Pulchinenoside B4 attenuates gouty arthritis by regulating NLRP3 inflammasome and macrophage polarization: a transcriptomics-based analysis

Abstract

Gouty arthritis (GA) is an inflammatory disorder characterized by the deposition of monosodium urate (MSU) crystals in joint tissues. Pulchinenoside B4 (B4) has broad-spectrum anti-inflammatory properties, but its role and potential mechanism in the pathogenesis of GA are still unclear. The purpose of this study is to comprehensively elucidate the therapeutic effect and mechanism of B4 on GA by integrating transcriptome analysis and in vitro and in vivo experiments. In the MSU-induced mouse GA model, B4 treatment significantly improved ankle edema and reduced inflammatory cell infiltration. Through the analysis of transcriptome sequencing results, we identified multiple differentially expressed long non-coding RNAs (lncRNAs), such as Nod1, Rbck1 and Pycard. In the in-depth exploration of the mechanism, we focused on the NOD-like receptor signaling pathway, NF-κB signaling cascade, and B4-regulated macrophage polarization. In vitro and in vivo models, we confirmed that B4 significantly inhibited the expression and activation of key components of NLRP3 inflammasome (such as ASC, Caspase-1 and IL-1β) by qPCR, Western blot and immunofluorescence. Flow cytometry and immunofluorescence analysis further showed that B4 could prevent MSU-induced macrophage polarization to pro-inflammatory M1 phenotype.Based on these results, this study elucidated the mechanism of B4 improving MSU-induced GA inflammatory response by inhibiting NLRP3 inflammasome activation and blocking M1 macrophage polarization. These results suggest that B4 has great potential as a candidate drug for the treatment of GA.

Keywords

Transcriptomics / Gouty arthritis / Pulchinenoside B4 / NLRP3 inflammasome / Macrophage polarization / Nuclear factor-κB

Funding

This work was supported by the National Natural Science Foundation of China (Nos. 82204590 and 82560758), Jiangxi Provincial Natural Science Foundation (Nos. 20224BAB216099 and 20252BAC240467), China Postdoctoral Science Foundation (No. 2023M731439), Key Technologies R & D Programme of Nanchang City (No. 2024zdxm023), and Jiangxi University of Chinese Medicine Science and Technology Innovation Team Development Program (No. CXTD22001).

Declaration of competing interests

These authors have no conflict of interest to declare.

References

[1] Dalbeth N, Gosling AL, Gaffo A, et al. Gout. Lancet. 2021; 397(10287):1843-1855. https://doi.org/10.1016/S0140-6736(21)00569-9.

[2] Patel U, Rajasingh S, Samanta S, et al. Macrophage polarization in response to epigenetic modifiers during infection and inflammation. Drug Discov Today. 2017; 22(1):186-193. https://doi.org/10.1016/j.drudis.2016.08.006.

[3] Lin S, Hu X, Li Y, et al. Stefin B alleviates the gouty arthritis in mice by inducing the M2 polarization of macrophages. Naunyn Schmiedebergs Arch Pharmacol. 2024; 397(8):5677-5688. https://doi.org/10.1007/s00210-023-02911-w.

[4] Zhou Q, Sun HJ, Zhang XW. Total saponin fraction of Dioscorea nipponica Makino improves gouty arthritis symptoms in rats via M1/M2 polarization of monocytes and macrophages mediated by arachidonic acid signaling. Chin J Integr Med. 2023; 29(11):1007-1017. https://doi.org/10.1007/s11655-022-3729-y.

[5] Liu L, Zhu XX, Zhao TY, et al. Sirt1 ameliorates monosodium urate crystal-induced inflammation by altering macrophage polarization via the PI3K/Akt/STAT6 pathway. Rheumatology (Oxford). 2019; 58(9):1674-1683. https://doi.org/10.1093/rheumatology/kez165.

[6] Qin DE, Liang W, Yu Y, et al. Modified Simiaowan prevents and treats gouty arthritis via the Nrf2/NLRP3 inflammasome signaling pathway. J Ethnopharmacol. 2024;318(Pt A):116906. https://doi.org/10.1016/j.jep.2023.116906.

[7] Wu YY, Zhou JW, Zuo XC, et al. Yanggan Jiangmei Formula alleviates hepatic inflammation and lipid accumulation in non-alcoholic steatohepatitis by inhibiting the NF-κB/NLRP3 signaling pathway. Chin J Nat Med. 2024; 22(3):224-234. https://doi.org/10.1016/S1875-5364(24)60595-9.

[8] Lin XY, Shao TJ, Huang L, et al. Simiao Decoction alleviates gouty arthritis by modulating proinflammatory cytokines and the gut ecosystem. Front Pharmacol. 2020; 11:955. https://doi.org/10.3389/fphar.2020.00955.

[9] Fan WM, Chen SX, Wu XH, et al. Resveratrol relieves gouty arthritis by promoting mitophagy to inhibit activation of NLRP 3 inflammasomes. J Inflamm Res. 2021; 14:3523-3536. https://doi.org/10.2147/JIR.S320912.

[10] Cao L, Zhao TY, Xue Y, et al. The anti-inflammatory and uric acid lowering effects of Si-Miao-San on gout. Front Immunol. 2022; 12:777522. https://doi.org/10.3389/fimmu.2021.777522.

[11] Luo ZW, Yin FF, Wang XB, et al. Progress in approved drugs from natural product resources. Chin J Nat Med. 2024; 22(3):195-211. https://doi.org/10.1016/S1875-5364(24)60582-0.

[12] Gong Q, Yin JL, Wang ML, et al. Anemoside B4 exerts hypoglycemic effect by regulating the expression of GLUT 4 in HFD/STZ rats. Molecules. 2023; 28(3):968. https://doi.org/10.3390/molecules28030968.

[13] Yuan RYK, He J, Huang LT, et al. Anemoside B4 protects against acute lung injury by attenuating inflammation through blocking NLRP3 inflammasome activation and TLR4 dimerization. J Immunol Res. 2020; 7502301. https://doi.org/10.1155/2020/7502301.

[14] Ma HM, Zhou MJ, Duan WB, et al. Anemoside B4 prevents acute ulcerative colitis through inhibiting of TLR4/NF-κB/MAPK signaling pathway. Int Immunopharmacol. 2020; 87:106794. https://doi.org/10.1016/j.intimp.2020.106794.

[15] Liu Y, Grimm M, Dai WT, et al. CB-Dock: a web server for cavity detection-guided protein-ligand blind docking. Acta Pharmacol Sin. 2020; 41(1):138-144. https://doi.org/10.1038/s41401-019-0228-6.

[16] Zeng XJ, Zhang Y. The interpretation of 2018 updated European League Against Rheumatism evidence based recommendations for the diagnosis of gout. Chin J Intern Med. 2019; 58(10):745-750. https://doi.org/10.3760/cma.j.issn.0578-1426.2019.10.005.

[17] So AK, Martinon F. Inflammation in gout: mechanisms and therapeutic targets. Nat Rev Rheumatol. 2017; 13(11):639-647. https://doi.org/10.1038/nrrheum.2017.155.

[18] Wu MM, Tian Y, Wang QQ, et al. Gout: a disease involved with complicated immunoinflammatory responses: a narrative review. Clin Rheumatol. 2020; 39(10):2849-2859. https://doi.org/10.1007/s10067-020-05090-8.

[19] Cunningham CC, Corr EM, McCarthy GM, et al. Intra-articular basic calcium phosphate and monosodium urate crystals inhibit anti-osteoclastogenic cytokine signalling. Osteoarthritis Cartilage. 2016; 24(12):2141-2152. https://doi.org/10.1016/j.joca.2016.07.001.

[20] Funes SC, Rios M, Escobar-Vera J, et al. Implications of macrophage polarization in autoimmunity. Immunology. 2018; 154(2):186-195. https://doi.org/10.1111/imm.12910.

[21] Liu YZ, Tang HL, Liu XX, et al. Frontline science: reprogramming COX-2, 5-LOX, and CYP4A-mediated arachidonic acid metabolism in macrophages by salidroside alleviates gouty arthritis. J Leukoc Biol. 2019; 105(1):11-24. https://doi.org/10.1002/JLB.3HI0518-193R.

[22] Li YX, Yuan JB, Deng W, et al. Buqi-Tongluo Decoction inhibits osteoclastogenesis and alleviates bone loss in ovariectomized rats by attenuating NFATc1, MAPK, NF-κB signaling. Chin J Nat Med. 2025; 23(1):90-101. https://doi.org/10.1016/S1875-5364(25)60810-7.

[23] Yang WQ, Wen W, Chen H, et al. Zhongfeng Xingnao Liquid ameliorates post-stroke cognitive impairment through sirtuin1 (SIRT1)/nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) pathway. Chin J Nat Med. 2025; 23(1):77-89. https://doi.org/10.1016/S1875-5364(25)60808-9.

[24] Xu XJ, Long JB, Jin KY, et al. Danshen-Chuanxiongqin Injection attenuates cerebral ischemic stroke by inhibiting neuroinflammation via the TLR2/TLR4-MyD88-NF-κB pathway in tMCAO mice. Chin J Nat Med. 2021; 19(10):772-783. https://doi.org/10.1016/S1875-5364(21)60083-3.

[25] Zhang Y. Awareness and ability of paradigm shift are needed for research on dominant diseases of TCM. Chin Herb Med. 2023; 15(4):475. https://doi.org/10.1016/j.chmed.2023.10.001.

[26] Li YH, Zou M, Han Q, et al. Therapeutic potential of triterpenoid saponin anemoside B4 from Pulsatilla chinensis. Pharmacol Res. 2020; 160:105079. https://doi.org/10.1016/j.phrs.2020.105079.

[27] Feng W, Zhang Y, Zhang YW, et al. Anemoside B4 ameliorates dextran sulfate sodium (DSS)-induced colitis through inhibiting NLRP3 inflammasome and modulating gut microbiota. Eur J Pharmacol. 2024; 963:176164. https://doi.org/10.1016/j.ejphar.2023.176164.

[28] He J, Yuan RYK, Cui XL, et al. Anemoside B4 protects against Klebsiella pneumoniae- and influenza virus FM1-induced pneumonia via the TLR4/Myd88 signaling pathway in mice. Chin Med. 2020; 15:68. https://doi.org/10.1186/s13020-020-00350-w.

[29] Ye Y. Pharmacokinetic study on the anti-hyperuricemia efficacy of Leukoderma saponin B4 and different administration routes. Nanning: University of Chinese Medicine in Guangxi; 2023. https://doi.org/10.27879/dcnki.ggxzy.2023.000665.

[30] Zhou C. Study on the efficacy and pharmacokinetics of saponin B4 suppository in the treatment of chronic colitis. Nanchang: Jiangxi University of Traditional Chinese Medicine; 2021. https://doi.org/10.27180/d.cnki.gjxzc.2021.000245.

[31] Ye YQ, Xue MZ, Tian XT, et al. Pharmacokinetic and metabolite profile of orally administered anemoside B4 in rats with an improved exposure in formulations of rectal suppository. J Ethnopharmacol. 2023; 315:116694. https://doi.org/10.1016/j.jep.2023.116694.

[32] Liu CY, Fu TT, Yang FF, et al. Comparative study on pulmonary pharmacokinetics and anti-inflammatory effects of Pulsatilla saponin B4 after tracheal and intravenous administration. Acta Pharm Sin. 2022; 57(9):2791-2797. https://doi.org/10.16438/j.0513-4870.2022.0307.

[33] Zhu W, Chai Y, Jin Y, et al. The effects of Cordyceps sinensis analgesic compounds on the transcriptome and pain-related genes such as Adora1 in gout rats. J Mycol. 2017; 36(1):48-59. https://doi.org/10.13346/j.mycosystema.160262.

[34] Zhang XH, Liu J, Sun YQ, et al. Chinese herbal compound Huangqin Qingrechubi Capsule reduces lipid metabolism disorder and inflammatory response in gouty arthritis via the LncRNA H19/APN/PI3K/AKT cascade. Pharm Biol. 2023; 61(1):541-555. https://doi.org/10.1080/13880209.2023.2191641.

[35] Fan QQ, Zhai BT, Zhang D, et al. Study on the underlying mechanism of Yinhua Gout Granules in the treatment of gouty arthritis by integrating transcriptomics and network pharmacology. Drug Des Devel Ther. 2024; 18:3089-3112. https://doi.org/10.2147/DDDT.S475442.

[36] Liu SL, Wang XH, Gao YG, et al. Transcriptomic analysis identifies differentially expressed genes (DEGs) associated with bolting and flowering in Saposhnikovia divaricata. Chin J Nat Med. 2018; 16(6):446-455. https://doi.org/10.1016/S1875-5364(18)30078-5.

[37] Geng QS, Liu RJ, Shen ZB, et al. Transcriptome sequencing and metabolome analysis reveal the mechanism of Shuanghua Baihe Tablet in the treatment of oral mucositis. Chin J Nat Med. 2021; 19(12):930-943. https://doi.org/10.1016/S1875-5364(22)60150-X.

[38] Gulzar F, Chhikara N, Kumar P, et al. ER stress aggravates NOD1-mediated inflammatory response leading to impaired nutrient metabolism in hepatoma cells. Biochem Biophys Res Commun. 2024; 735:150827. https://doi.org/10.1016/j.bbrc.2024.150827.

[39] Du JW, Peng LJ, Feng J, et al. RBCK1 overexpression attenuates inflammation and mobility of Derp1-induced nasal mucosal cells by downregulating NLRP3. Int Arch Allergy Immunol. 2023; 184(5):471-480. https://doi.org/10.1159/000527962.

[40] Yao Z, Li Y, Mai H, et al. Comprehensive multiomics analysis identifies PYCARD as a key pyroptosis-related gene in osteoarthritis synovial macrophages. Front Immunol. 2025; 16:1558139. https://doi.org/10.3389/fimmu.2025.1558139.

[41] Ouyang X, Li NZ, Guo MX, et al. Active flavonoids from Lagotis brachystachya attenuate monosodium urate-induced gouty arthritis via inhibiting TLR4/MyD88/NF-κB pathway and NLRP3 expression. Front Pharmacol. 2021; 12:760331. https://doi.org/10.3389/fphar.2021.760331.

[42] Latz E, Duewell P. NLRP3 inflammasome activation in inflammaging. Semin Immunol. 2018; 40:61-73. https://doi.org/10.1016/j.smim.2018.09.001.

[43] Lyu S, Ding RW, Liu P, et al. LC-MS analysis of serum for the metabolomic investigation of the effects of Pulchinenoside b4 administration in monosodium urate crystal-induced gouty arthritis rat model. Molecules. 2019; 24(17):3161. https://doi.org/10.3390/molecules24173161.

[44] Wang SH, Lei P, Feng Y, et al. Jinyinqingre Oral Liquid alleviates LPS-induced acute lung injury by inhibiting the NF-κB/NLRP3/GSDMD pathway. Chin J Nat Med. 2023; 21(6):423-435. https://doi.org/10.1016/S1875-5364(23)60397-8.

[45] Duez H, Pourcet B. Nuclear receptors in the control of the NLRP 3 inflammasome pathway. Front Endocrinol (Lausanne). 2021; 12:630536. https://doi.org/10.3389/fendo.2021.630536.

[46] Zhang XY, Liu YB, Deng GR, et al. A purified biflavonoid extract from Selaginella moellendorffii alleviates gout arthritis via NLRP3/ASC/Caspase-1 axis suppression. Front Pharmacol. 2021; 12:676297. https://doi.org/10.3389/fphar.2021.676297.

[47] Zhang YZ, Sui XL, Xu YP, et al. NLRP3 inflammasome and lipid metabolism analysis based on UPLC-Q-TOF-MS in gouty nephropathy. Int J Mol Med. 2019; 44(1):172-184. https://doi.org/10.3892/ijmm.2019.4176.

[48] Broz P.Immunology: caspase target drives pyroptosis. Nature. 2015; 526(7575):642-643. https://doi.org/10.1038/nature15632.

[49] Wu C, Li F, Zhang X, et al. (-)-Epicatechin ameliorates monosodium urate-induced acute gouty arthritis through inhibiting NLRP3 inflammasome and the NF-κB signaling pathway. Front Pharmacol. 2022; 13:799552. https://doi.org/10.3389/fphar.2022.799552.

[50] Teng L, Shen Y, Qu YH, et al. Cyasterone inhibits IL-1β-mediated apoptosis and inflammation via the NF-κB and MAPK signaling pathways in rat chondrocytes and ameliorates osteoarthritis in vivo. Chin J Nat Med. 2023; 21(2):99-112. https://doi.org/10.1016/S1875-5364(23)60388-7.

[51] Schroder K, Tschopp J. The inflammasomes. Cell. 2010; 140(6):821-832. https://doi.org/10.1016/j.cell.2010.01.040.

[52] Yang G, Lee HE, Moon SJ, et al. Direct binding to NLRP3 pyrin domain as a novel strategy to prevent NLRP3-driven inflammation and gouty arthritis. Arthritis Rheumatol. 2020; 72(7):1192-1202. https://doi.org/10.1002/art.41245.

[53] Lin W, Shen P, Huang Y, et al. Wutou Decoction attenuates the synovial inflammation of collagen-induced arthritis rats via regulating macrophage M1/M2 type polarization. J Ethnopharmacol. 2023; 301:115802. https://doi.org/10.1016/j.jep.2022.115802.

[54] Wang L, Zhao M. Suppression of NOD-like receptor protein 3 inflammasome activation and macrophage M1 polarization by hederagenin contributes to attenuation of sepsis-induced acute lung injury in rats. Bioengineered. 2022; 13(3):7262-7276. https://doi.org/10.1080/21655979.2022.2047406.

[55] Tian J, Zhou D, Xiang L, et al. MiR-223-3p inhibits inflammation and pyroptosis in monosodium urate-induced rats and fibroblast-like synoviocytes by targeting NLRP3. Clin Exp Immunol. 2021; 204(3):396-410. https://doi.org/10.1111/cei.13587.

[56] Ma WT, Gao F, Gu K, et al. The role of monocytes and macrophages in autoimmune diseases: a comprehensive review. Front Immunol. 2019; 10:1140. https://doi.org/10.3389/fimmu.2019.01140.

[57] Fan B, Liu QY, Yang Y, et al. Soufeng Sanjie Formula alleviates osteoarthritis by inhibiting macrophage M1 polarization and modulating intestinal metabolites. J Ethnopharmacol. 2025; 339:119147. https://doi.org/10.1016/j.jep.2024.119147.

[58] Mei JT, Zhou F, Qiao H, et al. Nerve modulation therapy in gouty arthritis: targeting increased sFRP2 expression in dorsal root ganglion regulates macrophage polarization and alleviates endothelial damage. Theranostics. 2019; 9(13):3707-3722. https://doi.org/10.7150/thno.33908.

[59] Chen YN, Hu MR, Wang L, et al. Macrophage M1/M2 polarization. Eur J Pharmacol. 2020; 877:173090. https://doi.org/10.1016/j.ejphar.2020.173090.

[60] Cutolo M, Campitiello R, Gotelli E, et al. The role of M1/M2 macrophage polarization in rheumatoid arthritis synovitis. Front Immunol. 2022; 13:867260. https://doi.org/10.3389/fimmu.2022.867260.

[61] Sloas C, Gill S, Klichinsky M. Engineered CAR-macrophages as adoptive immunotherapies for solid tumors. Front Immunol. 2021; 12:783305. https://doi.org/10.3389/fimmu.2021.783305.