Rheumatoid arthritis (RA) is a chronic inflammatory disease of the connective tissue and is characterized by synovial hyperplasia with local invasion of bone and cartilage. The disease is largely driven by the recruitment of activated T cells and B cells and macrophages to the afflicted joints. Pro-inflammatory cytokines such as IL-6, TNF-α produced by these activated cells contribute to the irreversible joint damage seen in RA. In view of the historical use and currently reported anti-inflammatory effects of pomegranate extract in several model systems (14
), we explored the potential of consumption of POMx for preventing joint damage in RA. In this study we demonstrate that consumption of POMx robustly prevents the development of CIA in mice (an animal model of RA) by selectively inhibiting a spectrum of signal transduction pathways and cytokines critical to the development and maintenance of inflammation in RA. Our results demonstrate that consumption of POMx reduces the incidence and severity of CIA in mice (). We also demonstrate that POMx abrogates the phosphorylation of JNK and production of pro-inflammatory cytokines and inhibits cytokine-induced NO production in mouse macrophages. The c-Jun N-terminal protein kinase mitogen-activated protein kinases (JNK) is a sub-group of the evolutionarily-conserved family of serine/threonine protein kinases. JNK is activated by treatment of cells with inflammatory cytokines (such as TNF-α) and by exposure of cells to endotoxin. Previous studies have demonstrated that inhibition of JNK inhibits joint damage in inflammatory arthritis (36
). Thus our in vitro
and in vivo
data collectively indicate that POMx potently inhibits a range of cellular responses, including the activation of JNK, that play critical roles in driving synovitis, pannus formation, and joint destruction in RA. In all experiments there were no signs of toxicity in any of the groups as evidenced by weight gain (), ALT or serum creatinine levels (data not shown). No mortality due to POMx in any of the groups was observed.
Dietary supplements are taken orally and hence our use of gavage feeding was chosen to maximize the delivery so that bioactivity of the administered dose could be determined independently of other factors. Examination of the data reveals that this regimen was effective in protecting mice from the development of CIA as statistically significant arthritis inhibition and joint sparing effects were demonstrated in POMx-fed mice compared to water-fed mice ( & ). These results provide the first in vivo evidence of antiarthritic efficacy of a chemically complex mixture of pomegranate fruit that is commercially available. Because in these studies pretreatment was required to inhibit the incidence and severity of joint inflammation, these data support the use of POMx for arthritis prevention and not for arthritis treatment in the face of active inflammation. Future studies will address the disease modifying aspect of POMx consumption.
Macrophages are present in RA joints and play an important role in the pathogenesis of RA (reviewed in 37
). These activated cells produce TNF-α, NO and other inflammatory mediators that exacerbate RA. Using biochemical techniques and immunoblotting we demonstrate that POMx inhibits LPS-induced activation of the JNK and NF-κB signal transduction pathways and downstream activation of transcription factors and the production of NO in mouse macrophages. NO is synthesized from L-arginine by the enzyme NO synthase (NOS). After induction of the inducible isoform of NOS (iNOS) by endotoxin or inflammatory cytokines, NO is overproduced resulting in the cyto- and bacteriotoxic effects. Role of NO in inflammation is also evident from its inhibitory effects on T cell activity, chemotaxis of monocytes and migration of neutrophils, production of PGE2
and thromboxane in macrophages (reviewed in 38
). Studies with animal models of inflammatory arthritis and in patients with rheumatoid arthritis have demonstrated an important role of high levels of NO in the pathogenesis of the disease (38
). Our novel data presented here suggest thats POMx-mediated inhibition of macrophages could contribute to its efficacy in inhibiting CIA as shown in this study and possibly in human RA.
Transcription factors NF-κB are ubiquitously expressed and can both induce and repress gene expression by binding to κB elements present in the promoters of target genes including the genes that control apoptosis, adhesion, inflammation, and tissue remodeling (reviewed in 39
). Activation of NF-κB involves the phosphorylation of its inhibitory protein IκB by IκB kinase (IKK) complex which results in its degradation and release of NF-κB. It has been shown that the incidence and severity of CIA were decreased significantly in transgenic mice constitutively expressing IκB compared with nontransgenic littermates (40
). Similar results were obtained when mice immunized to get CIA were treated with a small molecule inhibitor of NF-κB (41
). Attenuation of murine CIA by administration of a selective small molecule inhibitor of NF-κB correlated with the reduction in the levels of inflammatory cytokines such as IL-6 in the paw tissues of arthritic mice (42
). These data provide support to the results presented here showing that POMx inhibited the endotoxin-induced activation of NF-κB in mouse macrophages. Significantly low levels of IL-6 detected in the arthritic joints of POMx-fed mice compared to control mice (P
<0.005) may be related to the in vivo inhibitory effect of POMx on NF-κB activation. However, this remains to be investigated.
Additionally, low levels of inflammatory cytokines detected in the arthritic joints of the POMx-fed mice also support the hypothesis that the observed reduction in joint inflammation scores was likely due to the inhibition of inflammatory cytokine production in the joints. In contrast to the ineffectiveness of high dose POMx on the production of IL-1β in arthritic joints, low dose feeding of POMx was effective in suppressing the production of IL-1β () while high dose was needed to suppress the production of TNF-α (). These results suggest that the constituents of POMx are needed in different concentrations to suppress the damaging effects of IL-1β and TNF-α in arthritis and other diseases associated with abnormal production of these cytokines. Identification of these components of POMx will open new avenues for the development of effective preventive therapies against arthritis. Moreover, our demonstration of arthritis preventive and joint protective effects of POMx consumption provide support to the use of POMx or pomegranate containing dietary supplements for the prevention of inflammatory arthritis.