Anti-Arthritic Effect of Edaravone Against Complete Freund Adjuvant Induced Arthritis via Osteoclast Differentiation and HIF-1α–VEGF–ANG-1 Axis

Figures & data

Figure 1 Effect of Edaravone on the body weight and arthritis score of normal and CFA induced arthritis rats. (A): body weight and (B): arthritic score. The data are expressed as the mean ± standard error means (SEM) (n=10). Dunnett’s test was used for comparisons the data. Where *P<0.05, **P<0.01 and ***P<0.001 was considered as significant, more significant and extreme significant vs CFA control. ^###P<0.001 consider as significant and compared with the normal control.

Abbreviations: Ns, non-significant; NC, normal control; CFA, complete Freund adjuvant; ED, edaravone.

Figure 2 Effect of Edaravone on the spleen and thymus index of CFA induced arthritis rats. (A): Spleen index and (B): thymus index. The data are expressed as the mean ± standard error means (SEM) (n=10). Dunnett’s test was used for comparisons the data. Where *P<0.05, **P<0.01 and ***P<0.001 was considered as significant, more significant and extreme significant vs CFA control. ^###P<0.001 consider as significant and compared with the normal control.

Abbreviations: Ns, non-significant; NC, normal control; CFA, complete Freund adjuvant; ED, edaravone.

Table 1 Effect of Edaravone on Paw Swelling in CFA Induced Arthritis

S. No	Groups	Days
		Day 0	Day 7	Day 14	Day 21	Day 28
1	NC	0.03±0.01	0.03±0.01	0.05±0.02	0.04±0.02	0.04±0.01
2	CFA	1.59±0.63	2.12±0.69	2.76±0.72	3.12±0.81	3.64±0.85
3	CFA+ED (12.5 mg/kg)	1.47±0.32^ns	1.74±0.73**	1.63±0.73***	1.47±0.63***	1.32±0.57***
4	CFA+ED (25 mg/kg)	1.48±0.21^ns	1.64±0.43***	1.45±0.78***	1.23±0.54***	1.01±0.58***
5	CFA+ED (50 mg/kg)	1.45±0.34*	1.57±0.78***	1.36±0.79***	0.92±0.35***	0.34±0.21***
6	CFA+Indo (400 µg/kg)	1.42±0.83*	1.58±0.82***	1.48±0.83***	0.98±0.43***	0.38±0.28***

Notes: Dunnett’s test was used for comparisons the data. Where *P<0.05; **P<0.01 and ***P<0.001 was considered as significant; more significant and extreme significant.

Figure 3 Effect of Edaravone on the RF and CRP of CFA induced arthritis rats. (A): CRP and (B): RF. The data are expressed as the mean ± standard error means (SEM) (n=10). Dunnett’s test was used for comparisons the data. Where *P<0.05, **P<0.01 and ***P<0.001 was considered as significant, more significant and extreme significant vs CFA control. ^###P<0.001 consider as significant and compared with the normal control.

Abbreviations: Ns, non-significant; NC, normal control; CFA, complete Freund adjuvant; ED, edaravone; CRP, C-reactive protein; RF, Rheumatoid factor.

Figure 4 Effect of Edaravone on the haematological parameters of CFA induced arthritis rats. The data are expressed as the mean ± standard error means (SEM) (n=10). Dunnett’s test was used for comparisons the data. Where *P<0.05, **P<0.01 and ***P<0.001 was considered as significant, more significant and extreme significant vs CFA control. ^###P<0.001 consider as significant and compared with the normal control.

Abbreviations: Ns, non-significant; NC, normal control; CFA, complete Freund adjuvant; ED, edaravone; RBC, red blood cells; WBC, White blood cells; Hb, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; LYM, lymphocytes; MCHC, mean corpuscular hemoglobin concentration; PLT, platelet.

Figure 5 Effect of Edaravone on the antioxidant parameters of CFA induced arthritis rats. The data are expressed as the mean ± standard error means (SEM) (n=10). Dunnett’s test was used for comparisons the data. Where *P<0.05, **P<0.01 and ***P<0.001 was considered as significant, more significant and extreme significant vs CFA control. ^###P<0.001 consider as significant and compared with the normal control.

Abbreviations: Ns, non-significant; NC, normal control; CFA, complete Freund adjuvant; ED, edaravone; MDA, malonaldehyde; GSH, glutathione; CAT, catalase; SOD, superoxide dismutase.

Figure 6 Effect of Edaravone on the serum nitrate and total protein parameter of CFA induced arthritis rats. (A): total protein and (B): serum nitrate. The data are expressed as the mean ± standard error means (SEM) (n, 10). Dunnett’s test was used for comparisons the data. Where *P<0.05; **P<0.01 and ***P<0.001 was considered as significant; more significant and extreme significant vs CFA control. ^###P<0.001 consider as significant and compared with the normal control.

Abbreviations: Ns, non-significant; NC, normal control; CFA, complete Freund adjuvant; ED, edaravone.

Figure 7 Effect of Edaravone on the hepatic parameters of CFA induced arthritis rats. The data are expressed as the mean ± standard error means (SEM) (n, 10). Dunnett’s test was used for comparisons the data. Where *P<0.05; **P<0.01 and ***P<0.001 was considered as significant; more significant and extreme significant vs CFA control. ^###P<0.001 consider as significant and compared with the normal control.

Abbreviations: Ns, non-significant; NC, normal control; CFA, complete Freund adjuvant; ED, edaravone; ALT, alanine transaminase; AST, aspartate transaminase; ALP, alkaline phosphatase.

Figure 8 Effect of Edaravone on the pro-inflammatory parameters of CFA induced arthritis rats. The data are expressed as the mean ± standard error means (SEM) (n, 10). Dunnett’s test was used for comparisons the data. Where *P<0.05; **P<0.01 and ***P<0.001 was considered as significant; more significant and extreme significant vs CFA control. ^###P<0.001 consider as significant and compared with the normal control.

Abbreviations: Ns, non-significant; NC, normal control; CFA, complete Freund adjuvant; ED, edaravone; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; IL-2, interleukin-2; IL-6, interleukin-6; IL-10, interleukin-10; IL-16, interleukin-16; IL-17, interleukin-17; TGF-β, transforming growth factor beta.

Figure 9 Effect of Edaravone on the inflammatory parameters of CFA induced arthritis rats. (A): COX-2; (B): PGE₂ and (C): NF-κB. The data are expressed as the mean ± standard error means (SEM) (n, 10). Dunnett’s test was used for comparisons the data. Where *P<0.05; **P<0.01 and ***P<0.001 was considered as significant; more significant and extreme significant vs CFA control. ^###P<0.001 consider as significant and compared with the normal control.

Abbreviations: Ns, non-significant; NC, normal control; CFA, complete Freund adjuvant; ED, edaravone; COX-2, cyclooxygenase-2; PGE₂, prostaglandin; NF-κB, nuclear kappa B factor.

Figure 10 Effect of Edaravone on the angiogenesis parameters of CFA-induced arthritis rats. (A): HIF-1α; (B): VEGF and (C): ANG-1. The data are expressed as the mean ± standard error means (SEM) (n, 10). Dunnett’s test was used for comparisons the data. Where *P<0.05; **P<0.01 and ***P<0.001 was considered as significant; more significant and extreme significant vs CFA control. ^###P<0.001 consider as significant and compared with the normal control.

Abbreviations: Ns, non-significant; NC, normal control; CFA, complete Freund adjuvant; ED, edaravone; HIF-1α, hypoxia-inducible factor-1α; ANG-1, angiopoietin 1; VEGF, vascular endothelial growth factor.

Figure 11 Effect of Edaravone on the cell viability. (A): cell viability on osteoclast cells and (B): TRAP level. Data were presented as the mean ± SD of experiments. Where *P<0.05 and **P<0.01 compared with control group.

Figure 12 Effect of Edaravone on the level of cytokines. (A): TNF-α; (B): IL-1; (C): IL-6 and (D): IL-37. Data were presented as the mean ± SD of experiments. Where *P<0.05 and **P<0.01 compared with control group.

Abbreviations: Ns, non-significant; NC, normal control; CFA, complete Freund adjuvant; ED, edaravone; TNF-α, tumor necrosis factor-α; IL-1, interleukin-1; IL-6, interleukin-6; IL-37, interleukin-37.

Figure 13 Effect of Edaravone on the level of OPN; M-CSF and RANKL. (A): OPN; (B): M-CSF and (C): RANKL. Data were presented as the mean ± SD of experiments. Where *P<0.05 and **P<0.01 compared with control group.

Abbreviations: Ns, non-significant; NC, normal control; CFA, complete Freund adjuvant; ED, edaravone; OPN, osteopontin; RANKL, receptor activator for nuclear factor-κ B Ligand; M-CSF, macrophage colony stimulating factor.