Hideo Satsu

Anti-inflammatory effect of chlorogenic acid on the IL-8 production in Caco-2 cells and the dextran sulphate sodium-induced colitis symptoms in C57BL/6 mice

Chlorogenic acid (CHA) is an antioxidant polyphenol prevalent in human diet, with coffee, fruits, and vegetables being its main source. Effects of CHA and CHA metabolites were evaluated on the IL-8 production in human intestinal Caco-2 cells induced by combined stimulation with tumour necrosis factor alpha (TNFα) and H2O2. CHA and caffeic acid (CA) inhibited TNFα- and H2O2-induced IL-8 production. We also examined the in vivo effects of CHA and CA using dextran sulphate sodium (DSS)-induced colitis in mice. CHA attenuated DSS-induced body weight loss, diarrhea, fecal blood, and shortening of colon and dramatically improved colitis histological scores. Furthermore, increases in the mRNA expression of colonic macrophage inflammatory protein 2 and IL-1β, which were induced by DSS, were significantly suppressed by CHA supplementation. These results suggest that dietary CHA use may aid in the prevention of intestinal inflammatory conditions.

Histidine inhibits oxidative stress‐ and TNF‐α‐induced interleukin‐8 secretion in intestinal epithelial cells

We investigated the effect of several amino acids on the secretion of such inflammatory cytokines as interleukin‐8 (IL‐8) induced by hydrogen peroxide or tumor necrosis factor‐alpha (TNF‐α) in intestinal epithelial‐like Caco‐2 and HT‐29 cells. We found that histidine, one of the conditionally essential amino acids, significantly inhibited both hydrogen peroxide‐ and TNF‐α‐induced IL‐8 secretion and mRNA expression in Caco‐2 cells and HT‐29 cells. These inhibitions were dose dependent and the inhibition rate of hydrogen peroxide‐induced IL‐8 secretion reached more than 50% at a concentration of 25 mM, with over 95% inhibition at a concentration of 50 mM. TNF‐α increased the transcriptional activity of the IL‐8 promoter which was significantly inhibited by treating Caco‐2 cells with histidine. Histidine also abolished the NF‐κB‐dependent activation of the IL‐8 promoter induced by TNF‐α. These results indicate that histidine inhibited the hydrogen peroxide‐ and TNF‐α‐induced IL‐8 secretion at the transcriptional level in intestinal epithelial cells, suggesting that histidine has the potential to attenuate intestinal inflammation.

Producibility and digestibility of antihypertensive beta-casein tripeptides, Val-Pro-Pro and Ile-Pro-Pro, in the gastrointestinal tract: analyses using an in vitro model of mammalian gastrointestinal digestion.

Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) are antihypertensive tripeptides isolated from milk fermented with Lactobacillus helveticus and inhibit angiotensin-converting enzyme (ACE). We investigated whether these peptides were generated from beta-casein by digestive enzymes and whether they were resistant to enzymatic hydrolysis, using an in vitro model. VPP and IPP were not generated from beta-casein by gastrointestinal enzymes; instead, a number of longer peptides including VPP and IPP sequences were detected. The fermentation step would therefore be necessary to produce these antihypertensive tripeptides. VPP and IPP themselves were hardly digested by digestive enzymes, suggesting that orally administered VPP and IPP remain intact in the intestine, retaining their activity until adsorption. The present study also demonstrated that various functional peptide sequences in beta-casein were resistant to gastrointestinal enzymes. There may be a strong correlation between the resistance of peptides to gastrointestinal digestion and their real physiological effects after oral administration.

Activation of pregnane X receptor and induction of MDR1 by dietary phytochemicals.

The pregnane X receptor (PXR) is understood to be the key regulator for gene expression of such drug-metabolizing enzymes and transporters as multidrug-resistant protein 1 (MDR1) and the cytochrome P450 (CYP) family. We examined the effect of dietary phytochemicals on the PXR-dependent transcriptional activity in human intestinal LS180 cells by using a reporter assay. Among approximately 40 kinds of phytochemicals, tangeretin and ginkgolides A and B markedly induced the PXR-dependent transcriptional activity and also the activity of the human MDR1 promoter. The expression levels of MDR1 mRNA as well as of CYP3A4 mRNA, another gene regulated by PXR, were significantly increased by these phytochemicals. Furthermore, an increase was observed of the MDR1 protein and its functional activity by tangeretin and by ginkgolides A and B. These findings strongly suggest that tangeretin and ginkgolides A and B activated PXR, thereby regulating detoxification enzymes and transporters in the intestines.

5-Caffeoylquinic Acid and Caffeic Acid Down-Regulate the Oxidative Stress- and TNF-α-Induced Secretion of Interleukin-8 from Caco-2 Cells

Although chlorogenic acid (CHA) easily reaches a millimolar level in the gastrointestinal tract because of its high concentration in coffee and fruits, its effects on intestinal epithelial cells have been little reported. We investigated in this study the down-regulative effects of 5-caffeoylquinic acid (CQA), the predominant isomer of CHA, on the H(2)O(2-) or TNF-alpha-induced secretion of interleukin (IL)-8, a central pro-inflammatory chemokine involved in the pathogenesis of inflammatory bowel diseases, in human intestinal epithelial Caco-2 cells. After the cells had been pre- and simultaneously treated with CQA, the oversecretion of IL-8 and overexpression of its mRNA induced by H(2)O(2) were significantly suppressed in a dose-dependent manner in the range of 0.25-2.00 mmol/L. We further found that a metabolite of CQA, caffeic acid (CA), but not quinic acid, significantly inhibited the H(2)O(2)-induced IL-8 secretion and its mRNA expression in the same dose-dependent manner. Both CQA and CA suppressed the TNF-alpha-induced IL-8 secretion as well. Caffeic acid at 2.00 mmol/l was able to absolutely block the H(2)O(2)- or TNF-alpha-induced oversecretion of IL-8 in Caco-2 cells. However, CQA and CA did not suppress the TNF-alpha-induced increase in the IL-8 mRNA expression, indicating that the suppressive mechanisms are different between TNF-alpha-induced and H(2)O(2)-induced IL-8 production models. These results suggest that the habit of drinking coffee and/or eating fruits with a high CHA content may be beneficial to humans in preventing the genesis of inflammatory bowel diseases.

Hypertonicity stimulates taurine uptake and transporter gene expression in Caco-2 cells.

The osmoregulation of taurine transport in intestinal epithelial cells was investigated using human intestinal Caco-2 cells. The activity of taurine transport in the Caco-2 cells was increased by hypertonic conditions. This hypertonicity-induced up-regulation was dependent on both the culturing time and the osmotic pressure. Hypertonicity did not affect the activity of L-leucine, L-lysine, or L-glutamic acid transport, suggesting that osmoregulation was specific to taurine transport. The intracellular taurine content of Caco-2 cells was also increased by culturing in a hypertonic medium. These hypertonicity-induced changes in the intracellular taurine content and transport activity were reversible. A kinetic analysis of taurine transport in the control and hypertonic cells suggested that the up-regulation was associated with an increase in the amount of the taurine transporter. The mRNA level of the taurine transporter in hypertonic cells was markedly higher than that in the control cells, indicating that this osmotic regulation was due to the increased expression of the taurine transporter gene.

Inhibitory effect of a bitter melon extract on the P‐glycoprotein activity in intestinal Caco‐2 cells

Extracts of bitter melon, soybean, dokudami and welsh onion by 40% methanol increased the accumulation of rhodamine‐123 by Caco‐2 cells, suggesting that these extracts inhibited P‐glycoprotein (P‐gp). The extract of bitter melon was separated in a tC18 cartridge column and the eluate from 80% acetonitrile most markedly increased the [3H]‐daunomycin accumulation by Caco‐2 cells. The inhibitory compounds in the bitter melon fraction were isolated by HPLC with Pegasil C4 and Pegasil ODS columns. The HPLC fraction having the highest activity was analyzed by 1H‐NMR and FAB‐MS, and the active compound was identified as 1‐monopalmitin. The inhibitory activities of 1‐monopalmitin and its related compounds suggested that the inhibition of P‐gp activity was not dependent on the degree of unsaturation of fatty acid in the monoglyceride, but on the chain length. It was also suggested that the monoglyceride structure played an important role in the inhibition of P‐gp activity. Monoglycerides could therefore alter the pharmacokinetics of drugs by inhibiting the P‐gp‐mediated efflux.

Dietary flavonoid naringenin induces regulatory T cells via an aryl hydrocarbon receptor mediated pathway.

The aryl hydrocarbon receptor (AhR), a transcription factor mediating xenobiotic detoxification, plays a considerable role in regulatory T cell (Treg) induction. Tregs regulate the immune system, thus suppressing allergies and autoimmune diseases. This study aims to identify new types of antiallergic dietary factors, with focus on the flavonoids with potential AhR agonistic activity. Among 25 dietary flavonoid samples tested using a reporter assay, 8 showed marked induction of AhR-dependent transcriptional activity. The subsequent T cell proliferation suppression assay identified naringenin as the only sample capable of stimulating Treg induction; notably, this induction was eliminated by cotreatment with AhR antagonists. Indeed, naringenin induced CD4(+)Foxp3(+) Tregs, irrespective of the presence of the transforming growth factor-β (TGF-β), indicating that the conventional TGF-β-dependent signaling pathway might not be involved.

Protective effect of quercetin on ER stress caused by calcium dynamics dysregulation in intestinal epithelial cells

Quercetin, one of the flavonoids present in plants, expresses several physiological functions including antioxidative and anti-inflammatory properties. However, its effect on intestinal epithelia remains to be elucidated. Endoplasmic reticulum (ER) stress has been attracting considerable attention since ER stress triggers such disorders as inflammation and cancer. The effect of quercetin on ER stress was investigated in this present study. Several ER stress inducers (tunicamycin, A23187, thapsigargin and brefeldin A) were added to human colonic LS180 cells or Caco-2 cells with quercetin, and the GRP78 expression as an ER stress marker was determined. The results showed that quercetin suppressed the induction of GRP78 expression by these ER stressors, excepting brefeldin A, at both the mRNA and protein levels. Additionally, XBP-1 mRNA splicing was determined to evaluate the activation of IRE1. The phosphorylation of eIF2alpha and shutdown of protein synthesis were determined to evaluate the activation of PERK. Although quercetin activated IRE1 and PERK when added to LS180 cells alone, it suppressed the activation of IRE1 and PERK induced by A23187 or thapsigargin. The suppressive effect of quercetin on GRP78 mRNA induction was reproduced by PI3K inhibitors (LY294002 and wortmannin), but not by vitamin C and E. LY294002 failed to suppress the GRP78 mRNA induction in combination with quercetin. In conclusion, this study indicates for the first time that quercetin suppressed the ER stress caused by calcium dynamics dysregulation by the inhibition of PI3K. This study helps to clarify the mechanism for quercetin presenting its versatility.

Tumor necrosis factor α stimulates taurine uptake and transporter gene expression in human intestinal Caco‐2 cells

The effect of cytokines on the taurine uptake by human intestinal epithelial Caco‐2 cells was investigated. Among the various cytokines tested, tumor necrosis factor α (TNF‐α) markedly increased the taurine uptake by Caco‐2 cells, resulting in an increase in the intracellular taurine level. TNF‐α did not induce up‐regulation of the taurine uptake in hepatic HepG2, renal human embryo kidney 293, and macrophage‐like THP‐1 cells. The uptake of glycine, L‐leucine, and L‐glutamic acid by Caco‐2 cells was not affected by TNF‐α. A kinetic analysis of the taurine uptake by TNF‐α‐treated Caco‐2 cells suggests that this up‐regulation was associated with both an increase in the amount of the taurine transporter (TAUT) and an increase in its affinity. TNF‐α‐treated cells showed a higher mRNA level of the TAUT than did the control cells.