Grape seed proanthocyanidin extract (GSPE) attenuates collagen-induced arthritis

Abstract

To examine whether grape seed proanthocyanidin extract (GSPE) which is known to act as an antioxidant has therapeutic effect on collagen-induced arthritis (CIA) in mice, an animal model of rheumatoid arthritis. Mice were treated with an intraperitoneal injection of GSPE (10, 50, or 100 mg/kg) or saline. Clinical, histological, and biochemical parameters were assessed. The effects of GSPE on osteoclastogenesis were determined by tartrate-resistant acid phosphatase (TRAP) staining of the inflamed joints and bone-marrow cells cultured with the receptor activator of nuclear factor B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Intracellular levels of hydrogen peroxide were determined using carboxy-dichlorodihydrofluorescein diacetate. GSPE treatment significantly attenuated the severity of CIA in a dose-dependent manner and reduced the histology scores for synovial inflammation, cartilage erosion, bone erosion, and the number of TRAP⁺ osteoclasts. GSPE treatment significantly reduced the numbers of tumor necrosis factor alpha (TNF-α)- or interleukin 17 (IL-17)-producing cells in the synovial tissue and the spontaneous production of TNF-α and IL-17 by splenocytes compared with those in the control mice. The serum levels of type-II-collagen-specific IgG2a and plasma levels of 8-isoprostane in the GSPE-treated mice were significantly lower than those in the control mice. GSPE dose-dependently suppressed osteoclastogenesis in vitro. GSPE significantly reduced hydrogen peroxide production by anti-CD3-monoclonal-antibody-stimulated CD4⁺ splenocytes. These results indicate that intraperitoneal injection of GSPE attenuated CIA in mice. GSPE may be useful in the treatment of rheumatoid arthritis.

Introduction

Reactive oxygen species (ROS) such as the superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), and the hydroxyl radical, are continuously generated during normal cell metabolism. ROS can act as a physiological defense system against microbial infection and are involved in maintaining normal cellular functions, including proliferation, apoptosis, and intracellular signal transduction [1]. However, excessive amounts of ROS can damage lipids, proteins, DNA, and the extracellular matrix [2]. Therefore, ROS must be scavenged by antioxidants. Enzymatic antioxidants include superoxide dismutase, glutathione peroxidase, catalase, and thioredoxin reductase. Nonenzymatic antioxidants include glutathione, vitamin A, vitamin C, and vitamin E [3]. Oxidative stress refers to the cellular state in which the production of ROS is elevated and/or the levels of antioxidants reduced. Oxidative stress is considered to be involved in the pathogenesis of atherosclerosis, cancer, neurodegenerative disorders, diabetes, ageing, and autoimmune rheumatological diseases, including rheumatoid arthritis (RA), systemic lupus erythematosus, and systemic sclerosis [3].

RA is a chronic inflammatory autoimmune disease characterized by synovitis, bone destruction with pannus formation, and the degradation of the articular cartilage. The cause of RA is unknown, but several studies have suggested that oxidative stress is associated with the pathogenesis of RA. Epidemiological studies have demonstrated an inverse correlation between the dietary intake of antioxidants and the incidence of RA [4]. Inverse associations between serum antioxidant levels and the risk of developing RA have been reported [5]. The disease activity also correlates inversely with antioxidant levels and positively with the presence of oxidative stress in patients with RA [6], [7]. Increased oxidative enzyme activity has been demonstrated, together with reduced enzymatic and nonenzymatic antioxidant levels, in RA sera and synovial fluids [8]. Oxidative damage to proteins, lipids, DNA, cartilage, and extracellular collagen has been demonstrated in patients with RA [9]. Some drugs used to treat RA such as methotrexate, etancercept and infliximab are known to play essential roles as antioxidative agents [1]. Lipid peroxidation markers such as serum malondialdehyde and urine isoprostane are reported to be elevated in collagen-induced arthritis (CIA) compared with those in controls [10], [11]. The beneficial effects of antioxidants, including vitamin E, tempol, α-lipoic acid, N-acetylcysteine, the polyphenolic fraction of green tea, and (−)-epigallocatechin-3-gallate, have been demonstrated in mice with CIA [12], [13], [14], [15], [16], [17].

Grape seed extract is a natural plant constituent and contains lipids, proteins, carbohydrates, and polyphenols. Proanthocyanidins are the most abundant phenolic compounds in grape seeds, and are high-molecular-weight polymers comprised of dimers or trimers of (+)-catechin and (−)-epicatechin [18]. Grape seed proanthocyanidin extract (GSPE) has more powerful antioxidative activity than other well-known antioxidants, including vitamin C, vitamin E, and gallic acid [19]. GSPE has various biological functions such as antibacterial, antiviral, anti-inflammatory, antiallergic, and vasodilatory actions [20].

It has been suggested that GSPE is an effective therapy for oxidation-related diseases in animal models, including tumors, atherosclerosis, gastric ulcer, cataract, and diabetic retinopathy [21]. The beneficial effects of GSPE in reducing oxidized low-density lipoproteins and postprandial oxidative stress and improving chloasma have been demonstrated in human clinical trials [22], [23], [24]. However, little is known about the effects of GSPE on CIA and rheumatoid arthritis. To examine whether GSPE has therapeutic potentials in the treatment of RA, we examined the effects of GSPE on the severity of CIA in DBA/1 mice, using clinical, histological, and biochemical parameters. We found that GSPE treatment attenuates the severity of CIA in mice, reducing serum levels of type-II-collagen-specific IgG2a, inflammatory cytokine production, oxidative stress, bone destruction in vivo, and osteoclastogenesis in vitro.