Establishment of an in-vitro inflammatory bowel disease model using immunological differentiation of Caco-2 cells.

Studies on intestinal cell differentiation, particularly in dextran sodium sulfate (DSS)-induced inflammatory bowel disease (IBD), have predominantly focused on the disruption of intestinal crypts and suppressive effects on the intestinal microbiota; however, repeated administration of DSS is required to induce inflammatory pathology, and there is a lack of observation of early responses and consideration of differentiation stages. Although colonic adenocarcinoma (Caco-2) cells can be used as intestinal cell models, research on these cells in an immature state is limited. We, therefore, investigated the relationship between Caco-2 cell culture duration and immunological differentiation using α-defensin5 (DEFA5) as an indicator of intestinal immunity and differentiation. Changes in protein and gene expression levels in response to DSS were examined at each differentiation stage. Expression of immune- and differentiation-related proteins, including DEFA5 and lysozyme, was evident from Day 8 of culture. Immune responses to DSS varied with the differentiation stage, affecting cell viability and cytokine expression.•Caco-2 cell culture duration correlates with the differentiation stage of Paneth cells.•DSS exposure elicits different effects depending on the differentiation stage.•Our in-vitro model of IBD facilitates the characterization of the cell differentiation process and provides a methodology to help elucidate the causal mechanisms of IBD.

Curcumin effects on age-related changes in oral immunity: an in vivo study.

The current study aimed to investigate the effects of ageing on oral immunity using ß-defensin (DEFB) 1/2 as a marker and evaluate the effects of curcumin (CUR) on these processes. The study sample included thirty male C57BL/6J mice divided into three groups based on the treatment method used. The young control (YC) and old control (OC) groups received 0·5 % methylcellulose-400 (CUR vehicle) orally for 5 days, whereas the CUR group of older mice received a CUR solution suspended in 0·5 % methylcellulose-400 (dose: 3·0 mg/kg body). DEFB1/2 and immune indicator levels were measured in the saliva and salivary glands post-treatment. The saliva volume and protein content were significantly reduced in the OC group compared with the YC group. CUR administration restored these parameters, decreased DEFB1 expression in the salivary gland and increased DEFB1/2 secretion and DEFB2 expression. These findings were supported by epigenetic gene regulation and partial cytokine activation from changes in WD40 repeat protein 5, TNF alpha and IL-1beta. CUR can partially restore age-related changes in oral immune responses and promote oral health, thereby preventing frailty in the older population through a nutritional therapeutic pathway.

Regulation of Chloride Channels by Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor-Induced α-Defensin 5.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are used to treat non-small cell lung cancer with EGFR mutations. However, first-generation erlotinib and second-generation afatinib often cause diarrhea, which may develop because of the association between EGFR-TKIs and the chloride channel or abnormalities in the intestinal microbiota due to disruption of the intestinal immune system. As reports on the effects of EGFR-TKIs on intestinal immunity are lacking, we aimed to determine whether the intestinal immune system is involved in the molecular effects of EGFR-TKIs on chloride channels using Caco-2 cells. Initially, we evaluated the association of chloride channels with α-defensin 5 (DEFA5), a marker of intestinal immunity. Erlotinib and afatinib significantly increased the extracellularly secreted DEFA5 level and autophagy-related 16-like 1 and X-box binding protein 1 transcript levels, indicative of enhanced granule exocytosis. Conversely, intracellular DEFA5 and Toll-like receptor 4 protein expression and tumor necrosis factor-α transcript levels decreased significantly, suggesting that Toll-like receptor 4 suppression repressed DEFA5 production. Furthermore, among the chloride channels, DEFA5 was found to significantly increase the transcript levels of cystic fibrosis transmembrane conductance regulators. These results indicate that DEFA5 plays a significant role in the mechanism of chloride channel-mediated diarrhea induced by EGFR-TKIs. Therefore, we successfully elucidated the potential host action of DEFA5 in cancer therapy for the first time.

Association between α-defensin 5 and the expression and function of P-glycoprotein in differentiated intestinal Caco-2 cells.

α-Defensin 5 is known to be secreted by Paneth cells in the small intestine and plays an important role in eliminating pathogenic microorganisms. It has been reported that a decrease in α-defensin 5 level in the human small intestine is a risk of inflammatory bowel disease (IBD). Furthermore, P-glycoprotein (P-gp), a member of the ATP-binding cassette transporter superfamily, encoded by the ABCB1/MDR1 gene, plays an important role in the front line of host defense by protecting the gastrointestinal barrier from xenobiotic accumulation and may contribute to the development and persistence of IBD. Therefore, we examined the relationship between α-defensin 5 and the expression and function of P-gp using a human gastrointestinal model cell line (Caco-2). We found that MDR1 mRNA and P-gp protein level were increased in Caco-2 cells as well as α-defensin 5 secretion corresponded with the duration of cell culture. Exposure to α-defensin 5 peptide and recombinant tumor necrosis factor-α (TNF-α) significantly increased the expression and function P-gp. The mRNA levels of interleukin (IL)-8, IL-6, TNF-α, IL-1ß, and IL-2 were also increased following exposure to TNF-α, similar to α-defensin 5 treatment. These results suggest that α-defensin 5 regulates P-gp expression and function by increasing TNF-α expression in Caco-2 cells.

Gamma-Aminobutyric Acid (GABA) Attenuates Ischemia Reperfusion-Induced Alterations in Intestinal Immunity.

The aims of this study were to determine the effects of gamma-aminobutyric acid (GABA) on immunoglobulin A (IgA) secretion from Peyer's patch (PP) cells; to assess rat alpha-defensin-5 (RD-5) expression in the rat small intestine; and to determine the effect of GABA on intestinal ischemia reperfusion (I/R) injury-induced intestinal innate immunity. We found that GABA caused an increase in IgA secretion in the presence and absence of lipopolysaccharide (LPS). Moreover, GABA also significantly increased the mRNA levels of RD-5 and superoxide dismutase (Sod) 1, 3. Intestinal I/R was induced by a 30-min occlusion of the superior mesenteric artery followed by a reperfusion for 60-min. This led to a significant decrease in IgA secretion, and mRNA levels of RD-5 and Sod 1-3 in the ileum. On the other hand, administration of GABA before I/R induction had a significant protective effect against oxidative injury and attenuated the effects on intestinal immunity.

Inhibitory Effects of Baicalein Derived from Japanese Traditional Herbal Medicine on SN-38 Glucuronidation.

PURPOSE: The chemotherapeutic agent irinotecan is hydrolyzed to its active form SN-38 by human carboxyesterases, but SN-38 is converted into the inactive form SN-38G by hepatic UDP-glucuronosyltransferases (UGTs). The aim of the present study was to evaluate the inhibitory effects of two b-glucuronidase-treated Japanese traditional herbal medicines (kampo), Hange-Shashin-To (TJ-14) and Sairei-To (TJ-114) on SN-38 glucuronidation, and the deglycosylation of baicalin (BG) and glycyrrhizic acid (GL) derived from TJ-14 and TJ-114 to form their respective aglycones, baicalein (BA) and glycyrrhetinic acid (GA). METHODS: The inhibitory effects of b-glucuronidase-treated TJ-14 and TJ-114 on SN-38 glucuronidation by human liver microsomes were examined. BA and GA, which were enzymatically converted from BG and GL present in TJ-14 and TJ-114, were examined in the same manner. Furthermore, the enzymatic activities were measured by using recombinant UGT1A1 and UGT1A9 isoforms instead of human liver microsomes. BA, GA, SN-38, and their glycosides/glucuronides were analyzed with an LC-MS system. RESULTS: As regards the linear initial reaction rate, SN-38 glucuronidation by human liver microsomes was significantly inhibited by the addition of b-glucuronidase-untreated TJ-14 and TJ-114, but was more strongly inhibited by the addition of b-glucuronidase-treated TJ-14 and TJ-114. The results of LC-MS analysis and pharmacokinetic studies suggested that BA is the main inhibitor of SN-38 glucuronidation. In the Dixon plot, BA showed competitive inhibition of SN-38 glucuronidation, and the inhibition constant was 8.70 ± 3.24 mM. Previous reports, studies of recombinant UGT isoforms indicated that SN-38 glucuronidation was mainly catalyzed by UGT1A1. CONCLUSIONS: These findings strongly suggested that SN-38 glucuronidation is inhibited by BA. BA could act as a pharmacokinetic regulating factor associated with SN-38 glucuronidation. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.