XOD serves a crucial role in UA metabolism within the body, promoting the oxidation of hypoxanthine to xanthine and then further catalyzing the oxidation of xanthine to UA. Elevated UA concentrations can lead to hyperuricemia, potentially triggering attacks of gouty arthritis (28).

In mouse models treated with intraperitoneal injections of potassium oxonate and oral administration of xanthine, Morin, a principal component of Gouji [Maclura cochinchinensis (Lour.) Corner heartwood] extract, can inhibit XOD activity in a non-competitive manner, lowering serum UA levels (29). Sangye (Morus alba L.), the leaf of the mulberry tree, contains flavonoids as its main bioactive components (30). A previous study (24) has indicated that the key constituents of Sangye total flavonoids include myricetin, quercetin, rutin, kaempferol and isorhamnetin. Serum UA levels in hyperuricemic mice induced by potassium oxonate decreased after 1 week of administration of Sangye total flavonoids. In the same study, after 3 weeks of administration of Sangye total flavonoids, serum UA approached normal levels in rats, and XOD activity was reduced by 25.01% compared with that in the model group. Furthermore, serum triglyceride and free fatty acid levels were lowered, while total flavonoids of Sangye reduced the abnormal liver and heart coefficient caused by adenine in mice (24). This suggests that Sangye total flavonoids may regulate serum UA levels by inhibiting XOD and managing lipid disorders. A study has also shown that luteolin 4′-O-glucoside and its aglycones, two primary flavonoid compounds in Pseudognaphalium affine (D. Don) Anderb., can inhibit the activity of XOD in hyperuricemic mice, thereby preventing UA synthesis, and reducing the swelling and inflammation caused by MSU crystals (31). In animal research, a gouty arthritis rat model induced by MSU crystals revealed reductions in XOD activity and improvements in gouty arthritis symptoms after 5 days of rutin administration (Fig. 3) (25).

Furthermore, XOD generates a vast array of reactive oxygen species (ROS), including H₂O₂ and O₂, through cascade reactions, using molecular oxygen as an electron acceptor. These ROS are intricately linked to cell damage, inflammation, carcinogenesis and aging (32). Thus, inhibiting XOD activity not only potentially prevents gouty arthritis but also blocks inflammatory pathways, reduces pro-inflammatory cytokines, and offers protection against kidney injury, cardiovascular disease, aging and cancer (33).

The excretion of UA primarily occurs through UA transporters in the kidneys, which are responsible for the reabsorption and secretion of UA. The regulation of recombinant urate transporter 1 (URAT1) and recombinant ATP binding cassette transporter G2 promotes UA excretion and serves a crucial role in the treatment of elevated UA levels (34). The kidney is a key organ in UA excretion, and this process is divided into four stages (35): i) In total, >99% of serum UA is filtered by the glomerulus; ii) 98% of the filtered UA is actively reabsorbed in the S1 segment, the initial part of the proximal renal tubules; iii) the active reabsorption of UA gradually decreases in the S2 segment of the curvature of the proximal renal tubules, and 50% of UA is secreted into the renal tubules; and iv) in the straight S3 segment of the proximal renal tubules, the concentration of UA in the renal tubules exceeds that in the surrounding capillaries, resulting in passive reabsorption of UA into the surrounding capillaries. Various transporters are involved in this process. URAT1 is specifically expressed in human kidneys and is located on the luminal side of the proximal tubular epithelial cells in the renal cortex, mainly participating in the reabsorption of urate by the proximal renal tubules (36). Glucose transporter 9 (GLUT9) is primarily expressed in the kidney and liver, with two subtypes (GLUT9L and GLUT9S); GLUT9L is found in the basement membrane of proximal renal tubule cells and GLUT9S is located in the lateral membrane of the proximal renal tubule (37). Studies have indicated that GLUT9 serves a crucial role in the transport of UA from intracellular to extracellular spaces and participates in urate reabsorption at the apical membrane of renal proximal tubules (38,39). Furthermore, organic anion transporter (OAT)1 has been demonstrated to be involved in the transport of UA in a dose- and time-dependent manner, and it has been suggested to serve a role in the first step of urate secretion, specifically, the uptake of urate from the peritubular space into renal tubular cells (40,41). OAT3 is predominantly expressed in proximal curved tubules, thick ascending limbs of medullary loops and collecting ducts, and is involved in urate transport. While the precise mechanism of OAT3 in urate transport remains unclear, based on its expression site, it is speculated that it may participate in the uptake of urate in peripheral tubules, contributing to urate secretion, or in moving urate from the basement membrane side into the peritubular capillaries, thus engaging in urate reabsorption (42–44). The aberrant transport of UA in the kidneys is a significant pathogenic factor in gouty arthritis (45).

In a mouse model of hyperuricemia induced by oteracil potassium, the extract of Gnaphalium affine D. Don, specifically 7,4-dihydroxyflavone, could regulate murine (m)URAT1 and mGLUT9 to reduce serum UA levels. This also assisted in inhibiting the increase of urea nitrogen and creatinine levels (46). Gnaphalium pensylvanicum Willd., a traditional folk medicine used for relieving inflammation, coughs and rheumatoid arthritis, contains a high concentration of luteolin-7-O-glucoside, as identified by ultra-performance liquid chromatography-electrospray tandem mass spectrometry in prior studies (31,47). Extracts from Gnaphalium pensylvanicum Willd. have been demonstrated to alleviate foot swelling symptoms induced by MSU crystals and reduce the infiltration of inflammatory cells (48). Furthermore, western blotting results indicated that the extract primarily decreased serum UA by influencing GLUT9, OAT1 and URAT1, and by inhibiting XOD activity in mice (48). Corn silk, the style and stigma of the gramineous plant Zea mays L., also known as Yu Shu Li Rui, is both a traditional food and medicine in China, with flavonoids being its most effective components (49). These flavonoids, found in high concentrations in all parts of the corn plant (50), can reduce UA levels in hyperuricemia, effectively treating gout and gouty arthritis (51). In an in vitro experiment using HK-2 human renal tubular epithelial cells, the impact of total flavonoids from corn silk on UA absorption and related gene expression in HK-2 cells was assessed. After 48 h of incubation, each concentration of total flavonoids from corn silk inhibited UA absorption in HK-2 cells to varying degrees, with the inhibition rate increasing with increasing concentration. Furthermore, total flavonoids were able to reduce the UA-induced apoptosis rate in HK-2 cells. There was a marked decrease in GLUT9 mRNA expression, and an increase in OAT1 and OAT3 mRNA expression (52). α-Mangostin, the primary active component in mangosteen peel extract, was shown to decrease the serum UA level in hyperuricemic mice in a dose-dependent and time-dependent manner, and increased the UA clearance rate in hyperuricemic rats, indicative of the promotion of UA excretion in the kidney. The study also revealed a reduction in the expression levels of GLUT9 mRNA and protein in the kidneys of hyperuricemic mice, suggesting the involvement of α-mangostin in the downregulation of GLUT9 protein expression (53). Similarly, mangiferin, an active component found in Anemarrhena asphodeloides Bge., was capable of downregulating the mRNA and protein expression levels of urate transporters mURAT1 and mGLUT9 in mice with renal hyperuricemia induced by potassium oxonate. Mangiferin also upregulated the expression levels of mOAT1, indicating that it may promote UA excretion in hyperuricemic mice by inhibiting renal UA reabsorption and increasing UA secretion, thus reducing serum UA levels (54). These findings suggest that the regulation of UA transporters is one of the mechanisms through which flavonoids can improve hyperuricemia and gouty arthritis (Fig. 4).

The elevation of body UA levels due to abnormal purine metabolism, surpassing the normal physiological serum UA concentration, leads to a supersaturated state. This results in the precipitation of MSU crystals, which accumulate in the joints and surrounding tissues, causing inflammation (55). Inflammation and damage to the joints and surrounding tissues are driven by the release of inflammatory factors, which are regulated by numerous immune cells and signaling pathways (56). Phagocytes recognize the MSU crystals deposited in the joints through various mechanisms, including the formation of immune antibody complexes with the MSU crystals, promoting phagocytosis through fragment crystallizable (Fc) receptors. MSU crystals can also be directly recognized and phagocytosed by cell surface receptors. Key receptors involved in recognizing MSU crystals include CD16, CD11b, Toll-like receptor (TLR)2, TLR4 and CD14. The interactions between these receptors and MSU crystals activate downstream signaling pathways that mediate inflammation (57–59). It has been suggested that MSU crystals can also directly bind to cell membranes, causing tissue inflammatory damage (60,61). Following phagocytosis and recognition of MSU crystals in the joints, IL-1β expression is induced by signaling pathways such as TLR, NOD-like receptor 3 (NLRP3), P2X purinoceptor 7 and mitogen-activated protein kinase pathways, among others, leading to an inflammatory response. Activation of these signaling pathways results in the release of activated IL-1β into the cell, subsequently attracting and activating inflammatory cells, such as neutrophils, and releasing more inflammatory factors (62–64). This process triggers an inflammatory cascade amplification reaction (Fig. 5) (65).

i) NLRP3 inflammasome. The NLRP3 inflammasome comprises the effector protein pro-caspase-1, the receptor protein NLRP3 and the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC). The inflammasome can be activated through various stimuli, including danger-associated molecular patterns and pathogen-associated molecular patterns (66). Upon detection of a specific activator, NLRP3 undergoes a conformational change, which then promotes ASC oligomerization to form ASC ‘spots’ (67). Serving as a platform for macromolecular signaling, ASC attracts pro-caspase-1 through its CARD domain, enabling pro-caspase-1 to be cleaved and produce active caspase-1. Caspase-1 then processes pro-IL-18 and pro-IL-1β into mature IL-18 and IL-1β, respectively, leading to tissue damage and inflammation (68,69). The abnormal activation of the NLRP3 inflammasome is associated with various diseases, including gouty arthritis, cardiovascular disease and diabetes (70). A recent study (71) has demonstrated that amentoflavone (AM) and its total flavonoid (TF) extract from the Chinese fir [Cunninghamia lanceolata (Lamb.) Hook.] exhibited inhibitory effects on foot thickness, lymphocyte infiltration, synovial injury and cartilage destruction in mouse models of gouty arthritis. Further investigation revealed that AM and TF reduced IL-1β secretion and caspase-1 cleavage in a dose-dependent manner, suggesting that they inhibit NLRP3 inflammasome activation. Additionally, TF treatment notably decreased the formation of ASC spots, indicating that TF could prevent the assembly of the NLRP3 inflammasome, characterized by the formation of ASC spots and reduced NLRP3 expression (71,72). trans-Chalcone, a precursor to flavonoids found mainly in herbs such as licorice (Glycyrrhiza uralensis Fisch.) (73), exhibits anti-inflammatory and antioxidant biological activities. In an experiment investigating its protective effects in mice with gouty arthritis, trans-Chalcone pre-treatment was administered to mice that were then injected in the joints with MSU. This treatment was observed to inhibit MSU-induced edema, mechanical hyperalgesia, leukocyte recruitment and inflammatory cell recruitment in a dose-dependent manner. Additionally, it reduced the in vivo production of IL-1β, TNF-α and IL-6, while increasing the production of TGF-β. Notably, trans-Chalcone also decreased nuclear factor κB (NF-κB) activation and the mRNA expression of inflammasome components such as ASC, NLRP3, pro-IL-1β and pro-caspase-1 (74). Similarly, quail models with endogenous gout induced by a high-purine diet were treated with rutinum for 10 days. The results indicated that rutin could exert an anti-inflammatory effect by inhibiting the activation of the NLRP3 inflammasome (26).

ii) TLR4/myeloid differentiation factor 88 (MyD88)/NF-κB pathway. In models of gouty arthritis, TLR4 in the synovial tissue of rats is notably increased due to disturbances in purine metabolism. Such disturbances lead to elevated UA levels, which in turn activate the TLR4-mediated signaling pathway, promoting the production of inflammatory cytokines and chemokines (75). The TLR4/MyD88/NF-κB signaling pathway involves TLR4, MyD88 and NF-κB, serving a key role in immune and inflammatory responses (76). Activation by lipopolysaccharide leads TLR4 to recruit MyD88, further activating the IL-1 receptor-associated kinase, which associates with TNF receptor-associated factor 6. This sequence activates TGF-activated kinase 1, leading to the phosphorylation of the inhibitor of κB kinase, degradation of IκB, release of NF-κB and its translocation into the nucleus to regulate the expression of various inflammatory responses (77).

NF-κB is a crucial mediator of the inflammatory response, linking extracellular stimuli with intracellular signaling pathways, influencing the progression of gouty arthritis (78). Inhibiting NF-κB activation presents a valid strategy for improving gouty arthritis. Studies (79,80) have demonstrated that calycosin reduces knee joint swelling and neutrophil infiltration in a mouse model of gouty arthritis induced by MSU. Inflammatory markers such as IL-1β, IL-6, IL-10 and TNF-α showed notable decreases in peripheral blood mononuclear cells and THP-1 cells induced by 0.2 mg/ml MSU after a 24-h pre-treatment with calycosin. Further investigation revealed that calycosin decreased the levels of interferon-inducible protein AIM2 (AIM2), Kelch-like ECH-associated protein 1 (Keap1), phosphorylated (p-)p65 and p-IκBα proteins in MSU-challenged cells in vitro, and increased the protein expression levels of p62 (79). These findings suggest that calycosin may exert a protective role in gouty arthritis by inhibiting the AIM2 inflammasome-mediated inflammatory response via the NF-κB and p62-Keap1 pathways. Similar to the effects of calycosin, silencing of AIM2 also reversed MSU-induced apoptosis in monocytes and macrophages, indicating that calycosin can suppress apoptosis by deactivating the AIM2 inflammasome via certain pathways, thereby impacting MSU-induced gouty arthritis (79). Lagotis brachystachys Maxim. is recognized as an essential herb in the clinical treatment of ‘Huang-shui’ disease, symptoms of which are similar to those of arthritis, as understood in traditional Chinese medicine (81). In Tibet, China, this herb has been traditionally utilized for its anti-inflammatory properties, particularly in conditions such as gouty arthritis and alcoholic liver injury (82,83). Research indicates that its antigout effects are achieved by downregulating the expression levels of TLR4, MyD88 and NF-κB proteins in the synovial tissue of rats. Three active flavonoids, namely luteolin, luteolin-4′-O-glucoside and apigenin, have been isolated from Lagotis brachystachys Maxim. (84). These active flavonoids have been shown to exhibit anti-inflammatory activities in vivo (85–87). Recent studies have suggested that luteolin can reduce the inflammatory response in acute gouty arthritis by inhibiting the TLR/MyD88/NF-κB pathway, reducing the joint swelling index (88,89). Similarly, luteolin-4′-O-glucoside was demonstrated to reduce foot swelling in rats by lowering serum pro-inflammatory cytokines in MSU crystal-induced gouty arthritis (31). Previous research has also revealed that luteolin (90), luteolin-4′-O-glucoside (91) and apigenin (92) inhibit the TLR4 signaling pathway. Furthermore, a study employing molecular docking techniques to evaluate the binding effects of luteolin, luteolin-4′-O-glucoside and apigenin on TLR4 (93) found that these compounds interact with TLR4 through hydrophobic interactions and hydrogen bonding, with binding energy results less than-7 kcal/mol. This suggests a high affinity of luteolin, luteolin-4′-O-glucoside and apigenin with TLR4, indicating their potential therapeutic effects in inhibiting inflammation.

Under typical conditions, the body maintains a balanced and gradual oxidation equilibrium. However, certain stimuli can disrupt the antioxidant system of the body, leading to oxidative stress reactions triggered by factors such as ROS, resulting in localized or systemic damage (94). Currently, abnormal number and function of T lymphocyte subpopulations, the activation of inflammatory cytokines and the pathological loss of cell histology in the pathogenesis of gouty arthritis are closely linked to the extensive release of free radicals following oxidative stress. This connection indicates that oxidative stress serves an important role in the development of autoimmune diseases (95). Research has demonstrated that the accumulation and deposition of MSU crystals in the joint cavity can enhance oxidative stress, releasing large quantities of oxidants such as ROS, nitric oxide (NO) and malondialdehyde (MDA), while suppressing the activity of antioxidants such as superoxide dismutase (SOD) and glutathione (GSH). This exacerbates joint damage and causes symptoms such as redness, swelling, warmth, pain and restricted joint mobility (96). Catalase is abundantly found in the synovial cells of patients with gouty arthritis, with the inflammatory response it triggers being a result of oxidative stress. The marked increase in cellular NO⁻, Hydroxyl radical (OH⁻), Peroxyl Radical (ROO⁻) and Alkoxyl group (RO⁻) leads to an inflammatory response induced by ROS. In gouty arthritis, UA crystals enter endothelial cells through anion transporters in an exogenous pathogen-associated molecular pattern, becoming pro-oxidants and swiftly inducing oxidative stress. This promotes NO⁻ production by activating XOD and reduced nicotinamide adenine dinucleotide phosphate oxidase (97,98). The interaction between NO⁻ and O₂ releases peroxynitrite anion (ONOO⁻), affecting cell proliferation, leading to the degradation of connective tissue and joint tissue deterioration (Fig. 6) (98).

An animal study demonstrated that the administration of quercetin in a rat model of gouty arthritis, induced by injecting MSU crystal suspension into the right hind leg ankle, alleviated edema in a dose-dependent manner and reduced acute inflammatory histological characteristics in the treated animals. Quercetin treatment was found to inhibit leukocyte aggregation, decrease chemokine levels, lower the levels of MDA, a lipid peroxidation end product, and enhance the activity of antioxidant enzymes (27).

In rodents, such as rats and mice, UA resulting from purine metabolism is further degraded by uricase into allantoin, which has a higher solubility compared with UA and is excreted through urine. Quail, similar to humans, lack uricase in their UA synthesis and metabolism processes. The nucleic acids produced from nucleotide proteolytic hydrolysis are degraded into purine substances, which are then converted into UA by XOD and excreted as UA (99,100). An animal experiment in quail, involving a model of endogenous gout induced by a high-purine diet, examined administration of rutin for 10 days. The results indicated that rutin could improve gouty arthritis in quail by reducing XOD activity and UA levels. Rutin restored the oxidative stress balance by inhibiting the production of ROS and served a crucial anti-inflammatory role (26).

Another study on a rat model of gouty arthritis, induced by MSU crystals and followed by a 5-day administration of rutin, found that rutin reduced ankle swelling, and the levels of MDA and NO, and improved the activities of GSH-peroxidase, SOD and catalase in rats (25). These findings suggest that rutin could reduce gouty arthritis induced by MSU crystals in rats, likely through its anti-oxidative stress effects (25).