Therapeutic effect and mechanism of Daikenchuto in a model of methotrexate-induced acute small intestinal mucositis.

BACKGROUND: Daikenchuto (DKT) has positive therapeutic effects on improving various gastrointestinal disorders. The present study investigated whether or not DKT has a potential therapeutic effect on chemotherapy-induced acute small intestinal mucositis (CIM) in a rat model. METHODS: Intraperitoneal injection of 10 mg/kg methotrexate (MTX) every 3 days for a total of 3 doses was used for induction of CIM in a rat model. The MTX and DKT-MTX groups were injected with MTX as above from the first day, and the DKT-MTX and DKT groups were administered 2.7% DKT via the diet at the same time. The rats were euthanized on day 15. RESULTS: The DKT-MTX group showed an improvement in the body weight and conditions of gastrointestinal disorders as well as increased levels of diamine oxidase in plasma and in the small intestinal villi. The pathology results showed that small intestinal mucosal injury in the DKT-MTX group was less severe than that in the MTX group. Immunohistochemistry for myeloperoxidase and malondialdehyde and quantitative real-time polymerase chain reaction (RT-qPCR) for TGF-ß1 and HIF-1α showed that DKT attenuated peroxidative damage. The crypts in the DKT-MTX group contained more Ki-67-positive cells than MTX group. The zonula occluden-1 and claudin-3 results showed that DKT promoted repair of the mucosal barrier. RT-qPCR for the amino acid transporters EAAT3 and BO+AT also confirmed that DKT promoted mucosal repair and thus promoted nutrient absorption. CONCLUSION: DKT protected against MTX-induced CIM in a rat model by reducing inflammation, stimulating cell proliferation, and stabilizing the mucosal barrier.

Associations between intestinal microbiota, fecal properties, and dietary fiber conditions: The Japanese traditional medicine Junchoto ameliorates dietary fiber deficit-induced constipation with F/B ratio alteration in rats.

Changes in the intestinal microbiota are known to occur in constipated patients. Dietary fiber restriction presents obstacles to appropriate defecation and affects fecal properties, but the relationship between fecal microbiota and fecal morphological properties remains obscure. Therefore, we examined the influence of fiber diets on the fecal microbiome and properties in rats, and the effectiveness of the Japanese traditional medicine Junchoto (JCT) in rats with fiber deficit-induced constipation. Rats were fed three different fiber diets with varying cellulose contents (0 %, FFD; 5 %, ND; 15 %, HFD), respectively, as follows: study 1: 21 days of feeding; study 2: 14 days of feeding followed by 7 days of ND (fiber normalization in all groups); study 3: FFD for 21 days, followed by JCT administration from 14 days. Fecal properties and 16S rRNA amplicon sequencing results were examined. We observed that the fecal frequency, dry weight, and length were increased, and water ratio were decreased in a cellulose dose-dependent manner. The difference in several kinds of fecal microbiota, but not the α-diversity Chao1 index and the Firmicutes/Bacteroidetes ratio (F/B ratio), between groups were observed. The change in fecal property in both the HFD and FFD groups was ameliorated with fiber normalization, accompanied by alteration of the Chao1 index and/or F/B ratio. JCT administration reversed the fecal morphological changes in FFD group, accompanied by F/B ratio increasing. In conclusion, short-term dietary changes modulated microbial homeostasis, which is linked to fecal property. JCT may alter the F/B ratio and improve fecal properties to facilitate easier excretion.

Calcium-activated chloride channel is involved in the onset of diarrhea triggered by EGFR tyrosine kinase inhibitor treatment in rats.

EGFR tyrosine kinase inhibitors (TKIs) are mainly used to treat non-small cell lung cancer; however, adverse effects such as severe diarrhea represent a major obstacle towards the continuation of EGFR-TKIs therapy. Chloride channels, which control the fluid flow in the intestinal lumen, are proposed as an important target to remediate EGFR-TKIs-induced diarrhea, but the mechanism remains unclear. The aim of this study was to clarify the mechanism underlying EGFR-TKIs-induced diarrhea with a particular focus on the role of intestinal chloride channels. Here, we show that osimertinib-treated rats exhibit diarrhea and an increase in fecal water content without showing any severe histopathological changes. This diarrhea was attenuated by intraperitoneal treatment with the calcium-activated chloride channel (CaCC) inhibitor CaCCinh-A01. These findings were confirmed in afatinib-treated rats with diarrhea. Moreover, treatment with the Japanese traditional herbal medicine, hangeshashinto (HST), decreased fecal water content and improved fecal appearance in rats treated with EGFR-TKIs. HST inhibited the ionomycin-induced CaCC activation in HEK293 cells in patch-clamp current experiments and its active ingredients were identified. In conclusion, secretory diarrhea induced by treatment with EGFR-TKIs might be partially mediated by the activation of CaCC. Therefore, blocking the CaCC could be a potential new treatment for EGFR-TKI-induced diarrhea.

Weak acids induce PGE2 production in human oesophageal cells: novel mechanisms underlying GERD symptoms.

The role of weak acids with pH values in the range of 4-7 has been implicated in the symptoms of gastroesophageal reflux disease (GERD). Prostaglandin E2 (PGE2) is associated with heartburn symptom in GERD patients; however, the precise productive mechanisms remain unclear. In this study, we revealed that exposure to weak acids increases PGE2 production with a peak at pH 4-5, slightly in human normal oesophageal cells (Het-1A), and robustly in oesophageal squamous carcinoma cells (KYSE-270). Release of PGE2 from the oesophageal mucosa was augmented by weak acid treatment in rat. Chenodeoxycholic acid (CDCA), a bile acid, upregulated cyclooxygenase-2 (COX-2) expression in Het-1A and KYSE-270 and induced PGE2 production in KYSE-270 cells. Weak acid-induced PGE2 production was significantly inhibited by cytosolic phospholipase A2 (cPLA2), ERK, and transient receptor potential cation channel subfamily V member 4 (TRPV4), a pH-sensing ion channel, inhibitors. Hangeshashinto, a potent inhibitor of COX-2, strongly decreased weak acid- and CDCA-induced PGE2 levels in KYSE-270. These results indicated that weak acids induce PGE2 production via TRPV4/ERK/cPLA2 in oesophageal epithelial cells, suggesting a role in GERD symptoms like heartburn. Interventions targeting pH values up to 5 may be necessary for the treatment of GERD.

Preventive effect of oral hangeshashinto (TJ-14) on the development of reflux-induced esophageal cancer.

BACKGROUND: Prostaglandin E2 is one of the potential products that promotes development of tumors and also is a strong inducer of M2 phenotype macrophages, which contribute to tumor development in the immunosuppressed microenvironment. Hangeshashinto (TJ-14), a Japanese traditional medicine (Kampo medicine), has been reported to be effective in preventing chemotherapy-induced oral mucositis through the reduction of prostaglandin E2. We previously developed a surgical rat reflux model of esophageal cancer and used this well-established animal model to investigate the action of TJ-14 in preventing esophageal cancer. We also assessed the effect of TJ-14 on the downregulation of prostaglandin E2 production, utilizing esophageal squamous cell carcinoma cell line exposed to bile acid. METHODS: An end-to-side esophagojejunostomy was performed for the reflux model. A daily oral diet was subsequently administered, consisting of either diet-incorporated TJ-14 or standard diet as a control group. The rats were killed at 40 weeks after surgery. The incidence of esophageal cancer, Barrett's metaplasia, and proliferative hyperplasia were assessed histologically. CD163, a M2 phenotype macrophage marker, was assessed with immunohistochemistry. Prostaglandin E2 enzyme immunoassay and lactate dehydrogenase assay were performed on chenodeoxycholic acid or gastroesophageal reflux contents exposed to esophageal squamous cell carcinoma cell line. RESULTS: Sixty-seven percent of the controls (n = 12) developed esophageal cancer, but animals that received TJ-14 (n = 10) had a cancer incidence of 10% (P=.007). Barrett's metaplasia was found in 83% of the rats in the control group and 50% of the rats in the TJ-14 indicating a protective tendency of TJ-14 (P=.095). All of the rats developed proliferative hyperplasia. The number of M2 phenotype macrophage were significantly decreased in the TJ-14 group compared to the control group in both Barrett's metaplasia and esophageal cancer lesions. TJ-14 inhibited chenodeoxycholic acid or gastroesophageal reflux content-induced prostaglandin E2 production in esophageal squamous cell carcinoma cell. CONCLUSION: TJ-14 reduced the incidence of reflux-induced esophageal cancer and the infiltration of M2 macrophages in a surgical rat model or suppressed prostaglandin E2 production in esophageal squamous cell carcinoma cell. Further investigation is required regarding the potential clinical use of TJ-14 as an esophageal cancer chemopreventive agent.