western blotting.

RESULTS  Network pharmacology results showed that the key targets of TFPI for the treatment of HUA involved interleukin-6(IL-6), tumor necrosis factor-alpha(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 blotting 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 confirmes 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.