UA is a weak acid and its ionized form present in the body is urate at physiologic pH [31]. The pathological threshold of hyperuricemia is defined as 6.8 mg/dL (the in vitro solubility limit of MSU) [1,32]. When the level of serum UA is higher than the normal threshold, MSU crystals deposition begins to occur in tissues. Nevertheless, when the deposition of MSU crystals occurs in articular cartilage, synovial sacs and other tissues, it will cause inflammation with concomitant swelling and pain, which is called gouty arthritis. It can be said that hyperuricemia is the most important risk factor for gouty arthritis. The clinical picture of gout is divided into four stages: asymptomatic hyperuricemia, acute gouty arthritis, intercritical period, and chronic tophaceous gout [31]. Several studies disclosed MSU deposits in a proportion of asymptomatic hyperuricemia patients [33,34]. There are many separate animal models have been widely developed to investigate the causal mechanisms for hyperuricemia or gout but do not yet reliably and simultaneously simulate hyperuricemia and its complication gouty arthritis that occurs in humans. For protection, it is necessary to prevent UA from the formation of MSU crystals, which is effective in the treatment of gout. A combined hyperuricemia and gouty arthritis animal model will be in line with the population in the transition stage from asymptomatic hyperuricemia to acute gouty arthritis in clinical practice, which is a relatively new application model. Therefore, this combined model has more obvious research value and significance. In present study, marked ankle joint swelling and abundant inflammatory cell infiltration could be seen in ankle joint of model group. We also observed significant elevation of serum UA, CRE and BUN levels and reduction of urine UA and CRE levels in model mice. As our data indicated, we successfully induced a model combined hyperuricemia and gouty arthritis in mice by combination of intraperitoneal injection of PO and xanthine and intra-articular injection of MSU crystals [12,13,35,36].
Following kaempferol treatment, the photographs and H&E-staining of mice ankle joints demonstrated similar changes that kaempferol significantly alleviated MSU crystals-induced joint inflammation (ankle swelling and inflammatory cell infiltration), controlling the onset of gouty arthritis in mice. Also, kaempferol could also increase the excretion of UA and CRE in the kidney, which might ameliorate hyperuricemia and renal dysfunction, as well as caused the levels of serum UA, CRE and BUN tend to normal. Higher XOD activity can lead to excessive synthesis of UA [37,38]. Besides, treatment with kaempferol could significantly inhibit hepatic XOD activity, suggesting that the effect of kaempferol on reducing UA production might be due to the inhibition of XOD activity. Moreover, kaempferol showed a potential protective effect on renal injury by suppressing oxidative stress through activating the SOD, GSH and GSH-Px and reducing MDA. In short, the remarkable improvement in biochemical parameters and histopathologic changes were observed after kaempferol treatment, indicating the urate-lowering and decreasing inflammatory responses effects of kaempferol.
The transport and excretion of UA are complicated procedures which are related with various renal transporters, including GLUT9, ABCG2, OAT1, OCT2 and so on [39,40]. Specifically speaking, GLUT9 is expressed on basolateral membranes and is capable of transporting UA from renal tubules into the circulation [9]. ABCG2 is a high-capacity urate exporter and the reduced extra-renal UA excretion resulting from ABCG2 dysfunctional variants is a common cause of hyperuricemia [41,42]. OAT1 mediates urate secretion from the blood into the tubular lumen [42,43]. Elevated expression of OAT1 has been observed to promote the excretion of UA into urine, thereby leading to a decrease in the concentration of UA in serum. A decrease in the expression or malfunction of OAT1 has been shown to significantly elevate the likelihood of developing hyperuricemia [44,45]. Besides, OCT2 that exists on the basolateral membrane of renal tubules, plays the pivotal roles in absorption, distribution and excretion of hydrophilic organic cations [46]. According to previous studies, empagliflozin treatment was attributed to UA excretion promotion through up-regulating ABCG2 expression in KK-Ay mice with hyperuricemia [11]. Similarly, saponins induced the decrease of serum UA and increase the excretion of urine UA through up-regulating the GLUT9 expression level and down-regulating the OAT1 expression level in chronic hyperuricemia rats [47]. Astaxanthin also promoted UA excretion by down-regulating the protein expressions of GLUT9, as well as up-regulating the protein expressions of OAT1 and ABCG2 [48]. Consistent with previous findings, we found that kaempferol could effectively reverse the abnormal protein expressions of GLUT9, ABCG2, OAT1 and OCT2 in kidneys of mice. In brief, the anti-hyperuricemia effect, at least in part, is achieved by regulating renal transporter expressions [49–51]. Dysregulation of renal transporters can lead to excessive accumulation of UA in the body, which may activate inflammation.
As for inflammation, it is characterized by coordinated activation of various signaling pathways. NLRP3 interacts with the bridging molecule ASC to increase the expression of Caspase-1, which plays an important role in maturing IL-1β and IL-18 expressions [52]. The NLRP3 inflammasome activation triggers kidney and joint inflammatory response along with the up-regulation of IL-1β and IL-18 [53,54]. Moreover, NF-κB is produced by homologous or heterodimerization of Rel family proteins, mainly in the form of p50 and p65 subunits. Under normal conditions, NF-κB in the cytoplasm remains inactive and binds to the inhibitory protein IκB. Once stimulated, IκB kinase (IKK) is involved in the phosphorylation of IκBα, which then mediates the phosphorylation of NF-κB p65 [55]. In this study, the levels of inflammatory cytokines in model group were significantly higher than those in control group, and NLRP3 inflammasome and NF-κB pathway were activated, as evidenced by the up-regulated protein expressions of NLRP3, Caspase-1, ASC, p-IKKα/IKKα, p-p65/p65 and p-IκBα/IκBα. However, kaempferol treatment significantly reversed the elevations of inflammatory cytokines. Notably, different dosages of kaempferol could reduce the expression of the above proteins to varying degrees, which indicated that kaempferol might exert anti-inflammatory effects, including reducing inflammatory cytokine release and inflammatory cell infiltration into the lesion site, mediated by the suppression of NLRP3 inflammasome and NF-κB pathways. Previous studies also have shown that MAPK, TLR4 and AR/NOX2 signaling pathways are involved in the antioxidant or anti-inflammatory process of kaempferol [56,57]. Overall, the findings suggest that kaempferol possesses anti-inflammatory effects, potentially mediated through multiple signaling pathways.
It cannot be ignored that the development potential of dietary flavonoids might be limited due to their low solubility, absorption, and rapid metabolism. To address these limitations, various strategies have been developed to deliver poorly water-soluble flavonoids, including the use of an absorption enhancer, structural transformation (e.g., prodrugs, glycosylation), and pharmaceutical technologies (e.g., carrier complexes, cocrystals). Nanotechnology has shown promise in enhancing the bioavailability and systemic absorption of certain chemical compounds. It has been reported that kaempferol can be encapsulated using a coating of nanoparticles of poly (lactic acid-co-glycolic acid) and polyethylene oxide-poly propylene oxide-polyethylene oxide, which was effective in reducing cancer cell viability as compared to alone kaempferol [58]. In the study of Ilk, kaempferol was loaded into lecithin/chitosan nanoparticles to make antifungal activity more effective compared to pure kaempferol [59]. Moreover, kaempferol loaded chitosan nanoparticles enhanced the anti-quorum sensing activity [60]. However, whether the anti-inflammatory effects of kaempferol could be enhanced by these approaches remains to be studied.