Neoisoastilbin Ameliorates Acute Gouty Arthritis via Suppression of the NF-κB/NLRP3 Pathway

Acute gouty arthritis (AGA) is an acute inflammatory disease, whose occurrence and development mechanism are associated with inflammatory reaction of joint tissue. This study investigated the role of neoisoastilbin (NIA) in the treatment of AGA and explored the underlying mechanisms. C57BL/6 mice underwent intraarticular injection of monosodium urate (MSU) to establish an AGA model in vivo. Enzyme-linked immunosorbent assay, histopathological hematoxylin-eosin staining, western blotting, and other methods were used to observe the therapeutic effects of NIA on AGA and investigate the role of the NF-κB/NLRP3 pathway in the treatment. We found that NIA effectively reduced MSU-induced joint swelling and inflammatory cell infiltration in a concentration-dependent manner. NIA also significantly reduced interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) levels as compared with the respective values in the model mice group. In addition, administration of NIA significantly mitigated the phosphorylation of NF-κB-related proteins (IKKα, NF-κB, and IκBα) and the expression of NLRP3-related proteins (NLRP3, caspase-1, and ASC) in MSU-induced joint tissues. In conclusion, our research indicated that NIA significantly improved AGA, and its underlying mechanism was achieved by simultaneously inhibiting the NF-κB/NLRP3 pathway and the expression of inflammatory factors. This research preliminarily suggested the potential role of NIA in the treatment of AGA.


Introduction
Te incidence of gout is on the rise worldwide, with gouty arthritis accounting for the most prevalent type [1,2]. Its acute onset is an acute infammatory response caused by the deposition of monosodium urate (MSU) microcrystals in the periarticular tissues, which seriously afects the quality of life of patients [3]. Acute gouty arthritis (AGA) is responsible for an increased risk of developing hypertension, chronic kidney disease, obesity, diabetic kidney stones, myocardial infarction, heart failure, and stroke [4].
A critical role of MSU-induced infammation in the pathogenesis of AGA has been reported [5]. MSU remains in an ionic state under physiological conditions and precipitates in the peripheral tissues under excessive concentrations of blood uric acid. After MSU precipitation, monocytes drift towards the periarticular tissues and become activated. Once activated, NLRP3 transduces the recognition signal to the adapter apoptosis-relatedspeck-like protein (ASC) to facilitate activation of caspase-1, thereby stimulating the release of multiple infammatory factors including interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) [6,7]. Furthermore, infammatory factors cause severe pain and joint destruction of patients [8,9]. Terefore, inhibiting MSU-induced infammation could be an efective therapeutic strategy for AGA.
At present, nonsteroidal anti-infammatory drugs, colchicine, and corticosteroids are by far the most efcacious anti-AGA agents that exert their therapeutic efects by inhibiting infammation [10,11]. However, these drugs cause side efects and toxicity, which restrict their application [12][13][14]. Tese limitations have led to the search for new therapeutic drugs.
Neoisoastilbin (NIA), a kind of favonoid, holds antioxidant and anti-infammatory efects, and its molecular structure resembles that of astilbin [15,16]. It has been reported that Rhizoma smilacis glabrae water extract, which contains neoisoastilbin, efectively alleviates potassium oxonate-and MSU-induced hyperuricemia and gout in mice [17]. In addition, the total favonoids of Smilax glabra Roxb., which are mainly astilbin, neoastilbin, isoastilbin, and neoisoastilbin, have therapeutic activity against hyperuricemia [18]. Nevertheless, no studies in the literature have explored the efcacy of NIA in AGA. In the present study, we established an AGA model using intra-articular MSU injection in mice and evaluated the protective efects and potential mechanisms of NIA on the experimental mice.

Materials.
Neoisoastilbin with a purity of over 85% was prepared in the laboratory according to the procedures described previously [15]. Briefy, the Smilax glabra Roxb. was air-dried, ground, and extracted three times with ethanol/ water (60 : 40, v/v) at 80°C for 2 h each; then, the extract was fltered through Whatman No. 1 flter paper, and the ethanol from the extract was removed under vacuum. Ten, the residue was dissolved in distilled water and further fractionated with n-hexane, ethyl acetate, and n-butanol. Ten, isolation and preparation of neoisoastilbin were executed on  Mice were cultured in the SPF laboratory animal room at constant temperature and humidity with a 12 h dark/light cycle with adequate access to diet and water.
After adapting to the environment for one week, the C57BL/6 mice were randomly divided into fve groups: (a) control group (Con); (b) MSU treatment group (MSU); (c) MSU with colchicine treatment group (Col, 1 mg/kg); (d) MSU with low-dose NIA treatment group (NIA-L, 25 mg/ kg); (e) MSU with high-dose NIA treatment group (NIA-H, 50 mg/kg). All mice were given the appropriate drugs for 7 days. On the 6th of intragastric administration, the joints of the mice in the normal group were injected with 0.025 mL of physiological salt solution, and the joints of the remaining mice were injected with 0.025 mL of MSU at a concentration of 50 mg/mL to induce the AGA model. At the end of the experiment, all mice were executed, and joints were removed for subsequent experiments.

Measurement of Mouse Ankle Joint Swelling.
Before measurement, a line was drawn at the upside of the ankle joint as fducial marks demarcating the ankle joint areas to be analyzed. Te ankle joint was inserted into a specifc measuring cylinder flled with water. Te syringe was pushed to allow the water surface to reach the marked level of the labeled ankle and data were recorded. Te toe volumes of mice were measured by a toe volume measuring device before modeling and 2, 4, 6, 10, and 24 h after modeling.

Hematoxylin-Eosin Staining.
Te joint tissues were fxed in 4% paraformaldehyde at room temperature. Next, the tissues were parafn embedded and cut into 5 μm slices. After removal of parafn and endogenous peroxidase, the tissues were stained with hematoxylin and eosin. Finally, the sections were sealed with ultra-thin cover glass, and the histopathological changes of the diferent joint tissues were observed under an optical microscope.

Enzyme-Linked Immunosorbent Assay.
Te joint tissues were ground in liquid nitrogen and immersed in PBS. Tissue homogenates were collected and centrifuged at 4°C for 10 min under the condition of 12000 rpm. Te supernatant was taken to determine the levels of IL-1β, IL-6, and TNF-α according to the kit instructions.
2.6. Western-Blotting Analysis. Te joint tissues were ground in liquid nitrogen and lysed using RIPA lysis bufer with a 1% phosphatase inhibitor cocktail and a 1% protease inhibitor cocktail for 30 min. Ten, tissue homogenates were centrifuged at 12000 rpm for 10 min, and the supernatant was taken to determine the protein concentration. Meanwhile, the nuclear proteins and cytoplasmic proteins were isolated according to the procedure of the NE-PER ™ Kit.
After that, the same amounts of proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and subsequently separated proteins were transferred to PVDF membranes. Te membranes were then blocked in 5% nonfat milk for 2 h and incubated with the diferent primary antibodies overnight. Subsequently, the membranes were incubated with the HRPlabeled goat antimouse or rabbit IgG antibodies for 2 h. Te detection was performed by the chemiluminescence method. Te results of western blotting were obtained using the software Image J.

Statistical Analysis.
In this study, the results were analyzed by one-way analysis of variance (ANOVA), followed by multiple comparisons with the Dunnett test using the statistical software of SPSS 24.0 or a two-tailed unpaired Student's t-test using GraphPad Prism 8.0. All calculated data were expressed as mean ± standard deviation. In this study, p < 0.05 was considered statistically signifcant, and p < 0.01 was considered very statistically signifcant.

NIA Alleviates MSU-Induced AGA in Mice.
Te joint swelling is a principal marker of AGA. Figure 1 shows the swelling changes in all groups. Te rate of joint swelling in Con mice was consistently reduced over the period that was monitored. Mice in the MSU group exhibited a higher rate of joint swelling, which was signifcant over the measured time range (2-24 h) compared to the Con group. Te swellings reached their peak after 4 h and then showed a slight decrease within 4-24 h. Consistent with the previous study [19], the Col group signifcantly reduced MSU-induced joint swelling, as did the NIA low-dose and high-dose groups. Te result indicated that both NIA doses showed preventive efects on joint swelling, and such efects were dosedependent.
We also evaluated the histological status of all groups by hematoxylin-eosin staining. As shown in Figure 2, the MSU group had extensive infammatory cell infltration. However, infltration of infammatory cells was considerably improved in the NIA-treated group.

NIA Inhibited Infammatory Factor Expression in Mice.
In order to assess the role of NIA in AGA, the levels of infammatory factors (including IL-1β, IL-6, and TNF-α) in joint tissues were measured. As shown in Figure 3, the levels of IL-1β, IL-6, and TNF-α in the joints of mice were signifcantly increased in the MSU group, which were 2.3, 6.0, and 2.8 times higher than those in the Con group, respectively. Tis indicated that the accumulation of MSU crystals in the joints signifcantly promoted the expression of infammatory factors, and interestingly, NIA signifcantly modulated the levels of infammatory factors, and the efect of the high-dose group was better than that of the low-dose group.

NIA Modulated NF-κB/NLRP3 Pathway in Mice.
To investigate the possible mechanism of NIA in treating AGA, changes in the NF-κB/NLRP3 pathway were analyzed using western blotting. As demonstrated in Figure 4, MSU treatment signifcantly increased the expression of NLRP3, caspase-1, and ASC proteins, while these increases were reversed by NIA. As shown in Figure 5, MSU exhibited increased protein expression of phosphor-IKKα, phosphor-NF-κB, and phospho-IκBα without a change in total-IKKα, total-NF-κB, and total-IκBα. As expected, NIA plays a role in treating AGA by regulating the NF-κB/NLRP3 pathway.
To further elucidate the roles of the NF-κB/NLRP3 pathway in treating AGA, nuclear proteins and cytoplasmic proteins were isolated, and the expression of phospho-NF-κB was detected. As illustrated in Figure 6, MSU signifcantly increased the expression of phospho-NF-κB in the cytoplasm and nucleus, while Col and NIA both reverse the increased phospho-NF-κB expression, with high doses of NIA being more efective than low doses of NIA. NIA inhibited NF-κB from entering the nucleus to transcribe

Discussion
AGA has been widely considered as one of the main types of gout that causes decreased quality of life, and its clinical manifestation is a self-limited attack of synovitis [20,21]. With the upward trend in living standards, AGA has become a global health issue due to changes in dietary and lifestyle habits [22]. Currently, the drugs used to treat have severe side efects that make it possible to damage human health, necessitating to research agents that are efcacious and safe.
Previous studies had demonstrated that astilbin ameliorates osteoarthritis by suppressing TLR4/MD-2 and NF-κB signaling [23,24]. Cai et al. [25] reported that astilbin also improved collagen-induced arthritis. Astilbin has the potential to improve AGA, and NIA is the stereoisomer of astilbin, so we speculate that NIA has a protective efect against AGA. After the MSU injection, the mice began to Levels of TNF-α in mice joints. Te results shown are the means ± SD of ten independent experiments; # p < 0.05 or ## p < 0.01 compared with the Con group; * p < 0.05 or * * p < 0.01 compared with the MSU group.  Figure 4: NIA inhibited NLRP3 infammasome in mice joints. (a) Western blotting analysis of the NLRP3 pathway. Te relative protein expression levels of (b) NLRP3, (c) caspase-1, and (d) ASC were normalized to β-actin. Te results shown are the means ± SD of three independent experiments; # p < 0.05 or ## p < 0.01 compared with the Con group; * p < 0.05 or * * p < 0.01 compared with the MSU group.
show decreased activity and joint swelling. Te lesion severity of NIA-treated mice began to signifcantly decrease 2 h after modeling, and this trend lasted until the end of the daylong measurement. Furthermore, NIA treatment resulted in a signifcant reduction in infammatory cell infltration. Tese results clearly suggested that NIA is a potential therapeutic candidate for the treatment of AGA.
Te pathogenesis and mechanism of AGA are complicated. In addition to genetic and dietary factors, the increased production of infammatory cytokines plays a crucial role in AGA pathogenesis [26,27]. MSU crystals induce secretion of infammatory cytokines, including TNF-α, IL-6, and IL-1β in the progressive phase of AGA [28]. TNF-α, IL-6, and IL-1β are involved in the activation and maintenance of infammatory responses, which serve key roles in the development and sustenance of gouty arthritis [29][30][31]. Te study showed that chaetocin inhibits IL-1β secretion, thereby providing gout relief [32]. Curcumin prevents acute gout attacks by inhibiting the secretion of MSU-induced infammatory cytokines [33]. As in the abovementioned studies, our study indicated that NIA treatment decreased the expression of IL-6, TNF-α, and IL-1β. Terefore, we speculate that NIA reduced the severity of AGA by suppressing the increased infammatory response.
Te NF-κB/NLRP3 pathway plays a central role in infammation-related diseases, including gouty arthritis, and the blockade of the NF-κB/NLRP3 pathway is now proven to be an efective strategy to prevent and ameliorate AGA [34,35]. During AGA onset, the IKK complex is phosphorylated and then the NF-κB inhibitory protein IκBα is phosphorylated. It increases NF-κB phosphorylation levels and stimulates NF-κB translocation into the nucleus [36]. Ultimately, activation of NF-κB induces gene expression of IL-1β and NLRP3, which would induce the production of inactive IL-1β precursors [37]. NLRP3 frst binds ASC to form an infammasome that subsequently cleaves procaspase-1 to form active caspase-1 and, following cleavage, caspase-1 cleaves pro-IL-1β to the active form of IL-1β [38]. In addition to IL-1β, the core of gouty infammation, NF-κB/NLRP3 pathway involves activation of cytokines from a variety of cell types, including TNF-α and IL-6 [39]. Our study agreed with the abovementioned studies that the expression of NF-κB/NLRP3 pathwayrelated proteins was signifcantly increased by MSU crystals. NIA signifcantly inhibited the expressions of the above proteins, which suggested NIA ameliorated AGA via suppression of the NF-κB/NLRP3 pathway.
Inhibition of the NF-κB/NLRP3 pathway may reduce the release of infammatory factors such as IL-1β and consequently improve AGA. Our fndings are consistent with some but not all previous studies. Although both NIA and colchicine were found to decrease infammatory factors and inhibited NF-κB/NLRP3 pathway, NIA is slightly more potent than colchicine at inhibiting NF-κB/NLRP3 pathway,  Figure 5: NIA inhibited the NF-κB pathway in mouse joints. (a) Western blotting analysis of the NF-κB pathway. Te relative phosphorylation levels of (b) IKKα, (c) NF-κB, and (d) IκBα were normalized to the corresponding total protein. Te results shown are the means ± SD of three independent experiments; # p < 0.05 or ## p < 0.01 compared with the Con group; * p < 0.05 or * * p < 0.01 compared with the MSU group.
Evidence-Based Complementary and Alternative Medicine and colchicine is more potent than NIA in inhibiting the release of infammatory factors. Research has shown colchicine can inhibit AMPK pathways, the RhoA/Rho efector kinase (ROCK), and other infammation-related pathways [10,40]; NIA may be weaker in some infammation-related pathways, which was lacking in this experiment. Tese results indicate that NIA could be an efective agent in AGA treatment, which might also be used as an adjuvant in combination with other traditional anti-AGA drugs.

Conclusion
Te present study demonstrated in C57BL/6 mice that NIA could alleviate MSU-induced AGA. Te specifc regulatory mechanism may be through the inhibition of NF-κB/NLRP3 pathway, which may reduce the levels of infammatory factors (IL-1β, IL-6, and TNF-α) and the infammatory response in AGA.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request. Te results shown are the means ± SD of three independent experiments; # p < 0.05 or ## p < 0.01 compared with the Con group; * p < 0.05 or * * p < 0.01 compared with the MSU group.