Aliskiren, tadalafil, and cinnamaldehyde alleviate joint destruction biomarkers; MMP-3 and RANKL; in complete Freund's adjuvant arthritis model: Downregulation of IL-6/JAK2/STAT3 signaling pathway

Abstract

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease, which is accompanied by progressive joint damage and disability. The intolerability of conventional antirheumatic drugs by some patients necessitates the search for effective antirheumatic agents having better tolerability. In the current work, we aimed to investigate the efficacy of cinnamaldehyde, tadalafil, and aliskiren as potential antirheumatic candidates and to explore their modulatory effects on joint destruction, inflammatory response, and intracellular signaling. Arthritis was induced in female Wistar rats by complete Freund's adjuvant (CFA) 0.4 ml s.c. on days 1, 4, and 7. Treated groups received their respective drugs, starting from day 13, daily for 3 weeks. Methotrexate and prednisolone were the standard antirheumatic drugs, while cinnamaldehyde, tadalafil, and aliskiren were the test agents. Treatment with cinnamaldehyde, tadalafil, or aliskiren reduced serum levels of rheumatoid factor, and pro-inflammatory cytokines; tumor necrosis factor-alpha and interleukin-6 (IL-6), along with elevated level of IL-10 which is an anti-inflammatory cytokine. Besides, cartilage and bone destruction biomarkers; matrix metalloproteinase-3 (MMP-3) and receptor activator of nuclear factor-kappa B ligand (RANKL); were significantly reduced after treatment with the test agents, which was further confirmed by histopathological investigation. The elevated protein expressions of phosphorylated-Janus kinase 2 (p-JAK2), phosphorylated-signal transducer and activator of transcription 3 (p-STAT3), and inducible nitric oxide synthase (iNOS) in articular tissue were markedly attenuated after treatment with cinnamaldehyde, tadalafil, or aliskiren, while that of endothelial nitric oxide synthase (eNOS) was greatly enhanced. In addition, oxidative stress and inflammatory markers such as malondialdehyde, nitric oxide, and myeloperoxidase were reduced in joint tissue after treatment with the test agents, while glutathione content was elevated. Furthermore, the renin inhibitor aliskiren produced effects close to those of the normal and methotrexate, the gold standard antirheumatic drug, in most of the measured parameters. Collectively, these findings led to the assumption that the downregulation of IL-6/JAK2/STAT3 signaling by cinnamaldehyde, tadalafil, and aliskiren could alleviate joint destruction by MMP-3 and RANKL, reduce iNOS, and enhance eNOS expressions. Moreover, aliskiren could be a promising therapeutic agent for RA, because of its ability to normalize most of the measured parameters after CFA-induced arthritis.

Keywords: Aliskiren; CFA, complete Freund's adjuvant; CFA-induced arthritis; DMARD, disease-modifying antirheumatic drug; GSH, reduced glutathione; H&E, hematoxylin and eosin; IL-10, interleukin-10; IL-6, interleukin-6; IL-6/JAK2/STAT3 signaling; JAK2, Janus kinase 2; MDA, malondialdehyde; MMP-3; MMP-3, matrix metalloproteinase-3; MPO, myeloperoxidase; NO, nitric oxide; PDE, phosphodiesterase; RA, rheumatoid arthritis; RANKL; RANKL, receptor activator of nuclear factor-kappa B ligand; RAS, renin angiotensin system; STAT3, signal transducer and activator of transcription 3; TNF-α, tumor necrosis factor-alpha; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase.

Fig. 1

Effect on rheumatoid factor. Arthritic control group showed a marked elevation in serum level of RF, while treatment with methotrexate, prednisolone, cinnamaldehyde, tadalafil, or aliskiren significantly reduced it. Each column represents the mean of 8 rats ± SE. ^avs normal, ^bvs arthritic control, ^cvs methotrexate, and ^dvs prednisolone at p < 0.05.

Fig. 2

Effect on pro-inflammatory/anti-inflammatory cytokine balance. Levels of the pro-inflammatory cytokines IL-6 (A) and TNF-α (B) were significantly elevated after induction of arthritis by CFA, but these elevations were reduced after treatment with the test agents. On the other hand, level of the anti-inflammatory cytokine IL-10 (C) was reduced in arthritic control but elevated in treated groups. Each column represents the mean of 8 rats ± SE. ^avs normal, ^bvs arthritic control, ^cvs methotrexate, and ^dvs prednisolone at p < 0.05.

Fig. 3

Effect on markers of joint destruction. The levels of MMP-3 (A) and RANKL (B) were markedly elevated after induction of arthritis by CFA, but these elevations were significantly reduced after treatment with the test agents. Each column represents the mean of 8 rats ± SE. ^avs normal, ^bvs arthritic control, ^cvs methotrexate, and ^dvs prednisolone at p < 0.05.

Fig. 4

Effect on joint tissue oxidative stress and inflammatory markers. Articular tissue contents of MDA (A), GSH (B), MPO (C), and NO (D). MDA, MPO, and NO were significantly elevated in arthritic control group, while treatment with methotrexate, prednisolone, cinnamaldehyde, tadalafil, or aliskiren significantly reduced these parameters. On the other hand, the antioxidant GSH was reduced in arthritic control, but the reduction was ameliorated in treated groups. Each column represents the mean of 8 rats ± SE. ^avs normal, ^bvs arthritic control, ^cvs methotrexate, and ^dvs prednisolone at p < 0.05.

Fig. 5

Effect on protein expressions of p-JAK2, p-STAT3, iNOS, and eNOS. Western blots demonstrating the changes in protein expressions of p-JAK2, p-STAT3, iNOS, and eNOS (A). Graphical presentation for the relative quantification of p-JAK2 (B), p-STAT3 (C), iNOS (D), and eNOS (E). Protein expressions of p-JAK2, p-STAT3, and iNOS were significantly upregulated in arthritic control group, while downregulated in methotrexate, prednisolone, cinnamaldehyde, tadalafil, and aliskiren treated groups. Contrarily, the protein expression of eNOS was reduced in arthritic control, while enhanced in treated groups. ^avs normal, ^bvs arthritic control, ^cvs methotrexate, and ^dvs prednisolone at p < 0.05.

Fig. 6

Effect on histopathological alterations. Hematoxylin and eosin stained articular tissue sections of: Normal group (A) showing no inflammation and normal histological structure of the joint (bone, cartilage and fibrous joint capsule). Arthritic control (B) showing marked histopathological changes (+++) in the form of synovial hyperplasia with inflammatory cells infiltration, pannus formation and erosion in cartilage and bone. The groups of the standard antirheumatic drugs, methotrexate and prednisolone (C, D), showing mild degree (+) of articular changes. Cinnamaldehyde, tadalafil, and aliskiren treated groups (E, F, G) showing mild (+) articular changes for cinnamaldehyde and aliskiren and moderate (++) changes for tadalafil.