CRF receptor 1 antagonism and brain distribution of active components contribute to the ameliorative effect of rikkunshito on stress-induced anorexia

www.nature.com/scientificreports OPEN received: 16 September 2015 accepted: 09 May 2016 Published: 08 June 2016 CRF receptor 1 antagonism and brain distribution of active components contribute to the ameliorative effect of rikkunshito on stress-induced anorexia Sachiko Mogami1, Chiharu Sadakane1, Miwa Nahata1, Yasuharu Mizuhara1, Chihiro Yamada1, Tomohisa Hattori1 & Hiroshi Takeda2,3 Rikkunshito (RKT), a Kampo medicine, has been reported to show an ameliorative effect on sustained hypophagia after novelty stress exposure in aged mice through serotonin 2C receptor (5-HT2CR) antagonism. We aimed to determine (1) whether the activation of anorexigenic neurons, corticotropinreleasing factor (CRF), and pro-opiomelanocortin (POMC) neurons, is involved in the initiation of hypophagia induced by novelty stress in aged mice; (2) whether the ameliorative effect of RKT is associated with CRF and POMC neurons and downstream signal transduction; and (3) the plasma and brain distribution of the active components of RKT. The administration of RKT or 5-HT2CR, CRF receptor 1 (CRFR1), and melanocortin-4 receptor antagonists significantly restored the decreased food intake observed in aged male C57BL/6 mice in the early stage after novelty stress exposure. Seven components of RKT exhibited antagonistic activity against CRFR1. Hesperetin and isoliquiritigenin, which showed antagonistic effects against both CRFR1 and 5-HT2CR, were distributed in the plasma and brain of male Sprague-Dawley rats after a single oral administration of RKT. In conclusion, the ameliorative effect of RKT in this model is assumed to be at least partly due to brain-distributed active components possessing 5-HT2CR and CRFR1 antagonistic activities. Late-life anxiety and depression is a social problem in aging societies, and the recognition of this condition and adequate treatment are increasingly required1. Adverse life events are associated with the onset and persistence of depression2, which is characterized by feeding abnormalities (anorexia and bulimia). In particular, the co-occurrence of depression and anorexia may influence morbidity and progressive physical disability in the elderly3,4. Japanese traditional (herbal) or “Kampo” medicines are standardized with regard to the quality and quantity of their ingredients and have been approved by the Japanese Ministry of Health and Welfare. At present, almost 90% of physicians in Japan use Kampo medicines in their daily practice, sometimes as the first choice for treatment5,6. Rikkunshito (RKT) is a Kampo medicine that is often prescribed for anorexia and upper gastrointestinal disorders7–9. In addition, several multicenter, double-blind, randomized placebo-controlled studies have been conducted to examine its effects10,11. Various non-clinical studies of RKT have also been performed and have demonstrated its ameliorative effects on hypophagia and gastrointestinal dysmotility12. We have previously shown that RKT improves the sustained decrease in food intake that occurs after novelty stress exposure in aged mice and that its effect are associated with serotonin 2C receptor (5-HT2CR) antagonism13,14. It has also been reported that several RKT components possess 5-HT2CR antagonistic activities in vitro13,15. However, the inhibitory activities of these components are assumed to be insufficient to explain the complete set of effects of RKT, indicating the involvement of another mechanism of action. 1 Tsumura Research Laboratories, Tsumura & Co., Ibaraki 300-1192, Japan. 2Pathophysiology and Therapeutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido 060-0812, Japan. 3Hokkaido University Hospital Gastroenterological Medicine, Sapporo, Hokkaido 060-8648, Japan. Correspondence and requests for materials should be addressed to T.H. (email: ) Scientific Reports | 6:27516 | DOI: 10.1038/srep27516 1 www.nature.com/scientificreports/ Figure 1. Effect of RKT and a 5-HT2CR antagonist on novelty stress-induced hypophagia. Aged male mice (77 ± 8 weeks old) were deprived of food overnight and orally administered the 5-HT2CR antagonist SB242084 (6 mg kg−1), RKT (1000 mg kg−1) or DW. Immediately after administration, the mice were exposed to novelty stress and their cumulative food intake was measured. The data are presented as the mean ±  SEM (n =  5). *,***p <  0.05, 0.001, respectively, vs. the stress group based on Steel’s test at 1 h and Dunnett’s test at 3 h. DW, distilled water; RKT, rikkunshito. There is intimate interplay between 5-HT2CRs and anorexigenic corticotropin-releasing factor (CRF) neurons. The administration of 5-HT2CR agonists leads to increased levels of stress hormones in the blood16. Moreover, CRF-containing neurons in the paraventricular nucleus of the hypothalamus (PVN) co-express 5-HT2CR mRNA17. Conversely, the CRF-induced decrease in orexigenic hormone secretion is restored by 5-HT2CR antagonism18, indicating that CRF and 5-HT2CR signals influence each other. Furthermore, it has been reported that 5-HT2CR is also expressed in anorexigenic pro-opiomelanocortin (POMC) neurons in the arcuate nucleus and that 5-HT2CR activation promotes α-melanocyte-stimulating hormone (α-MSH) production19–21. This production contributes to appetite suppression via melanocortin-4 receptor (MC4R) activation. However, it remains to be determined whether the ameliorative effects of RKT are associated with these anorexigenic neurons and downstream signal transduction. Furthermore, whether RKT components exert any effects on CRF receptors (CRFRs) or MC4R is unclear to date. Various active components of RKT have been previously identified, and their 5-HT2CR antagonistic effects have been described13,15. Recently, the plasma levels of 32 RKT components were characterized in healthy volunteers after a single oral administration22. However, it is crucial to determine whether the active components of RKT penetrate the blood–brain barrier (BBB) if RKT exerts its effect on CRF and POMC neurons in the brain that are considered to be involved in the stress response. The brain distribution of RKT components in humans can only be estimated based on their distribution in the cerebrospinal fluid or determined through positron emission tomography. However, such analyses are difficult to perform for RKT because it contains various types of components. The present study was conducted to determine 1) whether the activation of anorexigenic CRF or POMC neurons is involved in the initiation of hypophagia induced by novelty stress in aged mice; 2) whether the ameliorative effect of RKT is associated with anorexigenic CRF or POMC neurons and downstream signal transduction; and 3) the plasma and brain distributions of RKT active components. Results Effects of RKT and a 5-HT2CR antagonist on the decrease in cumulative food intake in aged male mice after novelty stress exposure under fasting conditions. Cumulative food intake over 1 and 3 h was significantly decreas (...truncated)