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Anti-inflammatory effect of chlorogenic acid in intestinal epithelial cells SHIN, Hee Soon (Korea Food Research Institute) Chapter 1 Anti-inflammatory effect of chlorogenic aicd in vitro and in vivo Section 1-1 Synergistic effect of tumor necrosis factor-alpha and hydrogen peroxide on the induction of IL-8 production in human intestinal Caco-2 cells Oxidative stress and inflammatory cytokines such as TNF- are thought to be involved in mucosal inflammation. The intestinal epithelium may be concurrently stimulated by both oxidative stress and inflammatory cytokines during the inflammation process in the intestines. However, experimental models for intestinal inflammation still employ single stimulation, either by an oxidative stress or inflammatory cytokine. We therefore examined an in vitro inflammation study using human intestinal epithelial Caco-2 cells, in which the cells were stimulated both by hydrogen peroxide and TNF- and measured the IL-8 production as an index of inflammation. The IL-8 production (secretion, mRNA expression, and transcriptional activity) induced by both TNF- and H2O2 was significantly higher than that by single stimulation. The synergistic effect of TNF- and H2O2 on the NF-B signaling pathway (transcriptional activity and p65 nuclear translocation) was also observed. Oxidative stress and TNF- may cooperatively enhance IL-8 production via NF-B in Caco-2 cells. Section 1-2 Anti-inflammatory effects of chlorogenic acid and caffeic acid on the IL-8 production induced by combined stimuli of both tumor necrosis factor-alpha and hydrogen peroxide in human intestinal Caco-2 cells and on the dextran sulfate sodium-induced colitis in mice Pro-inflammatory cytokines and oxidative stress have been shown to play a pivotal role in the onset of inflammatory bowel diseases (IBDs). We evaluated the effect of dietary polyphenol, chlorogenic acid (CHA) and its metabolites caffeic acid (CA) and quinic acid (QA), on the IL-8 production induced by combined stimuli of both tumor necrosis factor alpha (TNF-) and hydrogen peroxide (H2O2) in human intestinal Caco-2 cells. As a result, CHA and its metabolite CA, but not QA, inhibited IL-8 production (secretion, mRNA expression, and transcriptional activity) induced by TNF- and H2O2. We also examined the effects of CHA and CA on the dextran sulfate sodium (DSS)-induced colitis in mice. Female C57BL/6 mice were treated as oral administration with 1mM (354 mg/kg diet, 3.54 mg/kg BW) CHA and 1mM (180 mg/kg diet, 1.8 mg/kg BW) CA for 15 days, and given 3% DSS in drinking water during for last 8 days to induce colitis. Dietary CHA and CA attenuated DSS-induced body weight loss, diarrhea, fetal bleeding, and shortening of the colon, and dramatically improved colitis histological scores. Further, DSS-induced increases in colonic MIP-2 and IL-1 mRNA expression were significantly suppressed by CHA and CA supplementation. In conclusion, the present study showed that dietary CHA and CA, its metabolite, inhibited both TNF- and H2O2-induced IL-8 production in vitro, and attenuated DSS-induced colitis in vivo. These indicate that CHA and CA can be important anti-inflammatory factors, and use of them may lead to prevention of intestinal inflammation like IBDs. Chapter 2 Anti-inflammatory mechanism of chlorogenic acid and caffeic acid in intestinal epithelial cells Section 2-1 Anti-inflammatory mechanism of chlorogenic acid and caffeic acid on the hydrogen peroxide-induced IL-8 production through scavenging intracellular reactive oxygen species (ROS) generation in human intestinal Caco-2 cells Reactive oxygen species (ROS) generation induced by oxidative stress leads to serious cellular injuries and the pathogenesis of several diseases. In intestinal epithelial cells, hydrogen peroxide (H2O2) induces the interleukine-8 (IL-8), pro-inflammatory cytokine, which plays a key role in the pathogenesis of inflammatory bowel diseases (IBDs), encompassing Crohn`s disease and ulcerative colitis. Chlorogenic acid (CHA) its metabolite caffeic acid (CA), antioxidants, often are exposed and absorbed in intestinal epithelial cells. We therefore investigated the effects of CHA and CA on the H2O2-induced IL-8 production in human intestinal epithelial Caco-2 cells. CHA and CA inhibited the IL-8 transcriptional activity induced by H2O2. They also significantly suppressed NF-B transcriptional activity, nuclear translocation of p65 subunit, and phosphorylations of IB Kinase and protein kinase D, up-stream signaling pathway of IL-8 production, induced by H2O2. Also, we examined intracellular ROS generation, and we found that CHA and CA scavenged H2O2-induced intracellular ROS generation. We suggest that the anti-inflammatory effects of CHA and CA were caused by scavenging capacity against H2O2-induced intracellular ROS generation in human intestinal epithelial cells. Section 2-2 Anti-inflammatory effect of chlorogenic acid and caffeic acid derivatives on the hydrogen peroxide-induced IL-8 production in human intestinal Caco-2 cells: Structure-activity relationships The antioxidant activity of phenolic compounds has attracted much attention in relation to their physiological functions such as prevention of coronary heart disease, cancer and inflammation. Our results showed that CHA and CA, phenolic compounds, inhibited H2O2-induced IL-8 production through scavenging intracellular ROS generation. These results mean that the anti-inflammatory effects of CHA and CA may be derived from their anti-oxidative effects. Generally, the radical scavenging activity of phenolic compounds has been known to depend on their molecular structure. We investigated the functional moieties of anti-inflammatory effects of CHA and CA using CA derivatives by structure-activity relationships. We found that anti-inflammatory effects of CHA and CA depended on catechol group through comparing phenolic compounds containing catechol group with them of non-containing catechol group. And also, the forces of inhibitory effect on the IL-8 production were sequentially presented DCA ≦ CHA < CA < PCA. We therefore suggest that although the anti-inflammatory activities of phenolic acids did not completely conform to the role of anti-oxidative activities, the capacities may be related lipid anti-oxidative activities, and CHA, CA, PCA and DCA, phenolic acids containing catechol group, can act as anti-inflammatory agents by inhibiting IL-8 production in human intestinal epithelial cells. <in vitro study> IL-8 Chlorogenic acid <in vivo study> Oxidative stress TNF- DSS誘導大腸炎マウスモデル membrane cytosol OH O P HO P IKK OH β α P O C HO HC OH CH O OH Chlorogenic acid NFB OH O P C P HO Chlorogenic acid OH O C HO nuclear C/EBP AP-1 HC CH OH O OH p50 P p65 P IL-8 S DS PKD Catechol group C IL-8 mRNA P ROS 3% P IB IL-1 mRNA NFB <腸管におけるクロロゲン酸の抗炎症作用> inflammation MIP-2 mRNA