Ameliorative Effect of Dabigatran on CFA-Induced Rheumatoid Arthritis via Modulating Kallikrein-Kinin System in Rats

Abstract

Rheumatoid arthritis is an autoimmune disease that affects joints, leading to swelling, inflammation, and dysfunction in the joints. Recently, research efforts have been focused on finding novel curative approaches for rheumatoid arthritis, as current therapies are associated with adverse effects. Here, we examined the effectiveness of dabigatran, the antithrombotic agent, in treating complete Freund's adjuvant (CFA)-induced arthritis in rats. Subcutaneous injection of a single 0.3 mL dosage of CFA into the rat's hind leg planter surface resulted in articular surface deformities, reduced cartilage thickness, loss of intercellular matrix, and inflammatory cell infiltration. There were also increased levels of the Anti-cyclic citrullinated peptide antibody (ACPA), oxidative stress, and tissue Receptor activator of nuclear factor-kappa B ligand (RANKL). Proteins of the kallikrein-kinin system (KKS) were also elevated. The inhibitory effects of dabigatran on thrombin led to a subsequent inhibition of KKS and reduced Toll-like receptor 4 (TLR4) expression. These effects also decreased RANKL levels and showed anti-inflammatory and antioxidant effects. Therefore, dabigatran could be a novel therapeutic strategy for arthritis.

Keywords: ACPA; Bradykinin; Kallikrein-Kinin system; RANKL; dabigatran; rheumatoid arthritis.

Figure 1

Histopathology. Photomicrographs of (a–c) knee joint samples of normal healthy rats showing well-organized intact chondrocytes inside lacuna (black arrow) in the presence of obvious demarcation between non-calcified and calcified zones (arrowhead); (d–f) knee joint sample of normal rats treated with dabigatran showing no anomalies; (g–i) knee joint samples of CFA-treated rats showing significant inflammatory cell infiltrates (yellow arrow) with a significant decrease in articular cartilage thickness; (j–l) knee joint samples of arthritic group treated with low dose of dabigatran showing minimized articular surface erosions and replacement with fibrous tissue (yellow arrow) with higher chondrogenic activity (black arrow); (m–o) knee joint samples of arthritic group treated with higher dose of dabigatran showing minor articular surface irregularities (yellow arrow), significant thickness recovery of articular cartilage (arrowhead), and the presence of mature chondrocytes all over articular surfaces (black arrow).

Figure 2

Effects of Dabigatran on serum ACPA and MDA levels. ELISA of serum ACPA levels (a), and MDA levels (b) in different groups as described in methods section. Not significant (ns); * p ≤ 0.05; **** p ≤ 0.0001, (n = 8).

Figure 3

Immunoblot analysis of tissue biomarkers. (a) Immunoblots of Kallikrein-1, LMW kininogen-1, Bradykinin RB2, and beta-Actin as a loading control. (b) Chemiluminescent analysis of band intensities of Kallikrein-1 in (a). (c) Chemiluminescent analysis of band intensities of LMW kininogen-1 in (a). (d) Chemiluminescent analysis of band intensities of Bradykinin RB2 in (a). (e) Heatmap depicting trend of changes in tissue biomarkers analyzed by immunoblots in (a). Not significant (ns); * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001, (n = 8).

Figure 4

Effects of Dabigatran on RANKL and TLR4 expression. qPCR analysis of tissue RANKL (a), and TLR4 levels (b) in different groups as described in methods section. ** p ≤ 0.01; **** p ≤ 0.0001, (n = 8).

Figure 5

Immunoblot analysis of tissue Collagen. (a) Immunoblots of Collagen-1 and beta-Actin as a loading control. (b) Chemiluminescent analysis of band intensities of Collagen-1 in (a). * p ≤ 0.05; **** p ≤ 0.0001, (n = 8).

Figure 6

Schematic illustration summarizing the anti-inflammatory/antiarthritic roles of Dabigatran.