Abstract: BACKGROUND: Qu Shi Jie Du Formula (QSJDF) is a clinical empirical prescription for preventing and treating hyperuricemia and gout based on the “dampness-toxin accumulation” theory. It exerts effects of eliminating dampness, detoxifying, and relaxing tendons and collaterals. However, its underlying mechanism remains unclear.
OBJECTIVE: To investigate the therapeutic effects and bone protection mechanism of QSJDF in hyperuricemia mice.
METHODS: (1) The chemical constituents of QSJDF were identified using ultra-high performance liquid chromatography – Q-Exactive mass spectrometry. Target genes of these components related to hyperuricemia were retrieved from relevant databases. Core targets for QSJDF in treating hyperuricemia were identified via network pharmacology and subjected to Kyoto Encyclopedia of Genes and Genomes pathway enrichment and Gene Ontology functional annotation. (2) Forty Balb/c mice were randomly divided into five groups (n=8 per group): blank control, model, allopurinol, QSJDF, and QSJDF + allopurinol combination. Except for the blank control group, hyperuricemia models were established. After 28 days of modeling, mice received daily intragastric administration for 4 weeks: blank control and model groups received sodium carboxymethyl cellulose; allopurinol group received allopurinol; QSJDF group received QSJDF; combination group received both QSJDF and allopurinol. Samples were collected 6 hours after the final dose. Serum levels of uric acid, creatinine, blood urea nitrogen, procollagen type I N-terminal propeptide, C-terminal telopeptide of type I collagen, and osteocalcin were measured. Renal pathology was observed using hematoxylin-eosin and Sirius Red staining. Bone mass of the knee joint was analyzed by Micro-CT. Osteoclast numbers were assessed via tartrate-resistant acid phosphatase staining. Immunofluorescence staining was performed to detect p53 and glucose transporter 9 in renal tissue and c-JUN expression in knee joints. Pearson correlation analysis was used to evaluate relationships between Micro-CT parameters and c-JUN fluorescence intensity.
RESULTS AND CONCLUSION: (1) A total of 75 active components were identified in QSJDF, with the top ones being curcumin, quercetin, apigenin, soy isoflavones, and genistein. A total of 198 predicted targets were obtained, with core targets including TP53, tumor necrosis factor, interleukin-6, JUN, interleukin-1B, and nuclear factor-KB1, mainly enriched in p53, hypoxia-inducible factor-1, and nuclear factor-κB signaling pathways. (2) Serum assays showed that QSJDF reduced renal function indicators (uric acid, creatinine, blood urea nitrogen) and bone metabolism indicators (procollagen type I N-terminal propeptide and C-terminal telopeptide of type I collagen) in hyperuricemia mice. Hematoxylin-eosin and Sirius Red staining demonstrated renal protective effects. Micro-CT and tartrate-resistant acid phosphatase staining revealed increased tibial bone mass and reduced osteoclast numbers. Immunofluorescence showed decreased expression of p53 and glucose transporter 9 in the kidney and c-JUN in the knee joint. Pearson analysis indicated a significant positive correlation between trabecular separation and c-JUN fluorescence intensity (r > 0, P < 0.05), while bone volume fraction and trabecular number were significantly negatively correlated with c-JUN fluorescence intensity (r < 0, P < 0.05). To conclude, QSJDF exerts urate-lowering and renal protective effects by regulating the p53/glucose transporter 9 pathway and alleviates bone loss by modulating c-JUN/activating protein 1 signaling to influence osteoclast activity in hyperuricemia mice

Key words: Qu Shi Jie Du Formula, hyperuricemia, liquid chromatography-mass spectrometry, toxin accumulation theory, network pharmacology, bone protection mechanism