Excavatolide B Attenuates Rheumatoid Arthritis through the Inhibition of Osteoclastogenesis

Abstract

Osteoclasts are multinucleated giant cells of macrophage/monocyte lineage, and cell differentiation with the upregulation of osteoclast-related proteins is believed to play a major role in the destruction of the joints in the course of rheumatoid arthritis (RA). Pro-inflammatory cytokines, such as interleukin-17A (IL-17A) and macrophage colony-stimulating factor (M-CSF), can be overexpressed in RA and lead to osteoclastogenesis. In a previous study, we found that cultured-type soft coral-derived excavatolide B (Exc-B) exhibited anti-inflammatory properties. In the present study, we thus aimed to evaluate the anti-arthritic activity of Exc-B in in vitro and in vivo models. The results demonstrated that Exc-B inhibits LPS-induced multinucleated cell and actin ring formation, as well as TRAP, MMP-9, and cathepsin K expression. Additionally, Exc-B significantly attenuated the characteristics of RA in adjuvant (AIA) and type II collagen-induced arthritis (CIA) in rats. Moreover, Exc-B improved histopathological features, and reduced the number of TRAP-positive multinucleated cells in the in vivo AIA and CIA models. Immunohistochemical analysis showed that Exc-B attenuated the protein expression of cathepsin K, MMP-2, MMP-9, CD11b, and NFATc1 in ankle tissues of AIA and CIA rats. Level of interleukin-17A and macrophage colony-stimulating factor were also decreased by Exc-B. These findings strongly suggest that Exc-B could be of potential use as a therapeutic agent by inhibiting osteoclast differentiation in arthritis. Moreover, this study also illustrates the use of the anti-inflammatory marine compound, Exc-B, as a potential therapeutic strategy for RA.

Keywords: (Exc-B); excavatolide B; osteoclast; osteoclastogenesis; rheumatoid arthritis (RA).

Figure 1

The effects of Exc-B in LPS-stimulated osteoclast-like cells. (A) RAW 264.7 cells were cultured for one, three and six days with 10 μM Exc-B in the presence of 10 ng/mL LPS. Haematoxylin and eosin staining was performed after fixation; (B) Immunofluorescence microscope photographs of TRAP in LPS-stimulated osteoclast-like cells treated with 10 μM Exc-B for six says and quantification of TRAP immunoreactivity in LPS-stimulated osteoclast-like cells; (C) Immunofluorescence microscope photographs of actin rings in LPS-stimulated osteoclast-like cells treated for six says with 10 μM Exc-B and the number of actin rings in LPS-stimulated osteoclast-like cells. Effect of Exc-B on MMP-9 (D); and cathepsin K mRNA (E) in LPS-stimulated osteoclast-like cells. RAW 264.7 cells were cultured for one, three and six days with 10 μM Exc-B in the presence of 10 ng/mL LPS. The mRNA levels of MMP-9 and cathepsin K were normalized to GAPDH levels. The data are representative of three independent experiments. Each experiment was repeated 4–6 times. Scale bar = 50 μm. Values reflect the mean ± SEM for each group. The data were analysed by one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls post hoc test. * p < 0.05 compared with the control group; # p < 0.05 compared with the LPS treatment alone.

Figure 1

The effects of Exc-B in LPS-stimulated osteoclast-like cells. (A) RAW 264.7 cells were cultured for one, three and six days with 10 μM Exc-B in the presence of 10 ng/mL LPS. Haematoxylin and eosin staining was performed after fixation; (B) Immunofluorescence microscope photographs of TRAP in LPS-stimulated osteoclast-like cells treated with 10 μM Exc-B for six says and quantification of TRAP immunoreactivity in LPS-stimulated osteoclast-like cells; (C) Immunofluorescence microscope photographs of actin rings in LPS-stimulated osteoclast-like cells treated for six says with 10 μM Exc-B and the number of actin rings in LPS-stimulated osteoclast-like cells. Effect of Exc-B on MMP-9 (D); and cathepsin K mRNA (E) in LPS-stimulated osteoclast-like cells. RAW 264.7 cells were cultured for one, three and six days with 10 μM Exc-B in the presence of 10 ng/mL LPS. The mRNA levels of MMP-9 and cathepsin K were normalized to GAPDH levels. The data are representative of three independent experiments. Each experiment was repeated 4–6 times. Scale bar = 50 μm. Values reflect the mean ± SEM for each group. The data were analysed by one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls post hoc test. * p < 0.05 compared with the control group; # p < 0.05 compared with the LPS treatment alone.

Figure 2

Effect of Exc-B in AIA rats. (A) Representative macroscopic photographs of the ankles and paws of the control, AIA, AIA + Exc-B (2.5 mg/kg), AIA + Exc-B (5 mg/kg), and Exc-B alone groups. The AIA and AIA + Exc-B (2.5 mg/kg) groups display significant oedema on the ankle joints and erythema on the hindpaws (red square) in comparison to the control group. The AIA + Exc-B (5 mg/kg) group demonstrate an apparent reduction in AIA-related oedema and erythema. Quantitative analysis of the effect Exc-B at doses of 2.5 or 5 mg/kg on: AIA-related paw oedema (B); arthritis scores (C); and body weight (D). Values reflect the mean ± SEM for each group. Scale bar = 1 cm. The data were analysed by Kruskal–Wallis One Way Analysis of Variance analysis. * p < 0.05, significantly different from the control group; # p < 0.05, significantly different from the AIA group.

Figure 3

Effect of Exc-B in CIA rats. (A) Representative macroscopic photographs of the ankles and paws of the control, CIA, and CIA + Exc-B (5 mg/kg) groups. The CIA group displays significant oedema on the ankle joints and erythema on the hindpaws (red square) in comparison to the control group. The CIA + Exc-B (5 mg/kg) group demonstrate an apparent reduction in CIA-related oedema and erythema. Quantitative analysis of the effect Exc-B at doses of 5 mg/kg on: CIA-related paw oedema (B); arthritis scores (C); and body weight (D). Values reflect the mean ± SEM for each group. Scale bar = 1 cm. The data were analysed by Kruskal–Wallis One Way Analysis of Variance analysis. * p < 0.05, significantly different from the control group; # p < 0.05, significantly different from the CIA group.

Figure 4

Histopathological assessments of the effect of Exc-B on the ankle joints in AIA rats. (A) Normal joint structure showing calcaneus-talus articulation with the distal tibia and normal synovial tissue in the control group; (B) Representative haematoxylin and eosin staining images of the ankle joint sections from the control, AIA, AIA + Exc-B (2.5 mg/kg), AIA + Exc-B (5 mg/kg), and Exc-B alone groups of similar areas as outlined by the boxes in the image of the normal joint structure. The AIA group exhibits synovial inflammation (arrows), bone erosion (b), and cartilage destruction (a). The representative histopathological scores for: synovial inflammation (C); cartilage destruction (D); and bone erosion (E) were assessed in ankle joints of the control, AIA, AIA + Exc-B (2.5 mg/kg), AIA + Exc-B (5 mg/kg), and Exc-B groups. Scale bar = 200 μm. Values reflect the mean ± SEM for each group. n = 6 rat per group. The data were analysed by one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls post hoc test. * p < 0.05, significantly different from the control group; # p < 0.05, significantly different from the AIA group.

Figure 5

Histopathological assessments of the effect of Exc-B on the ankle joints in CIA rats. (A) Normal joint structure showing calcaneus-talus articulation with the distal tibia and normal synovial tissue in the control group; (B) Representative haematoxylin and eosin staining images of the ankle joint sections from the control, CIA, and CIA + Exc-B (5 mg/kg) groups in similar areas as outlined by the boxes in the image of the normal joint structure. The CIA group exhibits synovial inflammation (arrows), cartilage destruction (a) and bone erosion (b). The representative histopathological scores for: synovial inflammation (C); cartilage destruction (D); and bone erosion (E) were assessed in ankle joints of the control, CIA, and CIA + Exc-B (5 mg/kg) groups. Scale bar = 200 μm. Values reflect the mean ± SEM for each group. n = 6 rat per group. The data were analysed by one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls post hoc test. * p < 0.05, significantly different from the control group. # p < 0.05, significantly different from the CIA group.

Figure 6

Effect of Exc-B on TRAP-positive multinucleated cell formation in vivo. Representative high-power field (400×) magnifications of specimens (A) from the: control; AIA; CIA; AIA + Exc-B (2.5 mg/kg); AIA + Exc-B (5 mg/kg); and CIA+ Exc-B (5 mg/kg) groups. The CIA and AIA groups show the TRAP-positive multinucleated cells (arrows) in the ankle joints sections. Quantitative analysis of the number of multinucleated cells (B). Values reflect the mean ± SEM for each group. Scale bar = 50 μm. n = 6 rat per group. The data were analysed by one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls post hoc test. * p < 0.05, significantly different from the control group; # p < 0.05, significantly different from the AIA or CIA group.

Figure 7

Effect of Exc-B on NFATc1 protein expression. (A) NFATc1 protein immunoreactivity is indicated in red-brown (arrows) in ankle joint sections from the control, AIA, CIA, AIA + Exc-B (2.5 mg/kg), AIA + Exc-B (5 mg/kg) and CIA + Exc-B (5mg/kg) groups. NFATc1 immunoreactivity in the articular cartilage, synovial tissue, and bone marrow, respectively, and the quantification of NFATc1 positive cells in the: articular cartilage (B); synovial tissue (C); and bone marrow (D) are shown. SB, subchondral bone; BM, bone marrow. Values reflect the mean ± SEM for each group. Scale bar = 100 μm. n = 6 rat per group. The data were analysed by one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls post hoc test. * p < 0.05, significantly different from the control group; # p < 0.05, significantly different from the AIA or CIA group.

Figure 8

Effect of Exc-B on cathepsin K, MMP-2, MMP-9, and CD11b protein expression in synovial tissue. (A) Immunoreactivity is indicated in red-brown (arrows) in ankle joint sections from the control, AIA, CIA, AIA + Exc-B (2.5 mg/kg), AIA + Exc-B (5 mg/kg) and CIA + Exc-B (5 mg/kg) groups. Quantitative analysis of: cathepsin K (B); MMP-2 (C); MMP-9 (D); and CD11b (E) positive cells in synovial cells is shown. Values reflec the mean ± SEM for each group. Scale bar = 100 μm. n = 6 rat per group. The data were analysed by one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls post hoc test. * p < 0.05, significantly different from the control group; # p < 0.05, significantly different from the AIA or CIA group.

Figure 8

Effect of Exc-B on cathepsin K, MMP-2, MMP-9, and CD11b protein expression in synovial tissue. (A) Immunoreactivity is indicated in red-brown (arrows) in ankle joint sections from the control, AIA, CIA, AIA + Exc-B (2.5 mg/kg), AIA + Exc-B (5 mg/kg) and CIA + Exc-B (5 mg/kg) groups. Quantitative analysis of: cathepsin K (B); MMP-2 (C); MMP-9 (D); and CD11b (E) positive cells in synovial cells is shown. Values reflec the mean ± SEM for each group. Scale bar = 100 μm. n = 6 rat per group. The data were analysed by one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls post hoc test. * p < 0.05, significantly different from the control group; # p < 0.05, significantly different from the AIA or CIA group.

Figure 9

Effect of Exc-B on cathepsin K, MMP-2, MMP-9 protein expression in cartilage. (A) Immunoreactivity is indicated in red-brown (arrows) in ankle joint sections from the control, AIA, CIA, AIA + Exc-B (2.5 mg/kg), AIA + Exc-B (5 mg/kg) and CIA + Exc-B (5 mg/kg) groups. Quantitative analysis of: cathepsin K (B); MMP-2 (C); and MMP-9 (D) positive cells in chondrocytes is shown. Values reflect the mean ± SEM for each group. Scale bar = 100 μm. n = 6 rat per group. The data were analysed by one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls post hoc test. * p < 0.05, significantly different from the control group. # p < 0.05, significantly different from the AIA or CIA group.

Figure 10

Effect of Exc-B on MAPK, HO-1, and HMGB-1 in homogenized knee synovial tissues. Western blot analysis of the effects of subcutaneous Exc-B administration on protein expression of HO-1, HMGB-1, and MAPK signalling, as well as GAPDH in knee synovial tissue homogenates from rats with: AIA (A); or CIA (B). Exc-B (2.5 or 5 mg/kg) significantly inhibits HMGB-1 and MAPK signalling and upregulates HO-1 protein expression after immunization. The There is no difference between groups in the protein expression of GAPDH in synovial tissues. Quantification values reflect the mean ± S.E.M. of three different experiments. n = 6 rat per group. The data were analysed by one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls post hoc test. * p < 0.05, significantly different from the control group. # p < 0.05, significantly different from the AIA or CIA group.

Figure 10

Effect of Exc-B on MAPK, HO-1, and HMGB-1 in homogenized knee synovial tissues. Western blot analysis of the effects of subcutaneous Exc-B administration on protein expression of HO-1, HMGB-1, and MAPK signalling, as well as GAPDH in knee synovial tissue homogenates from rats with: AIA (A); or CIA (B). Exc-B (2.5 or 5 mg/kg) significantly inhibits HMGB-1 and MAPK signalling and upregulates HO-1 protein expression after immunization. The There is no difference between groups in the protein expression of GAPDH in synovial tissues. Quantification values reflect the mean ± S.E.M. of three different experiments. n = 6 rat per group. The data were analysed by one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls post hoc test. * p < 0.05, significantly different from the control group. # p < 0.05, significantly different from the AIA or CIA group.

Figure 11

Effect of Exc-B on the levels of M-CSF and IL-17A in blood serum: (A) effect of Exc-B on the level of M-CSF in the blood serum of control, AIA, CIA, and Exc-B treated rats; and (B) effect of Exc-B on the level of IL-17A in the blood serum of control, AIA, CIA, and Exc-B treated rats. Values reflect the mean ± SEM for each group. n = 6 rat per group. The data were analysed by one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls post hoc test. * p < 0.05, significantly different from the control group; # p < 0.05, significantly different from the AIA or CIA group.