Mast cells are emerging key players in the erosive and inflammatory events leading to joint destruction in inflammatory arthritis. Accumulation of mast cells in rheumatoid synovial tissue and their activation and degradation associated with pro-inflammatory cytokines and matrix degrading enzymes at cartilage erosion sites suggest that they could be usefully selected as a therapeutic target . Mast cells are known to play a central role in inflammatory diseases as these cells contain potent inflammatory mediators, including histamine, heparin, proteinases, leukotrienes, and multifunctional cytokines, and their potential contributions to processes of inflammation and matrix degradation have become evident [27, 28]. In response to diverse stimuli, human basophilic KU812 cells release an array of inflammatory cytokines and chemokines especially IL-6 and IL-8 which have the potential to cause inflammation and tissue remodeling . IL-6 is a multifunctional cytokine that plays a key role in immune response, growth and differentiation of B- and T-cells, hematopoiesis and the induction of hepatic acute phase plasma proteins . Increased tissue levels of IL-6 in diseases like arthritis , psoriasis , scleroderma , and delayed pressure urticaria have been demonstrated . Binding of IL-6 to its receptor is known to induce IL-6-dependent signal transduction in mast cells and basophils . IL-6 is also an important co-factor in IL-4 dependent IgE synthesis. IL-6 is also produced by mast cells and basophils and its local accumulation in arthritic joints is associated with the chronic response . IL-8 is best known for its potent chemoattractant activity on neutrophils and T-cells and their functions . Like IL-6, IL-8 has been shown to be increased in various inflammatory diseases, including arthritis, that are characterized by elevated mast cells number . Interestingly, many reports have shown that human mast cells and basophils secrete both IL-6 and IL-8 [37, 38].
Polyphenols are plant molecules entering in our bodies through diet. The relationship between polyphenol-rich food consumption and reduced possibility of being affected by some diseases has attracted increasing interest from consumers, food manufacturers and nutritional scientists. Fruit and vegetable consumption may prevent cancers  and stroke , whereas wine consumption may have similar effect in preventing coronary heart disease , and prostate cancer . Soy consumption may have protective effects against cancerous cells  and osteoporosis  and tea polyphenols may prevent different cancers  and arthritis . Pomegranate fruit is a rich source of polyphenols. Hydrolysable tannins are predominant polyphenols found in pomegranate juice and account for 92% of its antioxidant activity . Pomegranate seeds are rich in sugars, polyunsaturated fatty acids, vitamins, polysaccharides, polyphenols, and minerals and have high antioxidant activity. When crushed and dried, the pomegranate seeds produce an oil with 80% punicic acid, the 18-carbon fatty acid, along with the isoflavone genistein, the phytoestrogen coumestrol, and the sex steroid estrone. The seed coat of the fruit contains delphinidin-3-glucoside, delphinidin-3,5-diglucoside, cyanidin-3-glucoside, cyanidin-3,5-diglucoside, pelargonidin-3-glucoside, and pelargonidin-3,5-diglucoside with delphinidin-3,5-diglucoside being the major anthocyanin in pomegranate juice . Pomegranate fruit extract are also rich in oligomers which upon hydrolysis produce ellagic acid, which is a potent antioxidant, anticancer and antiatherosclerotic agent [22, 49, 50]. Studies have also shown that the antioxidant capacity of pomegranate juice is three times that of the popular antioxidant-containing beverages such as red wine and green tea, presumably due to the presence of hydrolyzable tannins in the rind, along with anthocyanins and ellagic acid derivatives . In a comparative analysis, anthocyanins from pomegranate fruit were also shown to possess higher antioxidant activity than vitamin-E (α-tocopherol), ascorbic acid and β-carotene . Pomegranate extract has also been shown to protect from NSAID and ethanol-induced gastric ulceration . Repeated administration of high doses of a hydroalcoholic extract of pomegranate whole fruit or its constituent ellagitannin punicalagin were non toxic in the dosages commonly employed in traditional medicine systems [53, 54]. Flavonoid rich fractions of pomegranate fruit extract have also been shown to exert antiperoxidative effect as their administration significantly decreased the concentrations of malondialdehyde, hydroperoxides and enhanced the activities of catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase in the liver [55, 56]. We recently reported in vivo efficacy of pomegranate constituents and/or their metabolites that become bioavailable after oral ingestion pomegranate fruit extract . Here we show that POMx, a hydrolysable tannin rich extract of pomegranate, inhibited the gene expression and production of pro-inflammatory cytokines IL-6 and IL-8 by a human mast cell – like KU812 cells. The MAPK cascade is one of the important signaling pathways in an inflammatory response . The signaling pathways characterized by MAPKs p38, ERK, and JNK, are known to play a potential role in the regulation of inflammatory response . They are the key players in the molecular and cellular events associated with the pathogenesis of inflammatory arthritis and are being studied as a rational target of drug design for arthritis therapy . In the present study, POMx specifically inhibited the PMACI-induced activation of JNKp54/p46- and ERKp44/p42-sub-groups of MAPK and inhibited the production of IL-6 and IL-8 by KU812 cells. In addition, JNK- and ERK-specific inhibitors, SP600125 and PD98059 also reduced IL-6 and IL-8 gene expression, respectively, in KU812 cells. These data suggest that compounds present in POMx have the potential to inhibit the inflammatory stimuli-induced JNK- and ERK-MAPK activation and inhibit the downstream IL-6 and IL-8 gene and protein expression.
Activation of the master transcription factor NF-κB leads to the coordinated expression of many genes that encode cytokines, chemokines, enzymes, and adhesion molecules involved in mediator synthesis and the further amplification and perpetuation of the inflammatory reaction . Expression of IL-6 and IL-8 gene is dependent on the activation of transcription factor NF-κB . Because suppression of NF-κB activation has been linked with anti-inflammatary activity, we postulated that POMx mediates its inhibitory effects on IL-6 and IL-8 expressions at least in part, through the suppression of NF-κB activity. Activation of NF-κB requires phosphorylation and proteolytic degradation of the inhibitory protein IκBα, an endogenous inhibitor that binds to NF-κB in the cytoplasm and its degradation expose the nuclear localization signal (NLS) and allows the NF-κB to translocate to the nucleus and bind the promoter of target genes . In PMA plus A23187-stimulated KU812 cells, POMx inhibited the degradation of IκBα and nuclear translocation of the p65 NF-κB (Fig. 4A & 4B). In addition, DNA binding activity of NF-κB as demonstrated by the reporter assays (Fig. 4C) was also inhibited in these cells. These data indicate that POMx attenuates the inflammatory stimuli-induced activation and DNA binding activity of NF-κB in KU812 cells. As IL-6 and IL-8 genes are NF-κB dependent genes, this also inhibit their expression and production in PMACI-stimulated KU812 cells.
Cytokines produced by mast cell and basophils are associated with the progress of inflammation. Both basophils and mast cells play a major role in the pathogenesis of inflammatory diseases by releasing several pro-inflammatory mediators. Our results suggest that POMx may regulate the pro-inflammatory cytokine expression and production by mast cells through different mechanisms. In view of the increasing prevalence of allergic and inflammatory diseases such as arthritis, asthma, allergic rhinitis, and eczema worldwide [16, 28], there is a need for novel and safe treatment and or prevention option for the underlying inflammation caused by activation of mast cells and basophils . Mast cells play different roles in the inflammation by the release of various chemokines and cytokines via different intracellular signal transduction pathways . The results obtained in this study provide new evidence that POMx may contribute to the prevention and/or treatment of inflammatory diseases by inhibiting the activation of mast cells.
There is evidence from several studies that supplementation with POMx improves inflammatory symptoms in vivo and in vitro [21, 48–52, 56]. However, the molecular pharmacological basis for the observed effects has not been fully uncovered yet. Direct inhibitory effects of plant extracts or components in other systems have been reported [63, 64], but few have addressed the question of bioavailability and activity of bioavailable constituents. In this regard results reported by Schaffer, et al  are important as they showed that after oral ingestion of pycnogenol human plasma contained bioactive compounds that inhibited the activity of COX-1 and COX-2 in an in vitro assay. We also wish to point out that the in vivo efficacy of the extract used here has already been shown by us in an animal model of inflammatory arthritis  indicating that after oral consumption pomegranate metabolites can exert anti-inflammatory effect in vivo. This gets strength from our studies showing that after oral consumption of a pomegranate extract, its constituents/metabolites become bioavailable and inhibit COX-2 activity, PGE2 and NO production in chondrocytes . It is well documented that fruit or plant extracts are a complex mixture of various constituents and in most of the instances it is still not clear whether a single compound or a mixture of compounds is responsible for the reported effects . However, evidence is accumulating that often related compounds present in a fruit or herb extract augment each other's biological effect. For example, it has been reported that ellagic acid and quercetin (both are also present in pomegranate) together exert a more pronounced inhibitory effect against cancer cell growth than either compound alone .