WN1703 alleviates gout symptoms via inflammatory signaling pathways in an acute gout rat model

Abstract

We previously synthesized the xanthine oxidoreductase (XOR) inhibitor WN1703. In addition to showing XOR inhibitory effects, WN1703 also showed anti-inflammatory effects in a rat hyperuricemia model. Here, we studied WN1703's anti-inflammatory effects on gout and explored the underlying mechanisms. Tohoku Hospital Pediatrics-1 (THP-1) cells were stimulated by lipopolysaccharide/interferon-γ/monosodium urate (MSU). The levels of inflammatory cytokines in the supernatant and protein expression in THP-1 cells were detected using enzyme-linked immunosorbent assay (ELISA) kits and western blotting, respectively, to verify the inhibitory effects of WN1703 and its mechanism. Potassium oxonate, hypoxanthine, and MSU were administered to establish a hyperuricemia rat model complicated by acute gouty arthritis. At 1-24 h after MSU injection, the degree of ankle swelling was recorded to compare the anti-inflammatory effects at each time point. The potential mechanism was further explored using immunohistochemistry and ELISA. WN1703 significantly downregulated expression of nucleotide-binding oligomerization domain-like receptor thermal protein domain associated protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, toll-like receptor-4 (TLR4), myeloid differentiation primary response protein 88 (MyD88), nuclear factor-kappa B (NF-κB), and relevant cytokine levels in THP-1 cells. Identical doses of WN1703 and febuxostat had comparable effects on these proteins and cytokines. In the gout rats, the same dose of WN1703 and febuxostat showed equivalent inhibitory effects on NLRP3, ASC, and NF-κB; however, WN1703 showed weaker impacts on alleviating ankle swelling than febuxostat showed. In conclusion, WN1703 showed significant anti-inflammatory effects in hyperuricemic rats with acute gout. Such effects were related to the inhibition of the NLRP3/ASC/Caspase-1 and TLR4/MyD88/NF-κB signaling pathways, thereby downregulating inflammation-related protein expression and decreasing inflammatory cytokine secretion.

Keywords

Hyperuricemia / Acute gouty arthritis / Xanthine oxidoreductase inhibitors / Anti-inflammation

Author contributions

Fuyao Liu: Investigation, visualization, methodology, writing (original draft), and conceptualization.

Xiaodan Lu: Investigation and methodology.

Lei Zhang: Visualization and validation.

Jing Li: Conceptualization and supervision.

All the authors have read and approved the final version of the manuscript.

Conflicts of interest

The authors declare no conflict of interest.

Data availability

Data will be made available on request.

Ethics approval

The study protocol was approved (AEC: 2021003) by the Ethics Committee of South China University of Technology (Guangzhou, China).

Funding information

This work was supported by the Key-Area Research and Development Program of Guangdong Province (2020B010188001), Science and Technology Project of Guangzhou (202102080325), and Open Project supported by the Hebei Province Key Laboratory of Traditional Chinese Medicine Research and Development (ZSKF202302).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

Images of rats and THP-1 cell clusters in the Graphical Abstract were obtained from the Biorender website (https://www.biorender.com/). DNA and other signals that were observed in this study were drawn using Adobe Illustrator 2020 (Adobe, U.S.).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.pscia.2024.100039.

References

[1] R.T. Keenan, Limitations of the Current standards of care for treating gout and crystal deposition in the primary care setting: a review, Clin. Therapeut. 39 (2) (2017) 430-441. https://doi.org/10.1016/j.clinthera.2016.12.011.

[2] C. Chen, J.M. Lü, Q. Yao,Hyperuricemia-related diseases and xanthine oxidoreductase (XOR) inhibitors: an overview, Med. Sci. Mon. Int. Med. J. Exp. Clin. Res. 22 (2016) 2501-2512. https://doi.org/10.12659/msm.899852.

[3] C. Rakieh, P.G. Conaghan, Diagnosis and treatment of gout in primary care, Practitioner 255 (1746) (2011), 17-20, 2-3.

[4] K.S. Coutinho-Wolino, P.P. Almeida, D. Mafra, M.B. Stockler-Pinto, Bioactive compounds modulating Toll-like 4 receptor (TLR4)-mediated inflammation: pathways involved and future perspectives, Nutr. Res. 107 (2022) 96-116. https://doi.org/10.1016/j.nutres.2022.09.001.

[5] E.M. Reuven, A. Fink, Y. Shai, Regulation of innate immune responses by transmembrane interactions: lessons from the TLR family, Biochim. Biophys. Acta 1838 (6) (2014) 1586-1593. https://doi.org/10.1016/j.bbamem.2014.01.020.

[6] S. El-Sayed, S. Freeman, R.A. Bryce, A selective review and virtual screening analysis of natural product inhibitors of the NLRP3 inflammasome, Molecules 27 (19) (2022) 14. https://doi.org/10.3390/molecules27196213.

[7] F. Martinon, V. Pétrilli, A. Mayor, A. Tardivel, J. Tschopp, Gout-associated uric acid crystals activate the NALP3 inflammasome, Nature 440 (7081) (2006) 237-241. https://doi.org/10.1038/nature04516.

[8] L. Wilson, J.J. Saseen, Gouty arthritis: a review of acute management and prevention, Pharmacotherapy 36 (8) (2016) 906-922. https://doi.org/10.1002/phar.1788.

[9] J. Yu, Q. Qiu, L. Liang, X. Yang, H. Xu, Prophylaxis of acute flares when initiating febuxostat for chronic gouty arthritis in a real-world clinical setting, Mod. Rheumatol. 28 (2) (2018) 339-344. https://doi.org/10.1080/14397595.2017.1318467.

[10] C.Y. Man, I.T. Cheung, P.A. Cameron, T.H. Rainer, Comparison of oral prednisolone/paracetamol and oral indomethacin/paracetamol combination therapy in the treatment of acute goutlike arthritis: a double-blind, randomized, controlled trial, Ann. Emerg. Med. 49 (5) (2007) 670-677. https://doi.org/10.1016/j.annemergmed.2006.11.014.

[11] R.A. Terkeltaub, H.R. Schumacher, J.D. Carter, H.S. Baraf, R.R. Evans, J. Wang, S. King-Davis, S.P. Weinstein, Rilonacept in the treatment of acute gouty arthritis: a randomized, controlled clinical trial using indomethacin as the active comparator, Arthritis Res. Ther. 15 (1) (2013) R25. https://doi.org/10.1186/ar4159.

[12] J. Li, F. Wu, X. Liu, Y. Zou, H. Chen, Z. Li, L. Zhang, Synthesis and bioevaluation of 1-phenyl-pyrazole-4-carboxylic acid derivatives as potent xanthine oxidoreductase inhibitors, Eur. J. Med. Chem. 140 (2017) 20-30. https://doi.org/10.1016/j.ejmech.2017.08.047.

[13] L. Zhou, B. Wei, L. Wu, J. Li, B. Zhu, L. Zhang, Anti-hyperuricemia activity and toxicity prediction of a novel xanthine oxidoreductase inhibitor, Biomed. Chromatogr. 34 (2) (2020) e4727. https://doi.org/10.1002/bmc.4727.

[14] J.U. Song, S.P. Choi, T.H. Kim, C.-K. Jung, J.-Y. Lee, S.-H. Jung, G.T. Kim, Design and synthesis of novel 2-(indol-5-yl) thiazole derivatives as xanthine oxidase inhibitors, BMCL 25 (6) (2015) 1254-1258. https://doi.org/10.1016/j.bmcl.2015.01.055.

[15] Y. Li, X. Zhu, F. Liu, W. Peng, L. Zhang, J. Li, Pharmacodynamic evaluation of the XOR inhibitor WN1703 in a model of chronic hyperuricemia in rats induced by yeast extract combined with potassium oxonate, Curr. Res. Pharmacol. Drug. Discov. 3 (2022) 100098. https://doi.org/10.1016/j.crphar.2022.100098.

[16] M. Zhou, S. Li, L. Song, Q. Hu, W. Liu, 4-(2-(4-chlorophenyl) -1-((4-chlorophenyl) amino)ethyl)benzene-1, 3-diol is a potential agent for gout therapy as a dual inhibitor of XOD and NLRP3, Phytomedicine 42 (2018) 9-17. https://doi.org/10.1016/j.phymed.2018.03.007.

[17] R. Sun, C. Wang, Y. Wang, Y. Wu, P. Du, X. Sun, Q. Li, K. Bi, G. Jiang, Role of miRlet- 7c-5p/c-myc signaling axis in the committed differentiation of leukemic THP-1 cells into monocytes/macrophages, Oncol, Letture 26 (3) (2023) 403. https://doi.org/10.3892/ol.2023.13989.

[18] K. Wang, Y. Zhang, M. Zhou, Y. Du, P. Li, C. Guan, Z. Huang, HDAC inhibitors alleviate uric acid-induced vascular endothelial cell injury by way of the HDAC6/ FGF21/PI3K/AKT pathway, J. Cardiovasc. Pharmacol. 81 (2) (2023) 150-164. https://doi.org/10.1097/fjc.0000000000001372.

[19] Y.F. Qing, Q.B. Zhang, J.G. Zhou, L. Jiang, Changes in toll-like receptor (TLR)4- NFκB-IL1β signaling in male gout patients might be involved in the pathogenesis of primary gouty arthritis, Rheumatol. Int. 34 (2) (2014) 213-220. https://doi.org/10.1007/s00296-013-2856-3.

[20] Q. Wang, B. Lin, Z. Li, J. Su, Y. Feng, Cichoric Acid ameliorates monosodium urateinduced inflammatory response by reducing NLRP3 inflammasome activation via inhibition of NF-kB signaling pathway, Evid Based Complement Alternat Med 2021 (2021) 8868527. https://doi.org/10.1155/2021/8868527.

[21] B. Chen, H. Li, G. Ou, L. Ren, X. Yang, M. Zeng, Curcumin attenuates MSU crystalinduced inflammation by inhibiting the degradation of IκBα and blocking mitochondrial damage, Arthritis Res. Ther. 21 (1) (2019) 193. https://doi.org/10.1186/s13075-019-1974-z.

[22] C. Song, L. He, J. Zhang, H. Ma, X. Yuan, G. Hu, L. Tao, J. Zhang, J. Meng, Fluorofenidone attenuates pulmonary inflammation and fibrosis via inhibiting the activation of NALP3 inflammasome and IL-1β/IL-1R1/MyD88/NF-κB pathway, J. Cell Mol. Med. 20 (11) (2016) 2064-2077. https://doi.org/10.1111/jcmm.12898.

[23] C. Wu, F. Li, X. Zhang, W. Xu, Y. Wang, Y. Yao, Z. Han, D. Xia, (-)-Epicatechin ameliorates monosodium urate-induced acute gouty arthritis through inhibiting NLRP3 inflammasome and the NF-κB signaling pathway, Front. Pharmacol. 13 (2022) 799552. https://doi.org/10.3389/fphar.2022.799552.

[24] E.J. Giamarellos-Bourboulis, M. Mouktaroudi, E. Bodar, J. van der Ven, B.J. Kullberg, M.G. Netea, J.W. van der Meer, Crystals of monosodium urate monohydrate enhance lipopolysaccharide-induced release of interleukin 1 beta by mononuclear cells through a caspase 1-mediated process, Ann. Rheum. Dis. 68 (2) (2009) 273-278. https://doi.org/10.1136/ard.2007.082222.

[25] J.J. Jhang, J.H. Lin, G.C. Yen, Beneficial properties of phytochemicals on NLRP3 inflammasome-mediated gout and complication, J. Agric. Food Chem. 66 (4) (2018) 765-772. https://doi.org/10.1021/acs.jafc.7b05113.

[26] N. Busso, A. So, Mechanisms of inflammation in gout, Arthritis Res. Ther. 12 (2) (2010) 206, https://doi.org/10.1186/ar2952.

[27] N. Choi, G. Yang, J.H. Jang, H.C. Kang, Y.Y. Cho, H.S. Lee, J.Y. Lee, Loganin alleviates gout inflammation by suppressing NLRP3 inflammasome activation and mitochondrial damage, Molecules 26 (4) (2021) 1071. https://doi.org/10.3390/molecules26041071.

[28] C.J. Chen, Y. Shi, A. Hearn, K. Fitzgerald, D. Golenbock, G. Reed, S. Akira, K.L. Rock, MyD88-dependent IL-1 receptor signaling is essential for gouty inflammation stimulated by monosodium urate crystals, J. Clin. Invest. 116 (8) (2006) 2262-2271. https://doi.org/10.1172/jci28075.

[29] X. Sun, P. Li, X. Qu, W. Liu, Isovitexin alleviates acute gouty arthritis in rats by inhibiting inflammation via the TLR4/MyD88/NF-κB pathway, Pharm. Biol. 59 (1) (2021) 1326-1333. https://doi.org/10.1080/13880209.2021.1979595.

[30] N. Dalbeth, T.J. Lauterio, H.R. Wolfe, Mechanism of action of colchicine in the treatment of gout, Clin. Therapeut. 36 (10) (2014) 1465-1479. https://doi.org/10.1016/j.clinthera.2014.07.017.

[31] Y.Y. Leung, L.L.Y. Hui, V.B. Kraus, Colchicine-Update on mechanisms of action and therapeutic uses, Semin. Arthritis Rheum. 45 (3) (2015) 341-350. https://doi.org/10.1016/j.semarthrit.2015.06.013.

[32] K.A. Zoghebi, E. Bousoik, K. Parang, K.A. Elsaid, Design and biological evaluation of colchicine-CD44-targeted peptide conjugate in an in vitro model of crystal induced inflammation, Molecules 25 (1) (2019) 46. https://doi.org/10.3390/molecules25010046.

[33] J. Nomura, N. Busso, A. Ives, S. Tsujimoto, M. Tamura, A. So, Y. Yamanaka, Febuxostat, an inhibitor of xanthine oxidase, suppresses lipopolysaccharide-induced MCP-1 production via MAPK phosphatase-1-mediated inactivation of JNK, PLoS One 8 (9) (2013) e75527. https://doi.org/10.1371/journal.pone.0075527.

[34] W. Yan, Y. Zhang, L. Hu, Q. Li, H. Zhou, Febuxostat inhibits MPPt-induced inflammatory response through inhibiting the JNK/NF-κB pathway in astrocytes, Neurotox. Res. 39 (3) (2021) 566-574. https://doi.org/10.1007/s12640-020-00316-8.

[35] M. Raeispour, F. Talebpour Amiri, S. Farzipour, A. Ghasemi, S.J. Hosseinimehr, Febuxostat, an inhibitor of xanthine oxidase, ameliorates ionizing radiationinduced lung injury by suppressing caspase-3, oxidative stress and NF-κB, Drug Chem. Toxicol. 45 (6) (2022) 2586-2593. https://doi.org/10.1080/01480545.2021.1977315.

[36] K. Amirshahrokhi, Febuxostat attenuates ulcerative colitis by the inhibition of NF- κB, proinflammatory cytokines, and oxidative stress in mice, Int. Immunopharm. 76 (2019) 105884. https://doi.org/10.1016/j.intimp.2019.105884.

[37] P. Johnson, C. Loganathan, A. Iruthayaraj, K. Poomani, P. Thayumanavan, S-allyl cysteine as potent anti-gout drug: insight into the xanthine oxidase inhibition and anti-inflammatory activity, Biochimie 154 (2018) 1-9. https://doi.org/10.1016/j.biochi.2018.07.015.

[38] T.J. Coderre, P.D. Wall, Ankle joint urate arthritis (AJUA) in rats: an alternative animal model of arthritis to that produced by Freund’s adjuvant, Pain 28 (3) (1987) 379-393. https://doi.org/10.1016/0304-3959(87)90072-8.

[39] J. Sabán-Ruiz, A. Alonso-Pacho, M. Fabregate-Fuente, C. de la Puerta González-Quevedo, Xanthine oxidase inhibitor febuxostat as a novel agent postulated to act against vascular inflammation, Antiinflamm, Antiallergy Agents Med. Chem. 12 (1) (2013) 94-99. https://doi.org/10.2174/1871523011312010011.

[40] A. Ives, J. Nomura, F. Martinon, T. Roger, D. LeRoy, J.N. Miner, G. Simon, N. Busso, A. So, Xanthine oxidoreductase regulates macrophage IL1β secretion upon NLRP3 inflammasome activation, Nat. Commun. 6 (2015) 6555. https://doi.org/10.1038/ncomms7555.

[41] T.O. Crișan, M.C. Cleophas, M. Oosting, H. Lemmers, H. Toenhake-Dijkstra, M.G. Netea, T.L. Jansen, L.A. Joosten, Soluble uric acid primes TLR-induced proinflammatory cytokine production by human primary cells via inhibition of IL- 1Ra, Ann. Rheum. Dis. 75 (4) (2016) 755-762. https://doi.org/10.1136/annrheumdis-2014-206564.

[42] H. Yamanaka, S. Tamaki, Y. Ide, H. Kim, K. Inoue, M. Sugimoto, Y. Hidaka, A. Taniguchi, S. Fujimori, T. Yamamoto, Stepwise dose increase of febuxostat is comparable with colchicine prophylaxis for the prevention of gout flares during the initial phase of urate-lowering therapy: results from FORTUNE-1, a prospective, multicentre randomised study, Ann. Rheum. Dis. 77 (2) (2018) 270-276. https://doi.org/10.1136/annrheumdis-2017-211574.

[43] J. Nomura, T. Kobayashi, A. So, N. Busso, Febuxostat a xanthine oxidoreductase inhibitor, decreases NLRP3-dependent inflammation in macrophages by activating the purine salvage pathway and restoring cellular bioenergetics, Sci. Rep. 9 (2019) 10, https://doi.org/10.1038/s41598-019-53965-x.

[44] L.B. Ru, P. Scott, A. Sydlaske, D.M. Rose, R. Terkeltaub, Innate immunity conferred by toll-like receptors 2 and 4 and myeloid differentiation factor 88 expression is pivotal to monosodium urate monohydrate crystal-induced inflammation, Arthritis Rheum 52 (9) (2005) 2936-2946. https://doi.org/10.1002/art.21238.