Helicobacter pylori infection induces inflammation in gastric epithelial cells; this may play a significant role in the pathological events associated with infection by this organism [1, 2]. H. pylori induce inflammatory-associated gene expression in gastric epithelial cells, including that for ROS, COX-2, iNOS, and IL-8 [3-7]. ROS cause oxidative damage to DNA, proteins and lipids. Production of excessive ROS/reactive nitrogen species reportedly occurs in H. pylori-infected human gastric mucosa and, because of the resultant serious imbalance between production of ROS/reactive nitrogen species and antioxidant defense, the amount of ROS correlates with the degree of histological damage in the gastric mucosa. Oxidative stress caused by H. pylori contributes to inflammation, apoptosis and carcinogenesis .
Reactive oxygen species induced by H. pylori stimulate MAPKs, such as ERK, JNK and p38, and up-regulate transcription of NF-κB. NF-κB is a crucial regulator of many cellular processes, including control of the immune response and inflammation. Co-culture of H. pylori with cells can induce IL-8 via activation of the transcriptional regulator NF-κB. IL-8, a potent neutrophil-activating chemokine, is central to the immunopathogenesis of H. pylori-induced tissue injury .
Anthocyanins, polyphenols derived from many of the colors of fruit and flowers , are novel, safe, proven antioxidants and chemopreventive agents [11, 12]. Their basic skeletons are made up of 2-phenylbenzopyrylium or flavylium glycoside. These pigments act as powerful antioxidants that help to protect plants from various factors such as UV light damage. A broad spectrum of in vitro and in vivo studies has demonstrated that anthocyanins promote antioxidant status, healthy vision and anti-angiogenic, antibacterial and anti-inflammatory properties . Anthocyanins also inhibit the growth of some cancer cells . Anthocyanins are especially abundant in the epidermis palisade layer of the black soybean seed coat, which contains cyanidin-3-glucoside, delphinidin-3-glucoside and petunidin-3-glucoside [10, 14-17]. Cyanidin-3-glucoside is reportedly an effective antioxidant that helps prevent cerebrovascular and cardiac disorders; in this regard it is superior to other components of anthocyanins . Although many studies have demonstrated the beneficial effects of anthocyanins on antioxidants, the anti-inflammation mechanism has not yet been clarified. Recently, Kim et al. reported that anthocyanins inhibit secretion of H. pylori CagA and VacA .
The goal of this study was to determine the anti-inflammatory effects of anthocyanins against experimental H. pylori infection. In order to increase their clinical potential in gastrointestinal disease, we also hoped to clarify the mechanism of their protective effects against H. pylori-induced inflammation.
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- MATERIALS AND METHODS
In the present study, we investigated the effects on the responsiveness of H. pylori infected-human gastric epithelial cells of anthocyanins from black soybean (Glycine max [L.] Merr). This potential effect of anthocyanins was investigated by exploring various variables that are usually associated with inflammation, namely, (i) increase of ROS upon stimulation with H. pylori; (ii) phosphorylation of three MAPKs: ERK, JNK and p38; (iii) modulation of NF-κB and Iκβα activity; (iv) degree of expression of COX-2 and iNOS; and (v) secretion of the pro-inflammatory cytokine IL-8.
It has been shown that H. pylori induces increased ROS, MAPK and NF-κB activation in AGS cells [4, 9, 22]. ROS may also play a role as the initial molecules in the signaling and regulation of gene expression associated with the biological effects elicited by inflammation. Anthocyanins can alleviate these effects by their antioxidant actions and by modulating the downstream pathway. Our experiments showed that pretreatment of AGS cells with anthocyanins decreases ROS production.
Helicobacter pylori-induced ROS generation can also activate diverse downstream signaling molecules, such as MAPK. Lin et al. reported that ROS and inflammatory cytokines were associated with MAPK activation . MAPK signaling is one of the most important signaling pathways in immune responses. Numerous researchers have reported that H. pylori up-regulates NF-κB activity through the MAPK signaling pathway [24-28]. Indeed, H. pylori phosphorylates all three types of MAPK (ERK, JNK and p38). Moreover, H. pylori activates NF-κB and p-Iκβα in AGS cells. However, anthocyanins suppress H. pylori-induced ROS expression and inhibit successive MAPK signaling.
Nuclear factor-kappa B is an oxidant-sensitive transcriptional factor and ROS have an important role in its activation in H. pylori-infected AGS cells . It has previously been shown that H. pylori decreases cytosolic amounts of NF-κB and increases p-Iκβα in AGS cells [7, 29]. In response to various inflammatory stimuli, Iκβα is degraded and the NF-κB complex migrates into the nucleus and binds DNA recognition sites in the regulatory regions of target genes, including those for iNOS and COX-2 [5, 30]. All of the activation mechanisms that lead to NF-κB translocation may involve ROS [31, 32]. These findings suggest that, under the influence of ROS, activated MAPK augments activation of NF-κB and subsequent transcription of COX-2, and that iNOS is mediated by activation of NF-κB in H. pylori-infected AGS cells.
In this study, anthocyanins did decrease activation of MAPK, NF-κB, iNOS and COX-2 according to western blot analysis and RT-PCR. Being powerful antioxidants , anthocyanins affect inflammation by blocking ROS at the initial step and regulating the NF-κB and MAPK signaling pathways activated by H. pylori. According to the present results, anthocyanins simultaneously inhibit activation by H. pylori of all three MAPKs. The definitive one of these three types of MAPK signal pathway is not yet clear; however, there is definitely signal interactions among the MAPKs .
Antioxidants such as curcumin and β-carotene reportedly inactivate NF-κB by inhibiting phosphorylation of Iκβα [32, 34]. Thus, we investigated whether anthocyanins can prevent Iκβα phosphorylation and found that anthocyanins reduce H. pylori-induced phosphorylation of Iκβα in a dose-dependent manner. Therefore, the prevention by anthocyanins of Iκβα decrease indicates inhibition of NF-κB activation by H. pylori in AGS cells.
Because IL-8 secreted by gastric epithelial cells is likely to be an important host mediator of induction of neutrophil migration into infection sites, it may be important in regulation of the inflammatory and immune processes in response to H. pylori . NF-κB has a dominant role in H. pylori-induced IL-8 production in gastric epithelial cells . In this study, we found that anthocyanins inhibit H. pylori-induced IL-8 secretion, thus augmenting their benefit in regard to protection of gastric epithelial cells.
In summary, this study shows that anthocyanins from black soybean have both antioxidant effects and the ability to down-regulate ROS generation and decrease the activation of MAPKs, NF-κB, iNOS and COX-2 that is induced by H. pylori (Fig. 6). In addition, anthocyanins decrease production of H. pylori-induced cytokines such as IL-8, suggesting that they may prevent gastric damage. As a consequence, anthocyanins can reduce H. pylori-induced inflammation in gastric epithelial cells.
Figure 6. Anthocyanins from black soybean inhibit Helicobacter pylori-induced ROS in human gastric epithelial AGS cells.
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