Effect of the Japanese herbal medicine Hochuekkito for systemic inflammation, prognostic nutrition index, and body composition status in patients with gastrointestinal cancer.

BACKGROUND & AIMS: A systemic inflammatory response via host-tumor interactions is a cancer hallmark that plays a pivotal role in the pathogenesis of malnutrition and sarcopenia in patients with malignancies. Hochuekkito (TJ-41) is a traditional Japanese herbal medicine that modulates inflammation in patients with various chronic inflammatory diseases. However, the clinical efficacy of TJ-41 in patients with malignancies remains unclear. METHODS: We systemically analyzed chronological changes in levels of systemic inflammatory parameters, nutrition-related parameters, and body composition status in 99 patients who received TJ-41 treatment for more than 3 months. The cohort comprised 56 patients with gastrointestinal cancer (Cancer Cohort) and 43 with other diseases (Other Disease Cohort). We also performed in vivo experiments in mice to validate the clinical findings. RESULTS: Despite no significant changes in serum albumin concentration and prognostic nutrition index, the serum C-reactive protein (CRP) concentration significantly decreased in a time-dependent manner in all patients. However, the serum CRP concentration significantly decreased during TJ-41 treatment in the Cancer Cohort but not the Other Disease Cohort. Furthermore, downregulation of CRP during TJ-41 treatment occurred only in patients with metastases. The psoas muscle index, as a muscle quantity marker, was significantly lower in the CRP-increased group compared with the CRP-decreased group during TJ-41 treatment. In vivo experiments using a Colon-26 syngeneic model showed that the plasma CRP, amyloid A, and interleukin-6 concentrations were significantly lower in the TJ-41 group than the control group. CONCLUSION: TJ-41 might be useful as part of multimodality therapy for gastrointestinal cancer, especially in patients with metastases.

Rikkunshito improves anorexia through ghrelin- and orexin-dependent activation of the brain hypothalamus and mesolimbic dopaminergic pathway in rats.

BACKGROUND: Rikkunshito (RKT), a traditional Japanese medicine, can relieve epigastric discomfort and anorexia in patients with functional dyspepsia. RKT enhances the orexigenic hormone, ghrelin. Ghrelin regulates food motivation by stimulating the appetite control center in the hypothalamus and the brain mesolimbic dopaminergic pathway (MDPW). However, the effect of RKT on MDPW remains unclear. Here, we aimed to investigate the central neural mechanisms underlying the orexigenic effects of RKT, focusing on the MDPW. METHODS: We examined the effects of RKT on food intake and neuronal c-Fos expression in restraint stress- and cholecystokinin octapeptide-induced anorexia in male rats. KEY RESULTS: RKT treatment significantly restored stress- and cholecystokinin octapeptide-induced decreased food intake. RKT increased c-Fos expression in the ventral tegmental area (VTA), especially in tyrosine hydroxylase-immunoreactive neurons, and nucleus accumbens (NAc). The effects of RKT were suppressed by the ghrelin receptor antagonist [D-Lys3]-GHRP-6. RKT increased the number of c-Fos/orexin-double-positive neurons in the lateral hypothalamus (LH), which project to the VTA. The orexin receptor antagonist, SB334867, suppressed RKT-induced increase in food intake and c-Fos expression in the LH, VTA, and NAc. RKT increased c-Fos expression in the arcuate nucleus and nucleus of the solitary tract of the medulla, which was inhibited by [D-Lys3]-GHRP-6. CONCLUSIONS & INFERENCES: RKT may restore appetite in subjects with anorexia through ghrelin- and orexin-dependent activation of neurons regulating the brain appetite control network, including the hypothalamus and MDPW.

Effect of Goreisan, a Traditional Japanese Medicine, on Rat Hindlimb Lymphedema.

Secondary lymphedema occurs after cancer surgery involving lymph node dissection owing to the lymphatic system dysfunction. However, the pathophysiology of lymphedema and the molecular pathways involved remain unknown. This study aimed to develop a rat hindlimb lymphedema model and investigate the mechanisms that drive pathophysiology and the effects of the traditional Japanese medicine goreisan on lymphedema. The rat lymphedema model was induced by combination surgeries of popliteal lymph node dissection, skin cautery incision, and fascial ablation coagulation in the right hindlimb using male Wistar rats. The foot volume was significantly increased, and recovery was delayed by combination surgeries. Dermal thickness and dilated lymphatic vessels of the hindlimb were observed on postoperative day 2. The number of infiltrating leukocytes (CD45+ cells), including CD4+ T-cells, increased in the lymphedema group compared with that in the sham group. The relative mRNA expression and protein levels of interleukin-6 (IL-6), CC chemokine ligand 2 (CCL2), transforming growth factor ß1 (TGF-ß1), and Fms-related receptor tyrosine kinase 4 (FLT4) were significantly higher in the lymphedema group than in the sham group. Foot volume was decreased by goreisan, furosemide, and prednisolone treatments. Goreisan diminished the increase in CD4+ T-cells, and the same trend was observed for CCL2 and FLT4 expression. In conclusion, the rat hindlimb lymphedema model in this study exhibited increased foot volume, skin-infiltrating cells, and pathological changes accompanied by inflammatory and fibrotic responses, suggesting that the model presented significant clinical features of lymphedema. Goreisan may exert a therapeutic effect on lymphedema by inhibiting CD4+ T-cell infiltration.

Unkeito promotes follicle development by restoring reduced follicle-stimulating hormone responsiveness in rats with polycystic ovary syndrome.

Background: Polycystic ovary syndrome (PCOS) is a common disorder resulting in irregular menstruation and infertility due to improper follicular development and ovulation. PCOS pathogenesis is mediated by downregulated follicle-stimulating hormone receptor (FSHR) expression in granulosa cells (GCs); however, the underlying mechanism remains elusive. Unkeito (UKT) is a traditional Japanese medicine used to treat irregular menstruation in patients with PCOS. In this study, we aimed to confirm the effectiveness of UKT in PCOS by focusing on follicle-stimulating hormone (FSH) responsiveness. Methods: A rat model of PCOS was generated by prenatal treatment with 5α-dihydrotestosterone. Female offspring (3-week-old) rats were fed a UKT mixed diet or a normal diet daily. To compare the PCOS phenotype in rats, the estrous cycle, hormone profiles, and ovarian morphology were evaluated. To further examine the role of FSH, molecular, genetic, and immunohistological analyses were performed using ovarian tissues and primary cultured GCs from normal and PCOS model rats. Results: UKT increased the number of antral and preovulatory follicles and restored the irregular estrous cycle in PCOS rats. The gene expression levels of FSHR and bone morphogenetic protein (BMP)-2 and BMP-6 were significantly decreased in the ovarian GCs of PCOS rats compared to those in normal rats. UKT treatment increased FSHR staining in the small antral follicles and upregulated Fshr and Bmps expression in the ovary and GCs of PCOS rats. There was no change in serum gonadotropin levels. In primary cultured GCs stimulated by FSH, UKT enhanced estradiol production, accompanied by increased intracellular cyclic adenosine monophosphate levels, and upregulated the expression of genes encoding the enzymes involved in local estradiol synthesis, namely Cyp19a1 and Hsd17b. Furthermore, UKT elevated the expression of Star and Cyp11a1, involved in progesterone production in cultured GCs in the presence of FSH. Conclusions: UKT stimulates ovarian follicle development by potentiating FSH responsiveness by upregulating BMP-2 and BMP-6 expression, resulting in the recovery of estrous cycle abnormalities in PCOS rats. Restoring the FSHR dysfunction in the small antral follicles may alleviate the PCOS phenotype.

Role of p53 in Cisplatin-Induced Myotube Atrophy.

Chemotherapy-induced sarcopenia is an unfavorable prognostic factor implicated in the development of postoperative complications and reduces the quality of life of patients with cancer. Skeletal muscle loss due to cisplatin use is caused by mitochondrial dysfunction and activation of muscle-specific ubiquitin ligases Atrogin-1 and muscle RING finger 1 (MuRF1). Although animal studies suggest the involvement of p53 in age-, immobility-, and denervation-related muscle atrophy, the association between cisplatin-induced atrophy and p53 remains unknown. Herein, we investigated the effect of a p53-specific inhibitor, pifithrin-alpha (PFT-α), on cisplatin-induced atrophy in C2C12 myotubes. Cisplatin increased the protein levels of p53, phosphorylated p53, and upregulated the mRNA expression of p53 target genes PUMA and p21 in C2C12 myotubes. PFT-α ameliorated the increase in intracellular reactive oxygen species production and mitochondrial dysfunction, and also reduced the cisplatin-induced increase in the Bax/Bcl-2 ratio. Although PFT-α also reduced the cisplatin-induced increase in MuRF1 and Atrogin-1 gene expression, it did not ameliorate the decrease in myosin heavy chain mRNA and protein levels and muscle-specific actin and myoglobin protein levels. We conclude that cisplatin increases muscle degradation in C2C12 myotubes in a p53-dependent manner, but p53 has minimal involvement in the reduction of muscle protein synthesis.

Intestinal hypomotility due to longitudinal enterotomy can be alleviated by transverse closure.

BACKGROUND: Heineke-Mikulicz (HM) strictureplasty is commonly used to treat short stenoses in Crohn's disease. However, the degree to which intestinal motility is maintained remains unclear. We compared the peristalsis and transport capacity of the sutured intestines with HM configuration and transverse (TS) and longitudinal (LS) incisions. METHODS: The intestinal diameter, intraluminal pressure, and bead transit time of each sutured group were compared with that of the non-treatment (NT) group in the isolated proximal colon of rats. Propulsive contractions were induced using hydroxy-?-sanshool (HAS), a constituent of Japanese pepper. RESULTS: There was no change in the intestinal diameter between HM, TS, and NT groups ; however, it was significantly narrowed at the suture site and its distal side in the LS group. After HAS administration, the intestinal diameter at the suture site in the HM group was higher than that in the LS group. The intraluminal pressure was higher and the transit time was shorter in the HM group compared to those in the LS group. CONCLUSIONS: The HM configuration, which widens the incision site and distal diameter and shortens the cut surface of the circular muscle in the longitudinal direction, may help maintain basal and HAS-induced intestinal peristalsis and motility. J. Med. Invest. 70 : 180-188, February, 2023.

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.

Integrated analysis of effect of daisaikoto, a traditional Japanese medicine, on the metabolome and gut microbiome in a mouse model of nonalcoholic fatty liver disease.

Dysregulation of lipid metabolism and diabetes are risk factors for nonalcoholic fatty liver disease (NAFLD), and the gut-liver axis and intestinal microbiome are known to be highly associated with the pathogenesis of this disease. In Japan, the traditional medicine daisaikoto (DST) is prescribed for individuals affected by hepatic dysfunction. Herein, we evaluated the therapeutic potential of DST for treating NAFLD through modification of the liver and stool metabolome and microbiome by using STAM mice as a model of NAFLD. STAM mice were fed a high-fat diet with or without 3 % DST for 3 weeks. Plasma and liver of STAM, STAM with DST, and C57BL/6J ("Normal") mice were collected at 9 weeks, and stools at 4, 6, and 9 weeks of age. The liver pathology, metabolome and stool microbiome were analyzed. DST ameliorated the NAFLD activity score of STAM mice and decreased the levels of several liver lipid mediators such as arachidonic acid and its derivatives. In normal mice, nine kinds of family accounted for 94.1 % of microbiome composition; the total percentage of these family was significantly decreased in STAM mice (45.6 %), and DST administration improved this imbalance in microbiome composition (65.2 %). In stool samples, DST increased ursodeoxycholic acid content and altered several amino acids, which were correlated with changes in the gut microbiome and liver metabolites. In summary, DST ameliorates NAFLD by decreasing arachidonic acid metabolism in the liver; this amelioration seems to be associated with crosstalk among components of the liver, intestinal environment, and microbiome.

Saireito, a Japanese herbal medicine, alleviates leaky gut associated with antibiotic-induced dysbiosis in mice.

Antibiotics disrupt normal gut microbiota and cause dysbiosis, leading to a reduction in intestinal epithelial barrier function. Disruption of the intestinal epithelial barrier, which is known as "leaky gut", results in increased intestinal permeability and contributes to the development or exacerbation of gastrointestinal diseases such as inflammatory bowel disease and irritable bowel syndrome. We have previously reported on a murine model of intestinal epithelial barrier dysfunction associated with dysbiosis induced by the administration of ampicillin and vancomycin. Saireito, a traditional Japanese herbal medicine, is often used to treat autoimmune disorders including ulcerative colitis; the possible mechanism of action and its efficacy, however, remains unclear. In this study, we examined the efficacy of Saireito in our animal model for leaky gut associated with dysbiosis. C57BL/6 mice were fed a Saireito diet for the entirety of the protocol (day1-28). To induce colitis, ampicillin and vancomycin were administered in drinking water for the last seven consecutive days (day22-28). As previously demonstrated, treatment with antibiotics caused fecal occult bleeding, cecum enlargement with black discoloration, colon inflammation with epithelial cell apoptosis, and upregulation of pro-inflammatory cytokines. Oral administration of Saireito significantly improved antibiotics-induced fecal occult bleeding and cecum enlargement by suppressing inflammation in the colon. Furthermore, Saireito treatment ensured the integrity of the intestinal epithelial barrier by suppressing apoptosis and inducing cell adhesion proteins including ZO-1, occludin, and E-cadherin in intestinal epithelial cells, which in turn decreased intestinal epithelial permeability. Moreover, the reduced microbial diversity seen in the gut of mice treated with antibiotics was remarkably improved with the administration of Saireito. In addition, Saireito altered the composition of gut microbiota in these mice. These results suggest that Saireito alleviates leaky gut caused by antibiotic-induced dysbiosis. Our findings provide a potentially new therapeutic strategy for antibiotic-related gastrointestinal disorders.

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.

Daisaikoto improves fatty liver and obesity in melanocortin-4 receptor gene-deficient mice via the activation of brown adipose tissue.

Melanocortin 4 receptor gene-knockout (MC4R-KO) mice are known to develop obesity with a high-fat diet. Meanwhile, daisaikoto, one of Kampo medicines, is a drug that is expected to have therapeutic effects on obesity. Here, we report the efficacy of daisaikoto in MC4R-KO mice. Eight-week-old MC4R-KO male mice (n = 12) were divided into three groups as follows: the SD group, which is fed with a standard diet; the HFD group, fed a high-fat diet; and the DSK group, fed with a high-fat diet containing 10% of daisaikoto. After the four-week observation period, mice in each group were sacrificed and samples were collected. The body weights at 12 weeks were significantly higher in the HFD group than in the other groups, indicating that daisaikoto significantly reduced body weight gain and fat deposition of the liver. The metabolome analysis indicated that degradation of triglycerides and fatty acid oxidation in the liver were enhanced by daisaikoto administration. In MC4R-KO mice, the cytoplasm and uncoupling protein 1 expression of brown adipose tissue was decreased; however, it was reversed in the DSK group. In conclusion, daisaikoto has potentially improved fatty liver and obesity, making it a useful therapeutic agent for obesity and fatty liver.

Data of RNA-seq transcriptomes of liver, bone, heart, kidney and blood in klotho mice at a pre-symptomatic state and the effect of a traditional Japanese multi-herbal medicine, juzentaihoto.

We performed RNA-seq analyses of mRNA isolated from five organs, liver, bone, heart, kidney and blood at the pre-symptomatic state of klotho mice with/without administration of a Japanese traditional herbal medicine, juzentaihoto (JTT). Data of differentially expressed genes (DEG) with/without JTT was included. Intron retention (IR) is an important regulatory mechanism that affects gene expression and protein functions. We collected data in which retained-introns were accumulated in a particular set of genes of these organs, and showed that among these retained introns in the liver and bone a subset was recovered to the normal state by the medicine. All of the data present changes of molecular events on the levels of metabolites, proteins and gene expressions observed at the pre- symptomatic state of aging in klotho mice with/without JTT. The research article related to this Data in Brief is published in GENE entitled as "Intron retention as a new pre-symptomatic marker of aging and its recovery to the normal state by a traditional Japanese herbal medicine".

Role of Mitochondrial Dysfunction in the Pathogenesis of Cisplatin-Induced Myotube Atrophy.

Muscle atrophy is commonly observed during cisplatin chemotherapy, leading to a reduced QOL in cancer patients. Reduced skeletal muscle mass caused by cisplatin treatment results from the activation of ubiquitin ligases-Atrogin-1 and MuRF1, but the precise mechanisms are poorly understood. In this study, we investigated the possible involvement of mitochondrial dysfunction, including reactive oxygen species (ROS) generation and ATP production, in cisplatin-induced muscle atrophy. Skeletal C2C12 myotubes were treated with cisplatin, and gene and protein expression were evaluated. Mitochondrial mass, membrane potential, and ROS levels were measured using fluorescent dyes. Mitochondrial respiratory function, ATP production rates, and glycolytic capacity were also analyzed using an extracellular flux analyzer. Metabolomic analyses were performed using gas chromatography-tandem mass spectrometry. Cisplatin treatment reduced myosin heavy chain expression by activating the ubiquitin-proteasome system. Increased ROS production was observed after cisplatin treatment, followed by significant changes in apoptosis-related gene expression and decrease in mitochondrial mass, membrane potential, respiration, and ATP production. Glycolytic capacity and tricarboxylic acid (TCA) cycle metabolite levels were reduced with cisplatin treatment. Mitochondria-targeted antioxidant mitoquinone mesylate prevented up-regulation of Atrogin-1 gene expression and restored myosin heavy chain levels, accompanied by a decrease in ROS generation, but not mitochondrial ATP production. We concluded that cisplatin-induced myotube atrophy was associated with mitochondrial dysfunction. Reducing ROS generation, rather than promoting ATP production, could be a useful therapeutic strategy for preventing cisplatin-induced muscle atrophy.

Decline in Liver Mitochondria Metabolic Function Is Restored by Hochuekkito Through Sirtuin 1 in Aged Mice With Malnutrition.

Malnutrition impairs basic daily activities and leads to physical frailty, which is aggravated in the elderly compared with young adults. It is also well-known that the elderly are more vulnerable to metabolic stress. Therefore, in this study, using a food restricted (FR) mouse, we aimed to evaluate the effect of aging on locomotor activity and liver metabolic function. Further, we also investigated the involvement of hepatic mitochondria in liver metabolic function during aging, as well as the therapeutic benefit of the traditional Japanese medicine, hochuekkito (HET). Our findings indicated that following food restriction provided as 30% of ad libitum intake for 5 days, the locomotor activity was lower in 23-26-month-old (aged) mice than in 9-week-old (young) mice. Further, compared with young mice, aged mice exhibited significant decreases in the levels of metabolites related to the urea cycle, mitochondrial function, and anti-oxidative stress. The livers of the aged mice also showed a greater decrease in mitochondrial DNA copy number than young mice. Furthermore, the gene expression levels of sirtuin 1 (SIRT1) and mitochondrial biogenesis-related regulators were attenuated in aged mice. However, these changes were partially restored by HET treatment, which also improved locomotor activity, and combined treatment with alanine resulted in more significant effects in this regard. Therefore, our findings suggested that the decrease in locomotor activity in aged FR mice was associated with a decline in the metabolic function of hepatic mitochondria via decreased SIRT1 expression, which was restored by HET treatment. This implies that enhancing the metabolic function of liver mitochondria can contribute to alleviating energy deficiency in the elderly.

Combination therapy of Juzentaihoto and mesenchymal stem cells attenuates liver damage and regresses fibrosis in mice.

BACKGROUND: Liver cirrhosis is an end-stage multiple liver disease. Mesenchymal stem cells (MSCs) are an attractive cell source for reducing liver damage and regressing fibrosis; additional therapies accompanying MSCs can potentially enhance their therapeutic effects. Kampo medicines exhibit anti-inflammatory and anti-oxidative effects. Here, we investigated the therapeutic effect of MSCs combined with the Kampo medicine Juzentaihoto (JTT) as a combination therapy in a carbon tetrachloride (CCl4)-induced cirrhosis mouse model. METHODS: C57BL/6 mice were administered JTT (orally) and/or MSCs (one time, intravenously). The levels of liver proteins were measured in the sera. Sirius Red staining and hydroxyproline quantitation of hepatic tissues and immune cells were conducted, and their associated properties were evaluated. Liver metabolomics of liver tissues was performed. RESULTS: JTT monotherapy attenuated liver damage and increased serum albumin level, but it did not effectively induce fibrolysis. JTT rapidly reduced liver damage, in a dose-dependent manner, after a single-dose CCl4 administration. Furthermore, JTT-MSC combination therapy attenuated liver damage, improved liver function, and regressed liver fibrosis. The combination increased the CD4+/CD8+ ratio. JTT had stronger effects on NK and regulatory T cell induction, whereas MSCs more strongly induced anti-inflammatory macrophages. The combination therapy further induced anti-inflammatory macrophages. JTT normalized lipid mediators, and tricarboxylic acid cycle- and urea cycle-related mediators effectively. CONCLUSIONS: The addition of JTT enhanced the therapeutic effects of MSCs; this combination could be a potential treatment option for cirrhosis.

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.

Bcl-2-dependent autophagy disruption during aging impairs amino acid utilization that is restored by hochuekkito.

Chronic undernutrition contributes to the increase in frailty observed among elderly adults, which is a pressing issue in the sector of health care for older people worldwide. Autophagy, an intracellular recycling system, is closely associated with age-related pathologies. Therefore, decreased autophagy in aging could be involved in the disruption of energy homeostasis that occurs during undernutrition; however, the physiological mechanisms underlying this process remain unknown. Here, we showed that 70% daily food restriction (FR) induced fatal hypoglycemia in 23-26-month-old (aged) mice, which exhibited significantly lower hepatic autophagy than 9-week-old (young) mice. The liver expressions of Bcl-2, an autophagy-negative regulator, and Beclin1-Bcl-2 binding, were increased in aged mice compared with young mice. The autophagy inducer Tat-Beclin1 D11, not the mTOR inhibitor rapamycin, decreased the plasma levels of the glucogenic amino acid and restored the blood glucose levels in aged FR mice. Decreased liver gluconeogenesis, body temperature, physical activity, amino acid metabolism, and hepatic mitochondrial dynamics were observed in the aged FR mice. These changes were restored by treatment with hochuekkito that is a herbal formula containing several autophagy-activating ingredients. Our results indicate that Bcl-2 upregulation in the liver during the aging process disturbs autophagy activation, which increases the vulnerability to undernutrition. The promotion of liver autophagy may offer clinical therapeutic benefits to frail elderly patients.

Intron retention as a new pre-symptomatic marker of aging and its recovery to the normal state by a traditional Japanese multi-herbal medicine.

Intron retention (IR) is an important regulatory mechanism that affects gene expression and protein functions. Using klotho mice at the pre-symptomatic state, we discovered that retained-introns accumulated in several organs including the liver and that among these retained introns in the liver a subset was recovered to the normal state by a Japanese traditional herbal medicine. This is the first report of IR recovery by a medicine. IR-recovered genes fell into two categories: those involved in liver-specific metabolism and in splicing. Metabolome analysis of the liver showed that the klotho mice were under starvation stress. In addition, our differentially expressed gene analysis showed that liver metabolism was actually recovered by the herbal medicine at the transcriptional level. By analogy with the widespread accumulation of intron-retained pre-mRNAs induced by heat shock stress, we propose a model in which retained-introns in klotho mice were induced by an aging stress and in which this medicine-related IR recovery is indicative of the actual recovery of liver-specific metabolic function to the healthy state. Accumulation of retained-introns was also observed at the pre-symptomatic state of aging in wild-type mice and may be an excellent marker for this state in general.

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.

Usefulness of a Kampo Medicine on Stress-Induced Delayed Gastric Emptying in Mice.

Anxiety and depression often occur with gastrointestinal symptoms. Although the Japanese traditional medicine (Kampo medicine) bukuryoingohangekobokuto (BGH) is approved for treating anxiety, neurotic gastritis, and heartburn, its effect on gastrointestinal motility remains poorly known. This study aimed to examine the effect of BGH on delayed gastric emptying in stress model mice and clarified its action mechanism. Seven-week-old C57BL/6 male mice were acclimated for a week and fasted overnight. Stress hormone, corticotropin-releasing factor (CRF), was intracerebroventricularly injected to mice, and solid nutrient meal (ground chow and distilled water) was orally administered 1 hour after. Gastric contents were collected to evaluate gastric emptying rates by measuring its dry weight. Injection of CRF (0.3 or 1.0 µg/mouse) significantly delayed the 2-hour gastric emptying in mice. BGH (1.0 g/kg), which was administered 30 minutes before the CRF injection, significantly ameliorated the delayed gastric emptying induced by CRF (0.3 µg/mouse). BGH (0.5, 1.0 g/kg) significantly enhanced the 1-hour gastric emptying and slightly increased the 2-hour gastric emptying in mice without CRF injection. In vitro functional assays showed that components of BGH antagonized or inhibited CRF type-2, dopamine D2/D3, neuropeptide Y Y2 receptors, or acetylcholinesterase. In conclusion, the components of BGH may exert synergistic effects on improving gastric emptying via various targets. BGH is considered to be potentially useful for treating gastrointestinal dysmotility with psychological symptoms.