The ameliorating effects of tokishakuyakusan in a rat model of implantation failure involves endometrial gland leukemia inhibitory factor and decidualization.

ETHNOPHARMACOLOGICAL RELEVANCE: Tokishakuyakusan (TSS) is a Kampo medicine that is prescribed for the treatment of infertility in Japan. However, its precise mechanism of action remains unclear. AIM OF THE STUDY: Leukemia inhibitory factor (LIF) in the endometrium plays an indispensable role in embryo implantation and is linked to infertility or implantation failure. Previously, we demonstrated that TSS ameliorated implantation failure induced by mifepristone (RU-486), an antagonist of progesterone, in rats. Herein, we aimed to clarify whether the ameliorating effect of TSS on implantation failure in the rat model involves endometrial LIF. Additionally, we determined whether decidualization, the dysfunction of which is linked to infertility or implantation failure similar to LIF, progesterone, and other implantation-related factors, are involved in the effect of TSS. MATERIALS AND METHODS: The implantation failure rat model was developed via the subcutaneous administration of RU-486 (7 mg/kg) on day 3 post-coitus. Sesame oil was administered as the vehicle control. Rats were fed a diet containing 1% or 3% TSS or a control diet from day 13 pre-coitus. Subsequently, the implantation sites were assessed, and plasma progesterone levels were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on day 8 post-coitus. The LIF mRNA of the endometrial gland, which was segmented via laser-microdissection from the endometrial tissue, was measured, and endometrial LIF immunostaining was carried out on day 5. The gene expression of different factors related to implantation, including decidualization and progesterone-responsiveness on days 5 and 6, were measured. The human endometrial Ishikawa cell line derived from human adenocarcinoma was treated with TSS (30-300 µg/mL) for 24 h, and the LIF concentrations in the cell culture supernatants were measured. RESULTS: RU-486 decreased the number of implantation sites in the uterus of rats; however, the decrease was significantly alleviated by TSS (3%-diet), which tended to increase plasma progesterone. In rats with RU-486-induced implantation failure, endometrial gland LIF mRNA and endometrial LIF protein were markedly decreased while the gene expression of both decidualization-related factors such as interleukin-11, insulin-like growth factor binding protein-1, and cyclooxygenase-2, and progesterone responsive-related factors such as FK506 binding protein 5, were significantly decreased. These changes in the uterus of rats with implantation failure were significantly alleviated by TSS (3%-diet). Additionally, TSS significantly enhanced LIF protein production and LIF mRNA in Ishikawa cells. CONCLUSIONS: The mechanism whereby TSS ameliorates RU-486-induced implantation failure in rats may involve the alleviation of decreased LIF production derived from the endometrial gland, and a dysfunction of decidualization, including lower progesterone responsiveness in the model. These findings may partly contribute to the interpretation of the beneficial effects of TSS on infertility.

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.

Influence of Aging and Gender Differences on Feeding Behavior and Ghrelin-Related Factors during Social Isolation in Mice.

Psychological stress due to social isolation is known to cause abnormal feeding behaviors, but the influences of gender and aging on subchronic stress-induced changes in feeding behaviors are unknown. Thus, we examined the changes in body weight, food intake, and orexigenic ghrelin-related factors during 2 weeks of isolation stress in young and aged mice. Food intake increased significantly in young mice in the isolation group compared with the group-housed control throughout the experimental period. This isolation-induced increase in food intake was not observed in aged mice. In young mice, there were no significant differences in body weight between the isolated group and group-housed control up to 2 weeks. However, aged male mice exhibited significant weight loss at 2 weeks and a similar tendency was observed in aged female mice. Young male mice, but not female mice, had significantly increased (2.2-fold) plasma acylated ghrelin levels after 1 week of isolation compared with the group-housed control. A significant but lower increase (1.3-fold) was also observed in aged male mice. Hypothalamic preproghrelin gene expression decreased significantly with isolation in young male mice, whereas it increased significantly in female mice. The expression levels of NPY and AGRP in the hypothalamus, which are transmitted by elevated peripheral ghrelin signals, increased significantly in isolated young male mice, whereas the AGRP expression levels decreased significantly in young female mice. Isolation caused no significant differences in the expression levels of these genes in aged mice. In isolation, young female mice exhibited markedly increased dark- and light-phase locomotor activities compared with male mice, whereas male and female aged mice exhibited no obvious increases in activity immediately after the dark phase started. We conclude that the gender-specific homeostatic regulatory mechanisms required to maintain body weight operated during subchronic psychological stress in young mice but not in aged mice.

A New Strategy Using Rikkunshito to Treat Anorexia and Gastrointestinal Dysfunction.

Because the clinical condition of gastrointestinal dysfunction, including functional dyspepsia, involves tangled combinations of pathologies, there are some cases of insufficient curative efficacy. Thus, traditional herbal medicines (Kampo medicines) uniquely developed in Japan are thought to contribute to medical treatment for upper gastrointestinal symptoms. Rikkunshito is a Kampo medicine often used to treat dyspeptic symptoms. Over the past few years, several studies have investigated the efficacy of rikkunshito for dysmotility, for example, upper abdominal complaints, in animals and humans. Rikkunshito ameliorated the decrease in gastric motility and anorexia in cisplatin-treated rats, stress-loaded mice, and selective serotonin reuptake inhibitor-treated rats by enhancing plasma ghrelin levels via serotonin2B/2C receptor antagonism. In addition, rikkunshito ameliorated the decrease in food intake in aged mice and stress-loaded decreased gastric motility via enhanced ghrelin receptor signaling. Several clinical studies revealed that rikkunshito was effective in ameliorating upper gastrointestinal symptoms, including dyspepsia, epigastric pain, and postprandial fullness. In this review, we discuss these studies and propose additional evidence-based research that may promote the clinical use of Kampo medicines, particularly rikkunshito, for treating anorexia and gastrointestinal dysfunction.

Rikkunshito, a Japanese kampo medicine, ameliorates decreased feeding behavior via ghrelin and serotonin 2B receptor signaling in a novelty stress murine model.

We investigated the effects of rikkunshito (RKT), a ghrelin signal enhancer, on the decrease in food intake after exposure to novelty stress in mice. RKT administration (500 mg/kg, per os) improved the decrease in 6 h cumulative food intake. In control mice, the plasma acylated ghrelin levels significantly increased by 24 h fasting. In contrast, the acylated ghrelin levels did not increase by fasting in mice exposed to the novelty stress. RKT administration to the novelty stress mice showed a significant increase in the acylated ghrelin levels compared with that in the distilled-water-treated control mice. Food intake after administering serotonin 2B (5-HT(2B)) receptor antagonists was evaluated to clarify the role of 5-HT(2B) receptor activation in the decrease in feeding behavior after novelty stress. SB215505 and SB204741, 5-HT(2B) receptor antagonists, significantly improved the decrease in food intake after exposure to novelty stress. A component of RKT, isoliquiritigenin, prevented the decrease in 6 h cumulative food intake. Isoliquiritigenin showed 5-HT(2B) receptor antagonistic activity in vitro. In conclusion, the results suggested that RKT improves the decrease in food intake after novelty stress probably via 5-HT(2B) receptor antagonism of isoliquiritigenin contained in RKT.

Rikkunshito as a ghrelin enhancer.

Rikkunshito is a kampo herbal medicine which is widely used in Japan for the treatment of the upper gastrointestinal symptoms of patients with functional dyspepsia, gastroesophageal reflux disease, dyspeptic symptoms of postgastrointestinal surgery patients, and chemotherapy-induced dyspepsia in cancer patients. Recently, very unique characteristics of rikkunshito have been unveiled; oral administration of rikkunshito potentiates orexigenic action of ghrelin through several different mechanisms. In addition, several lines of evidence obtained from both animal and human studies indicate that rikkunshito can be an attractive and promising therapeutic option for the anorectic conditions including cisplatin-induced dyspepsia, anorexia of aging, stress-induced hypophagia, and cancer cachexia-anorexia syndrome. In this chapter, we highlight the orexigenic effect of rikkunshito with a special focus on its interaction with ghrelin signaling system.

Impaired ghrelin signaling is associated with gastrointestinal dysmotility in rats with gastroesophageal reflux disease.

Gastroesophageal reflux disease (GERD) is often associated with decreased upper gastrointestinal motility, and ghrelin is an appetite-stimulating hormone known to increase gastrointestinal motility. We investigated whether ghrelin signaling is impaired in rats with GERD and studied its involvement in upper gastrointestinal motility. GERD was induced surgically in Wistar rats. Rats were injected intravenously with ghrelin (3 nmol/rat), after which gastric emptying, food intake, gastroduodenal motility, and growth hormone (GH) release were investigated. Furthermore, plasma ghrelin levels and the expression of ghrelin-related genes in the stomach and hypothalamus were examined. In addition, we administered ghrelin to GERD rats treated with rikkunshito, a Kampo medicine, and examined its effects on gastroduodenal motility. GERD rats showed a considerable decrease in gastric emptying, food intake, and antral motility. Ghrelin administration significantly increased gastric emptying, food intake, and antral and duodenal motility in sham-operated rats, but not in GERD rats. The effect of ghrelin on GH release was also attenuated in GERD rats, which had significantly increased plasma ghrelin levels and expression of orexigenic neuropeptide Y/agouti-related peptide mRNA in the hypothalamus. The number of ghrelin-positive cells in the gastric body decreased in GERD rats, but the expression of gastric preproghrelin and GH secretagogue receptor mRNA was not affected. However, when ghrelin was exogenously administered to GERD rats treated with rikkunshito, a significant increase in antral motility was observed. These results suggest that gastrointestinal dysmotility is associated with impaired ghrelin signaling in GERD rats and that rikkunshito restores gastrointestinal motility by improving the ghrelin response.

10-Gingerol, a component of rikkunshito, improves cisplatin-induced anorexia by inhibiting acylated ghrelin degradation.

Rikkunshito (RKT), a Japanese traditional medicine, has been shown to stimulate food intake in rats with cisplatin-induced anorexia; however, the underlying mechanisms remain unknown. In this study, we investigated whether RKT is involved in the degradation of peripheral ghrelin. RKT inhibited decreases in plasma ghrelin level and enhanced acyl- to desacyl-ghrelin (A/D) ratio in cisplatin-treated rats. Several components of RKT demonstrated inhibitory activity against ghrelin deacylating enzymes. In addition, 10-gingerol, a component of RKT, inhibited exogenous ghrelin deacylation. Therefore, RKT may enhance plasma acyl-ghrelin level, at least in part, by inhibiting the circulating ghrelin degrading enzyme.

Decreased plasma ghrelin contributes to anorexia following novelty stress.

We hypothesized that anorexia induced by novelty stress caused by exposure to a novel environment may be due to activation of corticotropin-releasing factor (CRF) and subsequently mediated by decreasing peripheral ghrelin concentration via serotonin (5-HT) and melanocortin-4 receptors (MC4R). Each mouse was transferred from group-housed cages to individual cages to establish the novelty stress. We observed the effect of changes in feeding behavior in a novel environment using the method of transferring group-housed mice to individual cages. We investigated the effect of an intracerebroventricular injection of antagonists/agonists of CRF1/2 receptors (CRF1/2Rs), 5-HT(1B)/(2C) receptors (5-HT(1B)/(2C)R), and MC4R to clarify the role of each receptor on the decrease in food intake. Plasma ghrelin levels were also measured. The novelty stress caused a reduction in food intake that was abolished by administering a CRF1R antagonist. Three hours after the novelty stress, appetite reduction was associated with reduced levels of neuropeptide Y/agouti-related peptide mRNA, increased levels of proopiomelanocortin mRNA in the hypothalamus, and a decrease in plasma ghrelin level. Administering a CRF1R antagonist, a 5-HT(1B)/(2C)R antagonist, an MC4R antagonist, exogenous ghrelin, and an enhancer of ghrelin secretion, rikkunshito, resolved the reduction in food intake 3 h after the novelty stress by enhancing circulating ghrelin concentrations. We showed that anorexia during a novelty stress is a process in which CRF1R is activated at the early stage of appetite loss and is subsequently activated by a 5-HT(1B)/(2C)R and MC4R stimulus, leading to decreased peripheral ghrelin concentrations.