Sachiko Mogami

Roles of oxidative stress in stomach disorders

The stomach is a sensitive digestive organ that is susceptible and exposed to exogenous pathogens from the diet. In response to such pathogens, the stomach induces oxidative stress, which might be related to the development of gastric organic disorders such as gastritis, gastric ulcers, and gastric cancer, as well as functional disorders such as functional dyspepsia. In particular, the bacterium Helicobacter pylori plays a major role in eliciting and confronting oxidative stress in the stomach. The present paper summarizes the pathogenesis of oxidative stress in the stomach during the development of various stomach diseases.

Preventive effect of rikkunshito on gastric motor function inhibited by l-dopa in rats

We previously reported that ghrelin prevented l-dopa (LD)-induced inhibition of gastric emptying (GE) of a non-nutrient solution in rats. Parkinsons disease treatment involves the combined administration of l-dopa with the enzyme l-amino acid decarboxylase inhibitor, carbidopa (CD) to reduce peripheral formation of dopamine. We investigated the effect LD/CD given orogastrically (og) on GE of a non-nutrient or nutrient meal and whether og pretreatment with rikkunshito, a kampo medicine clinically used to treat gastroparesis, influenced LD/CD effect on GE and postprandial antral and duodenal motility in conscious rats. LD/CD (20/2 mgkg(-1)) decreased significantly GE to 26.3 ± 6.0% compared to 61.2 ± 3.2% in og vehicle monitored 20-min after a non-nutrient meal and to 41.9 ± 5.8% compared to 72.9 ± 5.2% in og vehicle monitored 60 min after a nutrient meal. Rikkunshito (0.5 or 1.0 g kg(-1)) reduced the LD/CD (20/2 mg kg(-1)) inhibition of GE of non-nutrient meal (36.9 ± 7.4% and 46.6 ± 4.8% respectively vs. 12.1 ± 7.4% in og vehicle plus LD/CD) while having no effect alone (56.6 ± 8.5%). The ghrelin antagonist, [d-Lys(3)]-GHRP-6 (1 mg kg(-1)) injected intraperitoneally partially reversed rikkunshito preventive effect on LD/CD-inhibited GE. Rikkunshito (1.0 g kg(-1)) blocked LD/CD (20/2 mg kg(-1))-induced delayed GE of a nutrient meal and the reduction of postprandial antral motility. In 6-hydroxydopamine-induced Parkinsons disease rat model, rikkunshito (1.0 g kg(-1), og) also prevented LD/CD-inhibited gastric emptying of a nutrient meal and enhanced fasting plasma levels of acylated ghrelin. These data indicate that oral rikkunshito alleviates the delayed GE induced by LD/CD in naïve and PD rat model in part through ghrelin-related mechanisms.

Impaired heme oxygenase-1 induction in the gastric antrum induces disruption of the interstitial cells of Cajal network in a rat model of streptozotocin-induced diabetes.

Streptozotocin (STZ) is known to induce type I diabetes and the loss of the interstitial cells of Cajal (ICC). However, the regulation of heme oxygenase‐1 (HO‐1) expression, which is reported to protect ICC, has not yet been elucidated in this model. The aim of this study was to investigate the alterations of HO‐1 expression and clarify the mechanism of ICC loss in the stomach using the rat model of STZ‐induced diabetes.

Reduced ghrelin production induced anorexia after rat gastric ischemia and reperfusion

The gastrointestinal (GI) tract is one of the most susceptible organs to ischemia. We previously reported altered gastric motility after gastric ischemia and reperfusion (I/R). However, there have also been few reports of alterations in the eating behavior after gastric I/R. Ghrelin is a GI peptide that stimulates food intake and GI motility. Although ghrelin itself has been demonstrated to attenuate the mucosal injuries induced by gastric I/R, the endogenous ghrelin dynamics after I/R has not yet been elucidated. The present study was designed to investigate the relationship between food intake and the ghrelin dynamics after gastric I/R. Wistar rats were exposed to 80-min gastric ischemia, followed by 12-h or 48-h reperfusion. The food intake, plasma ghrelin levels, gastric preproghrelin mRNA expression levels, and the histological localization of ghrelin-immunoreactive cells were evaluated. The effect of exogenous ghrelin on the food intake after I/R was also examined. Food intake, the plasma ghrelin levels, the count of ghrelin-immunoreactive cells corrected by the percentage areas of the remaining mucosa, and the expression levels of preproghrelin mRNA in the stomach were significantly reduced at 12 h and 48 h after I/R compared with the levels in the sham-operated rats. Intraperitoneal administration of ghrelin significantly reversed the decrease of food intake after I/R. These data show that gastric I/R evoked anorexia with decreased plasma ghrelin levels and ghrelin production, which appears to be attributable to the I/R-induced gastric mucosal injuries. The decrease in the plasma ghrelin levels may have been responsible for the decreased food intake after gastric I/R.

Patterns of Brain Activation and Meal Reduction Induced by Abdominal Surgery in Mice and Modulation by Rikkunshito

Abdominal surgery inhibits food intake and induces c-Fos expression in the hypothalamic and medullary nuclei in rats. Rikkunshito (RKT), a Kampo medicine improves anorexia. We assessed the alterations in meal microstructure and c-Fos expression in brain nuclei induced by abdominal surgery and the modulation by RKT in mice. RKT or vehicle was gavaged daily for 1 week. On day 8 mice had no access to food for 6–7 h and were treated twice with RKT or vehicle. Abdominal surgery (laparotomy-cecum palpation) was performed 1–2 h before the dark phase. The food intake and meal structures were monitored using an automated monitoring system for mice. Brain sections were processed for c-Fos immunoreactivity (ir) 2-h after abdominal surgery. Abdominal surgery significantly reduced bouts, meal frequency, size and duration, and time spent on meals, and increased inter-meal interval and satiety ratio resulting in 92–86% suppression of food intake at 2–24 h post-surgery compared with control group (no surgery). RKT significantly increased bouts, meal duration and the cumulative 12-h food intake by 11%. Abdominal surgery increased c-Fos in the prelimbic, cingulate and insular cortexes, and autonomic nuclei, such as the bed nucleus of the stria terminalis, central amygdala, hypothalamic supraoptic (SON), paraventricular and arcuate nuclei, Edinger-Westphal nucleus (E-W), lateral periaqueduct gray (PAG), lateral parabrachial nucleus, locus coeruleus, ventrolateral medulla and nucleus tractus solitarius (NTS). RKT induced a small increase in c-Fos-ir neurons in the SON and E-W of control mice, and in mice with surgery there was an increase in the lateral PAG and a decrease in the NTS. These findings indicate that abdominal surgery inhibits food intake by increasing both satiation (meal duration) and satiety (meal interval) and activates brain circuits involved in pain, feeding behavior and stress that may underlie the alterations of meal pattern and food intake inhibition. RKT improves food consumption post-surgically that may involve modulation of pain pathway.

Mashiningan Improves Opioid-Induced Constipation in Rats by Activating Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel

Opioid receptor stimulants are analgesics used in patients with and without cancer; however, they often cause constipation, resulting in poor adherence and deterioration of the quality of life. Hence, suitable treatments for constipation are required. In this study, we investigated the pharmacological mechanisms of action of mashiningan (MNG), a Kampo medicine used to treat constipation, and evaluated the effect of MNG on opioid-induced constipation in rats. MNG (100 or 300 mg/kg) was orally administered to normal or codeine phosphate (CPH)–induced constipation in rats, and its effect was evaluated on the basis of fecal counts, characteristics, and weight. Small intestinal fluid secretion was measured after treatment with MNG alone or coadministration with a cystic fibrosis transmembrane conductance regulator (CFTR)–specific inhibitor (CFTRinh-172). The effects of MNG on the CFTR and type-2 chloride channel were determined using patch-clamp or short-circuit current experiments, respectively. MNG increased the fecal weight and proportion of soft feces in normal rats. CPH-induced constipation in rats decreased fecal counts and weight, whereas MNG prevented these effects and increased the proportion of soft feces. MNG increased the electronic chloride current, and this effect was inhibited by the CFTRinh-172 in the CFTR assay. Furthermore, MNG increased small intestinal fluid secretion, and this effect was abolished by coadministration with the CFTRinh-172. MNG improved opioid-induced constipation in rats, and this improvement may have been mediated by increasing intestinal fluid secretion via CFTR chloride channel activation. Therefore, MNG is expected as a medicine of the treatment of constipation in patients taking opioids.

Peptide YY induces characteristic meal patterns of aged mice

Background & aim: Changes in eating behavior occur in the elderly due to oral and swallowing dysfunctions. We aimed to clarify the difference between basal meal patterns of young and aged mice in relation to appetite regulating hormones. Methods: Thirty two of young (7‐week‐old) and aged (23–25‐month‐old) C57BL/6 male mice were acclimated to a single housing and then transferred to a highly sensitive automated feeding monitoring device. Feeding behavior was monitored from the onset of the dark phase after habituation to the device. Plasma peptide YY (PYY) levels were assessed under the several feeding status or after treatment of PYY. PYY and its receptor (NPY Y2 receptor, Y2R) antagonist were intraperitoneally administered 30 min before the monitoring. Results: Although the basal 24‐h meal amounts did not differ by age, the total meal time and frequency of minimum feeding activity (bout) were significantly increased and the average bout size and time per bout were significantly decreased in aged mice. PYY dynamics were abnormal and the temporal reduction in food intake by exogenous PYY was more prominent in aged mice than in young mice. PYY administration to young mice induced aged‐like meal patterns, and Y2R antagonist administration to aged mice induced young‐like meal patterns. Conclusions: Aged mice exhibited characteristic meal patterns probably due to PYY metabolism dysfunction and/or enhanced PYY‐Y2R signaling, suggesting a novel method for assessing eating difficulties in aged animals and a potential target for the remedy. HIGHLIGHTSAged mice show characteristic meal patterns distinctive from young mice.Aged mice require more time to eat and eat by small amounts frequently.Aged mice show abnormal PYY dynamics and susceptibility to exogenous PYY.Enhanced PYY signaling may induce the characteristic meal patterns of aged mice.

Psychological stress exposure to aged mice causes abnormal feeding patterns with changes in the bout number

Stress responses are affected by aging. However, studies on stress-related changes in feeding patterns with aging subject are minimal. We investigated feeding patterns induced by two psychological stress models, revealing characteristics of stress-induced feeding patterns as “meal” and “bout” (defined as the minimum feeding behavior parameters) in aged mice. Feeding behaviors of C57BL/6J mice were monitored for 24 h by an automatic monitoring device. Novelty stress reduced the meal amount over the 24 h in both young and aged mice, but as a result of a time course study it was persistent in aged mice. In addition, the decreased bout number was more pronounced in aged mice than in young mice. The 24-h meal and bout parameters did not change in either the young or aged mice following water avoidance stress (WAS). However, the meal amount and bout number increased in aged mice for 0–6 h after WAS exposure but remained unchanged in young mice. Our findings suggest that changes in bout number may lead to abnormal stress-related feeding patterns and may be one tool for evaluating eating abnormality in aged mice.

Tu2030 A Ghrelin Enhancer, Rikkunshito, Ameliorates Anorexia in Cachexia Model of Pulmonary Fibrosis

BACKGROUND: Although the pathophysiology and management of dyssynergic defecation (DD) differs from that of other types of constipation, it shares many of the same symptoms. The diagnosis of DD relies largely on anorectal manometry, defecography, or balloon expulsion tests, which are not available in all practices. Ideally, a specific symptom from the history could suggest DD, leading the physician to efficiently utilize these resources. METHODS: For 5months, data was prospectively collected frompatients undergoing high resolution anorectal manometry (HRARM) for an indication of constipation. Patients completed the validated questionnaire “patient assessment of constipation” (PAC-SYM) and a 7 item bowel habits questionnaire (BHQ) prior to HRARM. The BHQ was designed to further characterize the evacuation process such as need and frequency for digital extraction of stool (TABLE 1). HRARM was performed using a solid state catheter with 10 circumferential sensors spaced at 0.6cm intervals. Sensation was assessed by inflating the rectal balloon with saline at 10cc increments. HRARMs were analyzed and mean anorectal pressures were collected. Presence of a dyssynergy pattern (either paradoxical contraction or inadequate relaxation of the anal sphincter) was noted. Statistical analysis was performed using Spearmans correlation coefficient and the point biserial correlation coefficient. RESULTS: Thirty patients presenting for HRARM fully completed the questionnaires. The median age was 39, 83.4% patients were female, 16.6% male, 70% were white, 23.3% black, and 6.7% other races. The mean total PAC-SYM score was 21 (range 7-41) of which 0 is the minimum and 48 the maximum score possible. The mean total BHQ score was 6.7 (range 2-12) of which 0 is the minimum and 28 the maximum score possible. On HRARM, a dyssynergy pattern was seen in 70% of the patients, of which 58.8%were characterized by paradoxical contraction of the sphincter and 41.2% by inadequate relaxation. Impaired rectal sensation was seen in 79% of patients. There was no statistically significant correlation between the total PAC-SYM score, total BHQ score, or score from any individual question and the presence of dyssynergy. There was no significant correlation between the total PAC-SYM score, total BHQ score, or score from any individual question and the percent anal relaxation during bear down maneuvers (TABLE 2). CONCLUSION: Even with the inclusion of a customized bowel habit questionnaire that aimed totarget anorectal symptoms, a significant correlation between symptoms and dyssynergy could not be found. This highlights the importance of a focused rectal exam and physiologic testing such as anorectal manometry in the diagnosis of DD. Bowel Habits Questionnaire (BHQ)

Enhanced gastric ghrelin-induced c-Kit protein expression in rats with gastric outlet obstruction.

Dear Editor, We reported in the April issue of Digestive Diseases and Sciences that rats with gastric outlet obstruction (GOO) had an enhanced mRNA expression of c-kit, a marker of interstitial cells of Cajal (ICC), in the stomach with an increased level of ghrelin production [1]. However, the distribution of c-Kit protein expression in the stomach was not shown. Now, we have completed the additional experiments to clarify this point. For histochemical staining of c-Kit, stomach tissue specimens were fixed with cold acetone for 30 min and embedded in Tissue-Tek OCT compound 4583 (Sakura Finetechnical, Tokyo, Japan), frozen in liquid isopentane cooled with liquid nitrogen, and cut into 5-lm sections with a cryostat (CM1850; Leica, Nussloch, Germany). The sections were placed on a microslide glass (Matsunami, Osaka, Japan) and incubated with Protein Block (DAKO Japan, Tokyo, Japan) containing 0.5 % Triton X-100 for 1 h at room temperature and incubated with rabbit anti-cKit polyclonal antibody (A4502; 1:200, Daco Japan) overnight at 4 C. Immunoreactivity was detected using Alexa Fluor 488 donkey anti-rabbit IgG (1:1,000; Invitrogen, Eugene, OR, USA). Coverslips were mounted with Permafluor (Beckman Coulter, Fullerton, CA, USA) and immunofluorescence was examined using a keyence BIOZERO (Keyence, Osaka, Japan) [2]. Now, we showed the enhanced levels of c-Kit immunoreactive cells in rats with GOO (Fig. 1a). The areas of the c-Kit-positive cells were quantified in 12 microscopic fields from each individual rat using the ImageJ program (National Institutes of Health). For the measurement of ICC-IM (intramuscular ICC)-positive levels, the area of the c-Kit-positive cells in the muscular layers was normalized with the whole area of inner circular muscular layers. For the measurement of ICC-MY (myenteric ICC)-positive levels, the area of the c-Kit-positive cells in the myenteric plexus was normalized with the whole area of intermuscular space. By the morphometric analyses, in the GOO group, the levels of ICC-IM as well as ICC-MY, in both the antrum and the corpus, were quantified (Fig. 1b). After intraperitoneal injection of [D-Lys3] GHRP-6, a ghrelin receptor antagonist, (6.0 mg/kg; Bachem, PA, USA), both the levels of ICC-IM and ICC-MY were decreased in the antrum and the corpus. It is concluded that GOO enhances c-Kit expression through the enhancement of ghrelin in the stomach. In the present experiment, we performed histopathologic sampling of the specimen 30 min after administration of ghrelin receptor antagonist, since Miyazawa et al. [3] reported that the biological half-time of c-Kit protein was 30–60 min in the cultured cells. The enhancement of immunoreativity of c-Kit protein was shown not only in the ICC-IM but also in the ICC-MY. Our results indicate that enhanced ghrelin keeps the expression of the mRNA and the protein expression of c-Kit at high levels in rats with GOO. E. Iwasaki H. Suzuki (&) S. Mogami T. Hibi Division of Gastroenterology and Hepatology, Department of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan e-mail: hsuzuki@a6.keio.jp