Abstract
IntroductionOxidative stress plays a role in the pathogenesis of rheumatoid arthritis (RA). Anthocyanin is a plant antioxidant. We investigated the therapeutic effects of anthocyanin extracted from black soybean seed coats (AEBS) in a murine model of collagen-induced arthritis (CIA) and human peripheral blood mononuclear cells (PBMCs) and explored possible mechanisms by which AEBS might exert anti-arthritic effects.Material and MethodsCIA was induced in DBA/1J mice. Cytokine levels were measured via enzyme-linked immunosorbent assays. Joints were assessed in terms of arthritis incidence, clinical arthritis scores, and histological features. The extent of oxidative stress in affected joints was determined by measuring the levels of nitrotyrosine and inducible nitric oxide synthase. NF-κB activity was assayed by measuring the ratio of phosphorylated IκB to total IκB via Western blotting. Th17 cells were stained with antibodies against CD4, IL-17, and STAT3. Osteoclast formation was assessed via TRAP staining and measurement of osteoclast-specific mRNA levels.ResultsIn the CIA model, AEBS decreased the incidence of arthritis, histological inflammation, cartilage scores, and oxidative stress. AEBS reduced the levels of proinflammatory cytokines in affected joints of CIA mice and suppressed NF-κB signaling. AEBS decreased Th17 cell numbers in spleen of CIA mice. Additionally, AEBS repressed differentiation of Th17 cells and expression of Th17-associated genes in vitro, in both splenocytes of naïve DBA/1J mice and human PBMCs. In vitro, the numbers of both human and mouse tartrate-resistant acid phosphatase+ (TRAP) multinucleated cells fell, in a dose-dependent manner, upon addition of AEBS.ConclusionsThe anti-arthritic effects of AEBS were associated with decreases in Th17 cell numbers, and the levels of proinflammatory cytokines synthesized by such cells, mediated via suppression of NF-κB signaling. Additionally, AEBS suppressed osteoclastogenesis and reduced oxidative stress levels.
Highlights
Oxidative stress plays a role in the pathogenesis of rheumatoid arthritis (RA)
The anti-arthritic effects of anthocyanin extracted from black soybean seed coats (AEBS) were associated with decreases in Th17 cell numbers, and the levels of proinflammatory cytokines synthesized by such cells, mediated via suppression of NF-κB signaling
Since methotrexate has been introduced as a RA treatment agent, several conventional disease-modifying anti-rheumatic drugs (DMARDs) have been used to treat RA
Summary
Osteoclast formation was assessed via TRAP staining and measurement of osteoclast-specific mRNA levels. Male DBA/1J mice aged 4–6 weeks were purchased from Orient Bio (Seongnam, South Korea) and fed food and water ad libitum. DBA/1J mice, 7 weeks of age, were intradermally immunized (Day 0) at the base of the tail with 100 μg amounts of bovine type II collagen (CII; Chondrex Inc., Redmond, WA) suspended in complete Freund’s adjuvant (Mycobacterium tuberculosis (H37Ra) 4mg/ml; CFA, Chondrex Inc). To explore the effects of AEBS on CIA, DBA/1J mice were fed daily for 7 weeks with 60 mg/kg AEBS (dissolved in saline, or saline as a control), commencing on day 7 after primary immunization and keep feeding for 7 weeks, and were monitored for a total of 8 weeks. All scoring was performed by two independent observers blinded to the identities of the experimental and control groups
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