RNA sequencing analysis reveals the potential therapeutic mechanisms of Huzhang Tongfeng granule in MSU-induced acute gouty arthritis mouse model

Abstract

BackgroundGout is one of the most prevailing inflammatory arthritis, in which joint pain and dysfunction may drastically reduce patients' quality of life, work efficiency, and increase the burden of medical treatment. The Huzhang Tongfeng granule (HZTFG) has been utilized clinically for more than 30 years with satisfactory effects and no significant adverse reactions. However, the mechanisms that underlie HZTFG's effect remain indeterminate. MethodsFirstly, the Liquid Chromatography-Mass Spectrometry (LC-MS) analyses were applied to clarify the multi-components of HZTFG to establish the quality control parameters. After the monosodium urate (MSU) crystals induced Acute gouty arthritis (AGA) model was established, behavioral testing, joint swelling, hematoxylin, and eosin (H&E) staining were applied to assess the effect of HZTFG on improving joint pain, swelling, synovial inflammation of the MSU crystals-injected mice. Moreover, to further understand the underlying mechanism of the action of HZTFG on AGA, RNA Sequencing (RNA-seq) analysis was performed to detect differentially expressed genes (DEGs) after HZTFG treatment. Thereafter, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were enriched via gene set enrichment analysis (GSEA) to assess the functions and pathways of DEGs. Furthermore, Reverse Transcription Polymerase Chain Reaction (RT-qPCR) analysis and enzyme-linked immunosorbent assay (ELISA) were applied for validation in vivo. ResultsAfter treatment with HZTFG, RNA-seq results revealed 44 DEGs, of which 20 genes were remarkably down-regulated and 24 genes were up-regulated. Besides, the expression of these assayed genes were noticeably decreased, including Cxcl1, Cxcl2, Cebpb, Lcn2, Ptgs2, Mmp13, Fos, Socs3, Sele, and Junb. Moreover, animal experiments also demonstrated that TNF signaling pathway and IL-17 signaling pathway are key pathways in HZTFG treated MSU-induced AGA mouse model. ConclusionHZTFG attenuated MSU crystals-induced inflammatory response through TNF and IL-17 signaling pathways by inhibiting the activation of related chemokines, inflammatory proteins, transcription factors, suppressor of cytokine signaling, and adhesion molecule in AGA. This study further provides the potential therapeutic targets for AGA treatment.