Qu-zhuo-tong-bi decoction exerts gouty arthritis therapy by skewing macrophage polarization through butanoate metabolism

Abstract

Background: Qu-zhuo-tong-bi decoction (QZTBD), a traditional Chinese medicine (TCM), has demonstrated efficacy in the treatment of gouty arthritis. However, to date, the precise pharmacological mechanisms remain unclear.

Purpose: The study aims to ascertain the therapeutic effects and the underlying mechanisms of QZTBD in the treatment of gouty arthritis.

Methods: The efficacy and safety of different doses of QZTBD were investigated in Uox-KO mice. Candidate active ingredients were identified using UHPLC-MS/MS. The potential therapeutic pathways of the active ingredients were predicted through network pharmacology. The mechanisms of QZTBD in alleviating gouty arthritis were explored via comprehensive analyses of gut microbiota, combined with RT-qPCR, western blot, immunofluorescence, ELISA, flow cytometry, and Seahorse assay. Fecal microbiota transplantation (FMT), bacterial culture experiment, butyrate-producing bacteria (BPB) and butyrate administration, and 2-DG intervention were conducted to explore the roles of BPB and butanoate metabolism in gout progression and therapeutic mechanisms of QZTBD. In vitro studies further validated the regulatory effects of butyrate and QZTBD on macrophage polarization through glycolysis modulation.

Results: 18.0 g/kg/d of QZTBD effectively alleviated the symptoms of gouty arthritis with excellent hepatic and renal safety. UHPLC-MS/MS analysis and network pharmacology revealed that QZTBD exerts its effects on butanoate metabolism during gouty arthritis inflammation. QZTBD treatment increased the abundance of BPB, the levels of serum and colon butyrate, and the expression levels of Buk and But. The transplantation of QZTBD-treated microbiota reproduced the therapeutic effects of QZTBD. M1 macrophage polarization was suppressed after QZTBD intervention. The administration of BPB and butyrate attenuated gouty arthritis and orchestrated macrophage polarization. Inhibition of glycolysis regulated the phenotype of macrophage and attenuated inflammatory processes. In vitro analysis unveiled that QZTBD and butyrate modulated glycolysis to regulate macrophage polarization, thereby alleviating gouty arthritis.

Conclusion: QZTBD targeted butanoate metabolism to regulate macrophage polarization, thereby effectively alleviating intestinal inflammation and restoring immune homeostasis in gouty arthritis. These findings establish a mechanistic foundation for developing precision therapeutic strategies leveraging QZTBD to combat gouty arthritis.

Keywords: Butanoate metabolism; Glycolysis; Gouty arthritis; Gut microbiome; Macrophage polarization; Qu-zhuo-tong-bi decoction.

Fig.1

Therapeutic effects of QZTBD on disease symptoms of Uox-KO mice. A, B Serum and urinary UA levels after QZTBD and BBR treatment. C Representative images and H&E-stained sections of footpads 24 h post-MSU injection (Scale bar: 100 µm). D, E Footpad swelling index and mechanical pain threshold after treatment. F H&E-stained liver (top) and kidney (bottom) histological sections (Scale bar: 100 µm). G QZTBD (18.0 g/kg/d) attenuated hepatic and renal pathological damage. H Colon sections stained with H&E (top) and IHC staining for ZO-1 (middle) and occludin (bottom) (Scale bar: 100 µm). I Serum IL-1β, IL-6, and TNF-α concentrations measured by ELISA (n = 6). n = 7–9 mice per group. Values are expressed as mean ± SEM. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001

Fig.2

Chemical profiling of QZTBD aqueous extracts by UHPLC-MS/MS. A Total ion chromatography in positive ion mode. B Total ion chromatography in negative ion mode. C Classification of the 344 identified or tentatively characterized compounds. D KEGG pathway enrichment analysis of QZTBD bioactive components

Fig.3

QZTBD restores M1/M2 macrophage balance in Uox-KO mice. A, B Representative flow cytometry plots of intestinal lamina propria M1 macrophages (CD86⁺ F4/80⁺ CD45⁺) and M2 macrophages (CD163⁺ F4/80⁺ CD45⁺). C Percentage of intestinal M1 and M2 macrophages and M1/M2 ratio (n = 7). D, E Representative flow cytometry plots of splenic M1 and M2 macrophages. F Percentage of splenic M1 and M2 macrophages and M1/M2 ratio (n = 7). G–J Immunofluorescence of M1 and M2 macrophages in footpad tissue sections (Scale bar: 50 μm) (n = 4). K, L Cytokine levels (IL-1β, IL-6, TNF-α, IL-18, IL-10) in serum and intestinal tissue (n = 5). Values are expressed as mean ± SEM. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001

Fig.4

QZTBD regulates butyrate metabolism in Uox-KO mice. A, B Fecal and serum butyric acid levels after QZTBD and BBR treatment (n = 5–7). C Relative abundance of metabolites in the butyrate metabolism pathway post-QZTBD treatment (n = 6). D Relative abundance of butyric acid-producing bacteria (n = 7). E, F Relative expression of Buk and But in intestinal contents after treatment (n = 7). G Bacterial growth density measured by OD₆₀₀ (n = 3). H, I Relative expression of Buk and But in bacteria cultured with QZTBD (n = 3–4). Values are expressed as mean ± SEM. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001

Fig.5

Effects of BPB and butyrate supplementation on macrophage polarization. A, B Fecal and serum butyric acid after BPB and butyrate treatment (n = 7–8). C, D Relative mRNA expression of Buk and But expression after BPB and butyrate treatment (n = 7). E Serum UA levels after BPB and butyrate treatment (n = 6–7). F Mechanical pain threshold after BPB and butyrate treatment (n = 6–7). G Percentage of intestinal M1 and M2 macrophages and M1/M2 ratio after BPB and butyrate treatment (n = 7). H Percentage of splenic M1 and M2 macrophages and M1/M2 ratio after BPB and butyrate treatment (n = 7). I, J Immunofluorescence of M1 and M2 macrophages in footpad tissue after BPB and butyrate treatment (n = 4). K, L Cytokine levels in serum and intestinal tissue after BPB and butyrate treatment (n = 5–6). Values are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001

Fig.6

Fecal microbiota transplantation regulates butyrate metabolism in Uox-KO mice. A Schematic of FMT experimental design. B Serum UA level after QZTBD and QZ-FMT treatment. C, D Footpad swelling index after QZTBD and QZ-FMT treatment. E Mechanical allodynia thresholds after QZTBD and QZ-FMT treatment. F Relative abundance of the butyric acid-producing bacteria after QZTBD and QZ-FMT treatment. G qPCR quantification of representative butyrate-producing bacteria after QZTBD and QZ-FMT treatment. H, I Fecal and serum butyric acid levels after QZTBD and QZ-FMT treatment. J, K Relative mRNA expression of Buk and But after QZTBD and QZ-FMT treatment. n = 6–8 mice per group. Values are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001

Fig.7

Fecal microbiota transplantation reprograms macrophage polarization in Uox-KO mice. A, B Representative flow cytometry plots of M1 macrophages and M2 macrophages in intestinal lamina propria. C Percentage of intestinal M1 and M2 macrophages and M1/M2 ratio (n = 7). D, E Representative flow cytometry plots of M1 and M2 macrophages in spleen. F Percentage of splenic M1 and M2 macrophage and M1/M2 ratio (n = 7). G–J Immunofluorescence of M1 and M2 macrophages in footpad (Scale bar: 50 μm) (n = 4). K, L Cytokine levels in serum and intestinal tissue (n = 5–6). Values are expressed as mean ± SEM. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001

Fig.8

QZTBD and butyrate regulate macrophage metabolic reprogramming. A PFK-1, G6PI, and LDH protein expression in colon tissue after QZTBD and BBR treatment. B PFK-1, G6PI, and LDH protein expression in colon tissue post-QZTBD and QZ-FMT treatment. C PFK-1, G6PI, and LDH protein expression in the colon tissue following BPB and butyrate intervention. D–E PFK-1, G6PI, and LDH protein expression in BMDMs. F–I ECAR and OCR in BMDMs treated with QZTBD-containing serum (10%) and butyrate (0.5 mM, 1.0 mM, 1.5 mM) treatment. J–M Phenotypic shift from M1 to M2 macrophage after treatment with QZTBD-containing serum and butyrate in vitro. n = 3 mice per group. Values are expressed as mean ± SEM. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 9

Glycolytic inhibition ameliorates macrophage polarization imbalance and gouty arthritis. A Volcano plot of differentially expressed genes (DEGs) in gout (n = 3). B KEGG pathway enrichment of upregulated DEGs in Gout. C Serum UA levels after 2-DG treatment (n = 5–6). D–F 2-DG attenuates joint inflammation in Uox-KO mice (Scale bar: 100 µm) (n = 5). G, H Representative flow cytometry plots, the percentage and the ratio of M1 and M2 macrophages in intestine and spleen after 2-DG treatment (n = 5). I, J Serum and intestinal cytokine levels after 2-DG treatment (n = 5). K In vitro M1/M2 polarization in BMDMs after 2-DG treatment (n = 4). L The levels of cytokines in BMDMs supernatant after 2-DG treatment (n = 4). Values are expressed as mean ± SEM. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 10

Mechanistic illustration of the QZTBD-mediated alleviation of gouty arthritis. The absence of butyrate-producing bacteria in Uox-KO mice results in a reduction of butyrate levels. Butyrate deficiency drives pro-inflammatory M1 macrophage polarization via glycolytic reprogramming, exacerbating the progression of gouty arthritis. QZTBD treatment restores gut microbial homeostasis, elevates butyrate production, and reprograms macrophage metabolism from glycolysis to OXPHOS. BPB and butyrate supplementation mimics the therapeutic effects of QZTBD, confirming the critical role of microbiota-derived butyrate in metabolic-immune crosstalk. Investigating the impact of QZTBD on bacterial butanoate metabolism represents a promising avenue for understanding its beneficial effects. Targeting the gut microbiota-butyrate-macrophage axis provides a novel therapeutic paradigm for the management of gouty arthritis