Rhaponticin Alleviates Collagen-induced Arthritis by Inhibiting NLRP3/GSDMD-mediated Neutrophil Extracellular Traps

Abstract

Neutrophil extracellular traps (NETs) play an important role in the inflammatory response and progressive joint destruction in rheumatoid arthritis (RA). Rhaponticin (Rha) is a stilbene glycoside compound with antioxidant and anti-inflammatory effects. This study aimed to investigate the therapeutic potential of Rha in RA, with a specific focus on its effects on NETs and on the underlying mechanisms of Rha. NETs formation induced by phorbol 12-myristate 13-acetate (PMA) and a collagen-induced arthritis (CIA) mouse model were implemented to evaluate the pharmacological effects of Rha in vitro and in vivo. The potential mechanism of Rha in improving RA was screened and verified using the SuperPred and DisGeNET databases. Disulfiram (a GSDMD inhibitor) and S100a8^cre GSDMD^fl/fl mice were used to confirm whether GSDMD is key to the role of Rha. The findings demonstrate that Rha significantly inhibited reactive oxygen species and NETs production in PMA-activated neutrophils. In vivo, Rha treatment significantly relieved joint symptoms in CIA mice and NETs production. Mechanistically, Rha reduced NETs production via inhibition of NLRP3/GSDMD activation. Neutrophil-specific GSDMD depletion eliminated the effects of Rha on NETs production in vitro. Disulfiram eliminated the effects of Rha on the inhibition of NETs production and alleviated joint inflammation in mice in vivo and in vitro. Overall, our results indicated that Rha exerts a protective effect against CIA by inhibiting NETs production through the NLRP3/GSDMD pathway. The results of this study provide new strategies for treating RA.

Keywords: Gasdermin-D; Neutrophil extracellular traps; Rhaponticin; Rheumatoid arthritis.

Fig. 1

Rha inhibited PMA-stimulated NETs production in vitro. a Chemical structure of Rha. b Detection of neutrophil ROS fluorescence intensity after PMA stimulation and treatment with different concentrations of Rha (n = 8 for each group). c Quantification of cfDNA in the supernatant of neutrophils after PMA stimulation and Rha-treatment (n = 6 for each group). d Quantification of NE in NETs after PMA stimulation and Rha-treatment (n = 6 for each group). e Neutrophil MPO flow cytometry detection and MFI quantification after PMA stimulation and Rha treatment (n = 3 for each group). f–g Visualization of NETs in vitro and quantification of NETs formation. Immunofluorescence staining was performed with DAPI (blue), MPO (green), and CitH3 (red). Scale bar = 10 μm. (n =  = 6 for each group). h-i Immunoblotting and quantification of neutrophil lysates from PMA and Rha treatments. (n = 3 for each group). The data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, no significance; Rha, rhaponticin; NC, normal control; PMA, phorbol 12-myristate 13-acetate. NE, neutrophil elastase

Fig. 2

Rha improved joint inflammation in CIA mice. a Flow chart of animal experiments conducted on CIA mice. b-c Arthritis scores and paw thickness of different groups (n = 6 for each group). d Representative image of joint swelling in mice on the day of dissection. e Hematoxylin and eosin (H&E) staining of joints in different groups. f Safranin O and Fast Green staining of mouse joints in different groups. Scale bars = 100 μm. g-i Histopathological scores for synovial inflammation, bone erosion, and cartilage depletion in different groups (n = 6 for each group). j Weight of different groups (n = 6 for each group). k Levels of serum AST in different groups (n = 6 for each group). (l) Levels of serum ALT in different groups (n = 6 for each group). The data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, no significance. WT, wild type; CIA, collagen-induced arthritis; Rha, rhaponticin; MTX, methotrexate

Fig. 3

Rha reduced NETs production in CIA mice. a Representative immunofluorescence image of CitH3 (red). Scale bar = 100 μm. b Quantification of NETs formation by immunofluorescence (n = 6 for each group). c Serum cfDNA quantification in different groups (n = 6 for each group). d-g Serum from CIA mice were detected for TNF-α, IL-18, IL-1β, IL-6 using Elisa kits (n = 6 for each group). The data were presented as mean ± SEM. *#P < 0.05, **##P < 0.01, ***###P < 0.001, ****####P < 0.0001. ns, no significance. WT, wild type; CIA, collagen-induced arthritis; Rha, rhaponticin

Fig. 4

Network pharmacology analysis and validation of NLRP3/GSDMD as a key pathway for Rha. a Overlapping Venn diagram of RA-related targets and Rha-related targets. b KEGG pathway enrichment map. c Representative IHC staining of NLRP3 and GSDMD in joints. Scale bars = 50 μm. d–g Western blotting and quantification of NLRP3, caspase-1, and GSDMD of PMA-stimulated neutrophil lysates (n = 3 for each group). h-j The mRNA levels of NLRP3, caspase-1, and GSDMD in neutrophils stimulated by PMA (n = 4 for each group). The data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, no significance. WT, wild type; CIA, collagen-induced arthritis; RA, rheumatoid arthritis; Rha, rhaponticin

Fig. 5

The effect of Rha on NETs production was eliminated in neutrophil-specific GSDMD knockout cells. a The diagram of S100a8^cre GSDMD^fl/fl mice breeding scheme. b Genotyping of S100a8^cre GSDMD^fl/fl mice by routine PCR. P: Positive Control, B6: Negative Control (C57BL/6 J), N: No-Template Control. c Quantification of cfDNA in the neutrophil supernatant of S100a8^cre GSDMD^fl/fl mice in vitro (n = 6 for each group). d Flow cytometry detection and MFI quantification of MPO in the neutrophils of S100a8^cre GSDMD^fl/fl mice (n = 6 for each group). e Quantification of NE in NETs after PMA stimulation and Rha-treatment in the neutrophils of S100a8^cre GSDMD^fl/fl mice (n = 6 for each group). f–g Visualization and quantification of NETs in vitro in S100a8^cre GSDMD^fl/fl mice (n = 6 for each group). Scale bar = 10 μm. The data were presented as mean ± SEM. ****P < 0.0001. ns, no significance. NC, normal control; PMA, phorbol 12-myristate 13-acetate; Rha, rhaponticin

Fig. 6

DSF eliminated the inhibitory effect of Rha on NETs production. a Quantification of cfDNA in the supernatant of neutrophils treated with PMA and DSF + Rha (n = 6 for each group). b Quantification of NE in NETs after PMA stimulation and DSF + Rha treatment (n = 6 for each group). C Flow cytometry detection and MFI quantification of MPO in neutrophils treated with PMA and DSF + Rha (n = 4 for each group). d–e Visualization and quantification of NETs in vitro (n = 6 for each group). Scale bar = 10 μm. The data were presented as mean ± SEM. **P < 0.01, ****P < 0.0001. ns, no significance; NC, normal control; PMA, phorbol 12-myristate 13-acetate; Rha, rhaponticin; DSF, disulfiram

Fig. 7

DSF eliminated the protective effect of Rha against arthritis in CIA mice. a–b Arthritis scores and paw thickness of different groups (n = 6 for each group). c Representative image of mouse joint swelling on the day of dissection. d Hematoxylin and eosin (H&E) staining of joints in different groups. e Safranin O and Fast Green staining of mouse joints in different groups. Scale bars = 100 μm. f Histopathological scores for synovial inflammation, bone erosion, and cartilage depletion in different groups (n = 6 for each group). The data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, no significance. WT, wild type; CIA, collagen-induced arthritis; RA, rheumatoid arthritis; Rha, rhaponticin; DSF, disulfiram

Fig. 8

DSF eliminated the protective effect of Rha reduced NETs in CIA mice. a Representative immunofluorescence image of CitH3 (red). Scale bar = 100 μm. b Quantification of NETs formation by immunofluorescence (n = 6 for each group). c Serum cfDNA quantification in different groups (n = 6 for each group). The data were presented as mean ± SEM. ****P < 0.0001. ns, no significance. WT, wild type; CIA, collagen-induced arthritis; RA, rheumatoid arthritis; Rha, rhaponticin; DSF, disulfiram