Junichi Koseki

Acid modulates the squamous epithelial barrier function by modulating the localization of claudins in the superficial layers

Acid is a major cause of gastro-esophageal reflux disease. However, the influence of acid on the esophageal stratified epithelial barrier function and tight junction (TJ) proteins is not fully understood. Here, we explore the influence of acid on barrier function and TJ proteins using a newly developed model of the esophageal-like squamous epithelial cell layers that employs an air–liquid interface (ALI) system. Barrier function was determined by measuring trans-epithelial electrical resistance (TEER) and diffusion of paracellular tracers. TJ-related protein (claudin-1, claudin-4, occludin and ZO-1) expression and localization was examined by immunofluorescent staining, and by western blotting of 1% NP-40 soluble and insoluble fractions. We also examined the influence of acid (pH 2–4) on the barrier created by these cells. The in vitro ALI culture system showed a tight barrier (1500–2500 Ω·cm2) with the expression of claudin-1, claudin-4, occludin and ZO-1 in the superficial layers. Claudin-1, claudin-4, occludin and ZO-1 were detected as dots and whisker-like lines in the superficial layers, and as a broad line in the suprabasal layers. These localization patterns are similar to those in the human esophagus. On day 7 under ALI culture, TJ proteins were detected in the superficial layers with functional properties, including decreased permeability and increased TEER. Dilated intercellular spaces were detected at the suprabasal cell layers even under the control conditions of ALI cells. pH 2 acid on the apical side significantly reduced the TEER in ALI-cultured cells. This decrease in TEER by the acid was in parallel with the decreased amount of detergent-insoluble claudin-4. Claudin-4 delocalization was confirmed by immunofluorescent staining. In conclusion, TJs are located in the superficial layers of the esophagus, and acid stimulation disrupts barrier function, at least in part by modulating the amount and localization of claudin-4 in the superficial layers.

Rikkunshito, a traditional Japanese medicine, may relieve abdominal symptoms in rats with experimental esophagitis by improving the barrier function of epithelial cells in esophageal mucosa.

BackgroundA traditional Japanese medicine, rikkunshito, has been reported to relieve dyspepsia symptoms. We investigated the effect of rikkunshito on RE-induced abdominal dyspepsia, and performed experiments to elucidate the mechanism of that effect.Methods RE model rats were prepared using 8-week-old male Wistar rats, and rikkunshito was administered in drinking water. Voluntary movement was used as an index of RE-induced abdominal dyspepsia, which was monitored by an infrared sensor. On the tenth day after surgery, the total area of esophageal erosion was measured, and samples of nonerosive mucosa were collected. Using those samples, intercellular spaces of epithelial mucosa were examined by transmission electron microscopy, and the NP-40-soluble and -insoluble levels of the tight junction proteins claudin-1, -3 and -4 and their mRNAs were determined.ResultsRikkunshito did not reduce the average total area of erosive lesions in the esophageal mucosa of RE model rats. On day 10, voluntary movement was significantly decreased in the RE model rats and rikkunshito significantly increased it. Nonerosive esophageal mucosa from RE rats showed dilation of intercellular spaces in epithelium, and significantly decreased claudin-3 mRNA and protein levels. Rikkunshito significantly suppressed intercellular space dilation and significantly increased the level of NP-40-insoluble claudin-3, but it did not affect the mRNA level, suggesting that it promoted tight junction formation by facilitating the translocation of proteins.ConclusionRikkunshito increased voluntary movement in RE model rats. This may have been because rikkunshito ameliorated the symptoms of RE by improving the barrier function of esophageal mucosa.

Establishment of esophageal-like non-keratinized stratified epithelium using normal human bronchial epithelial cells

Current experimental models of esophageal epithelium in vitro suffer from either poor differentiation or complicated culture systems. We have established a model to study stratified squamous epithelium in vitro, which is very similar to esophageal epithelium in vivo. A stratified squamous multilayer epithelium was formed by seeding primary normal human bronchial epithelial (NHBE) cells onto collagen- and fibronectin-coated trans-well inserts and then cultivating the cells under air-liquid interface (ALI) conditions in the presence of growth factors and low levels of all-trans-retinoic acid. Trans-epithelial electrical resistance (TEER) measurements revealed the presence of a tight barrier, previously only achievable with esophageal biopsies mounted in Ussing chambers. Molecular markers for desmosomes, cornified envelope, tight junctions, and mature esophageal epithelium were upregulated in the differentiating culture in parallel with functional properties, such as decreased permeability and acid resistance and restoration. Acid exposure resulted in a decrease in TEER, but following 1-h recovery the TEER values were fully restored. Treatment with all-trans-retinoic acid decreased TEER and inhibited the recovery after acid challenge. PPAR-delta agonist treatment increased TEER, and this temporary increase in TEER was consistent with an increase in involucrin mRNA. Global gene expression analysis showed that ALI-differentiated NHBE cells had expression profiles more similar to epithelial biopsies from the esophageal tissue of healthy volunteers than to any other cell line. With respect to morphology, molecular markers, barrier properties, and acid resistance, this model presents a new way to investigate barrier properties and the possible effects of different agents on human esophagus-like epithelium.

Plasma Pharmacokinetics of Polyphenols in a Traditional Japanese Medicine, Jumihaidokuto, Which Suppresses Propionibacterium acnes-Induced Dermatitis in Rats

Most orally administered polyphenols are metabolized, with very little absorbed as aglycones and/or unchanged forms. Metabolic and pharmacokinetic studies are therefore necessary to understand the pharmacological mechanisms of polyphenols. Jumihaidokuto (JHT), a traditional Japanese medicine, has been used for treatment of skin diseases including inflammatory acne. Because JHT contains various types of bioactive polyphenols, our aim was to clarify the metabolism and pharmacokinetics of the polyphenols in JHT and identify active metabolites contributing to its antidermatitis effects. Orally administered JHT inhibited the increase in ear thickness in rats induced by intradermal injection of Propionibacterium acnes. Quantification by LC-MS/MS indicated that JHT contains various types of flavonoids and is also rich in hydrolysable tannins, such as 1,2,3,4,6-penta-O-galloyl glucose. Pharmacokinetic and antioxidant analyses showed that some flavonoid conjugates, such as genistein 7-O-glucuronide and liquiritigenin 7-O-glucuronide, appeared in rat plasma and had an activity to inhibit hydrogen peroxide-dependent oxidation. Furthermore, 4-O-methylgallic acid, a metabolite of Gallic acid, appeared in rat plasma and inhibited the nitric oxide reaction. JHT has numerous polyphenols; it inhibited dermatitis probably via the antioxidant effect of its metabolites. Our study is beneficial for understanding in vivo actions of orally administered polyphenol drugs.

Saireito probably prevents mesangial cell proliferation in HIGA mice via PDGF-BB tyrosine kinase inhibition

BackgroundIn this study, we administered saireito to high serum IgA (HIGA) mice and investigated its inhibitory effect on platelet-derived growth factor (PDGF) receptor tyrosine kinase (which causes mesangial proliferation) as one of the possible antinephritic mechanisms of saireito.MethodsFemale HIGA/NscSlc mice, aged 10 weeks, were divided into five groups (each, n = 12; a control group, three saireito-mixed feed groups, and a captopril-mixed feed group) so that the plasma IgA levels were comparable among the groups. After the grouping, the animals were administered the saireito or captopril, mixed in the feed, until the age of 45 weeks.ResultsAt the age of 45 weeks, the glomerular cell number was 47.8 ± 3.9 / cross section in the HIGA mice in the control group, but 41.6 ± 2.3 / cross section in the 1.3% saireito-mixed feed group and 38.7 ± 3.5 / cross section in the captopril-mixed feed group, being significantly lower in both these treatment groups than in the control group. At the age of 45 weeks, the sclerosis score in the HIGA mice in the control group was 0.92 ± 0.23. However, the sclerosis scores in the 0.26% (0.59 ± 0.26) and 1.3% (0.58 ± 0.16) saireito-mixed feed groups were significantly lower than that in the control group. In the captopril-mixed feed group, the sclerosis score was 0.64 ± 0.34, significantly lower than that in the control group. It was clarified that saireito suppressed mesangial cell proliferation without showing any cytotoxicity. Furthermore, as a result of investigating the mesangial cell proliferation-suppressing effect similarly with the 23 substances constituting saireito, a proliferation-suppressing effect was recognized with isoliquiritigenin (a component of Glycyrrhizae Radix) and oroxylin A (a component of Scutellariae Radix). Oroxylin A and isoliquiritigenin showed an inhibitory effect on PDGF receptor tyrosine kinase. Furthermore, the inhibitory effects of oroxylin A and isoliquiritigenin on tyrosine kinase were found to be specific to the PDGF receptor, and showed no influence on the tyrosine kinase activities of other growth-factor receptors examined.ConclusionThese results suggest that the antinephritic effects of saireito in HIGA mice may be partly due to the inhibiton of PDGF tyrosine kinase by oroxylin A and isoliquiritigenin, components of saireito.

Effect of rikkunshito on the expression of substance P and CGRP in dorsal root ganglion neurons and voluntary movement in rats with experimental reflux esophagitis.

While there are reports that the herbal medicine rikkunshito (RKT) relieves upper gastrointestinal disease symptoms, the effect of RKT on primary afferent neurons is unknown.

Role of transient receptor potential ankyrin 1 in gastric accommodation in conscious guinea pigs

We report the establishment of a new model for measuring gastric tone and liquid meal-induced accommodation in conscious guinea pigs and the role played by transient receptor potential ankyrin 1 (TRPA1). An indwelling polyethylene bag was placed in proximal stomachs of 5-week-old male Hartley guinea pigs. Gastric tone was measured by distending the bag and recording changes in intrabag pressure at various volumes. Gastric accommodation was measured by administering liquid meals and recording intrabag pressure over time. Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME) (a nitric-oxide synthase inhibitor), atropine sulfate (atropine) (a muscarinic receptor antagonist), allyl isothiocyanate (AITC) (a TRPA1 agonist), or theophylline-7-(N-4-isopropylphenyl) acetamide (HC-030031) (a selective TRPA1 antagonist) was administered 15 to 60 min before measurement. Gastric tone was increased by stepwise distension of the bag and was further significantly increased by l-NAME and significantly decreased by atropine. A liquid meal (15% w/v; 1.7 kcal) significantly decreased intrabag pressure 5 to 20 min after administration, indicating gastric accommodation; this was completely suppressed by l-NAME and further enhanced by atropine. AITC significantly increased gastric tone; this increase was decreased by HC-030031 and atropine. A combination of AITC and l-NAME significantly increased gastric tone compared with l-NAME alone. HC-030031 alone significantly decreased gastric tone. Liquid meal-induced gastric accommodation was significantly suppressed by pretreatment with AITC. We established a new model for measuring gastric tone and accommodation in conscious guinea pigs. TRPA1 activation suppresses gastric accommodation by increasing gastric tone through cholinergic neuronal pathways.

NK-1 receptor is involved in the decreased movement in a rat chronic acid reflux oesophagitis model

Background  We previously reported that rats with reflux oesophagitis (RE) show a decrease in voluntary movement, which could be used as a measure of chronic visceral symptoms. However, what mediates these symptoms is still unknown, and pain‐related neuropeptides or their receptors in oesophageal mucosa are possibly related to the symptom generation of oesophagitis. In the present study, we investigated the role of NK‐1 receptor (NK‐1R) as a mediator of oesophagitis symptoms.

Impairment of gastric accommodation induced by water-avoidance stress is mediated by 5-HT2B receptors.

Psychological stress has been shown to impair gastric accommodation (GA), but its mechanism has not been elucidated. This study was conducted to clarify the role of 5‐HT2B receptors in a guinea pig model of stress‐induced impairment of GA.

Suppression of Propionibacterium acnes-Induced Dermatitis by a Traditional Japanese Medicine, Jumihaidokuto, Modifying Macrophage Functions

Purpose. Macrophages serve as sweepers of microbes and inflammation-derived wastes and regulators of inflammation. Some traditional Japanese medicines are reported to have adjuvant effects by modifying macrophages. Our aim was to characterize the actions of jumihaidokuto (JHT) for treatment of skin inflammations including acne vulgaris, in which Propionibacterium acnes has pathogenic roles. Methods. Dermatitis was induced in rat ears by intradermal injection of P. acnes. JHT or prednisolone (PDN) was given orally, and ear thickness and histology were evaluated. The effects of constituents and metabolites of JHT on monocytes were tested by cell-based assays using the human monocytic THP-1 cell. Results. JHT and PDN suppressed the ear thickness induced by P. acnes injection. Histological examinations revealed that JHT, but not PDN, promoted macrophage accumulation at 24 h after the injection. PDN suppressed the macrophage chemokine MCP-1 in the inflamed ears, while JHT did not affect it. The JHT constituents liquiritigenin and isoliquiritin increased expression of CD86 (type-1 macrophage marker) and CD192 (MCP-1 receptor) and enhanced phagocytosis by THP-1. Conclusions. JHT suppressed dermatitis, probably by enhancing type-1 macrophage functions, with an action different from PDN. JHT may be a beneficial drug in treatment of skin inflammation induced by P. acnes.