Multidisciplinary research systematically elucidates the mechanism of Huangqin Qingre Chubi capsule in attenuating inflammation of gouty arthritis.

The Jianpi Qingre Tongluo method-Huangqin Qingre Chubi capsule (HQC) is a featured herbal drug that was created based on traditional Chinese medicine theory. HQC is approved as a clinical evidence-based prescription in China and has been used to treat gouty arthritis (GA) for many years. However, interdisciplinary and diversified research on HQC against GA inflammation is still lacking. This study aims to integrate real-world clinical analysis, bioinformatics, virtual screening, and in vitro and in vivo experiments to elucidate the potential mechanism of HQC in improving GA inflammation. A retrospective analysis was conducted of laboratory metrics and self-perception of patients (SPP) scale of 1226 patients with GA based on real-world. The association rule algorithm was used to calculate the associations between HQC and laboratory metrics and SPP. Bioinformatics analyses were applied to predict key targets and key signaling pathways of HQC against GA. High-throughput virtual screening was adopted to screen the core active ingredients of HQC. GA rat model was constructed to evaluate the effects of HQC on inflammation in GA rats by joint observations, ELISA, and immunohistochemical staining (IHC). The effects of HQC on fibroblast-like synoviocytes (FLSs) of GA was evaluated using CCK8 assay, ELISA, WB, and immunofluorescence (IF). Significant improvements in inflammatory metrics and SPP were observed in patients with GA treated with HQC; these improvements were strongly associated with HQC. The bioinformatics analyses revealed that the key targets of HQC against GA inflammation were the NFKB1, TLR4, IL1B, IL10, CXCL2, CXCL8, PTGS2, NFE2L2, CYP19A1, and PPARA, while NF-κB signaling pathway was the key signaling pathway. Two core active molecules were identified by high-throughput virtual screening, i.e., 2-monoolein and 5,7,2,5-tetrahydroxy-8,6-dimethoxyflavone. HQC significantly improved the joint conditions of GA rats and down-regulated the levels of a series of pro-inflammatory markers. IHC showed that HQC was able to down-regulate TLR4/MyD88/NF-κB signaling pathway in the synovium. The cellular experiments indicated that HQC drug-containing serum significantly lowered the IL-1β, IL-6, TNF-α, and NLRP3 levels and elevated the IL-10 level. WB and IF demonstrated that HQC inhibited TLR4/MyD88/NF-κB signaling pathway expression. This study proved that HQC treated GA by attenuating joint and systemic inflammatory responses, primarily by inhibiting the TLR4/MyD88/NF-κB signaling pathway. This study was conducted solely on a single batch of HQC. Given the natural variability of herbs, the generalizability of these specific findings to all batches of HQC requires further confirmation.