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.

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.

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.

Role of Bitter Taste Receptors in Regulating Gastric Accommodation in Guinea Pigs.

Taste stimulants play important roles in triggering digestion and absorption of nutrients and in toxin detection, under the control of the gut-brain axis. Bitter compounds regulate gut hormone secretion and gastrointestinal motility through bitter taste receptors (TAS2Rs) located in the taste buds on the tongue and in the enteroendocrine cells. Gastric accommodation (GA) is an important physiologic function. However, the role of TAS2R agonists in regulating GA remains unclear. To clarify whether GA is influenced by bitter stimulants, we examined the effect of TAS2R agonist denatonium benzoate (DB), administered intraorally and intragastrically, by measuring the consequent intrabag pressure in the proximal stomach of guinea pigs. Effects of the Kampo medicine rikkunshito (RKT) and its bitter components liquiritigenin and naringenin on GA were also examined. Intraoral DB (0.2 nmol/ml) administration enhanced GA. Intragastric DB administration (0.1 and 1 nmol/kg) promoted GA, whereas higher DB doses (30 µmol/kg) inhibited it. Similar changes in GA were observed with intragastric (1000 mg/kg) and intraoral (200 mg/ml) RKT administration. Liquiritigenin and naringenin also promoted GA. These findings suggest that GA is affected by the stimulation of TAS2Rs in the oral cavity or gut in guinea pigs.

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 30min 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.

Boiogito, a Kampo medicine, improves hydrarthrosis in a rat model of knee osteoarthritis.

BACKGROUND: Hydrarthrosis, which is associated with knee pain and limited range of motion, decreases the quality of life (QOL) of patients with osteoarthritis (OA). The Kampo medicine boiogito is prescribed for the treatment of knee OA with hydrarthrosis; however, its precise mechanisms of action remain unknown. The purposes of this study were to assess the pharmacological effects of boiogito and its mechanisms of action on joint effusion in rats with surgically induced OA. METHODS: A rat OA model was produced by transecting the anterior (cranial) cruciate ligament, medial collateral ligament, and medial meniscus in the right knee joints of 7-week-old female Wistar rats. The rats were given chow containing boiogito (1 or 2%) or indomethacin (0.002 %) for 4 weeks after surgical transection. Levels of interleukin-1ß (IL-1ß) and hyaluronic acid (HA) were measured by enzyme-linked immunosorbent assay. Knee joint pain was assessed using an incapacitance tester. Osmotic water permeability in cultured rabbit synovial cells was assessed using stopped-flow analysis. RESULTS: Increased synovial fluid volume and knee joint pain were observed in rats with surgically induced OA. In rats with OA, levels of IL-1ß and HA in the articular cavity were higher but concentration of HA in synovial fluid was lower than in sham-operated rats, suggesting excessive synovial fluid secretion. Administration of boiogito improved hydrarthrosis, IL-1ß, and HA concentrations and alleviated knee joint pain in rats with OA. Indomethacin reduced IL-1ß and knee joint pain but failed to improve hydrarthrosis or HA concentration in rats with OA. Osmotic water permeability in synovial cells, which is related to the function of the water channel aquaporin, was decreased by treatment with boiogito. CONCLUSION: Boiogito ameliorates the increased knee joint effusion in rats with OA by suppressing pro-inflammatory cytokine IL-1ß production in the articular cavity and regulating function of water transport in the synovium. The improvement of hydrarthrosis by boiogito results in the increased HA concentration in synovial fluid, thus reducing joint pain. Boiogito may be a clinically useful treatment of QOL in patients with OA with hydrarthrosis.

Cancer cachexia pathophysiology and translational aspect of herbal medicine.

About half of all cancer patients show a syndrome of cachexia, characterized by anorexia and loss of adipose tissue and skeletal muscle mass. Numerous cytokines have been postulated to play a role in the etiology of cancer cachexia. Cytokines can elicit effects that mimic leptin signaling and suppress orexigenic ghrelin and neuropeptide Y signaling, inducing sustained anorexia and cachexia not accompanied by the usual compensatory response. Furthermore, cytokines have been implicated in the induction of cancer-related muscle wasting. In particular, tumor necrosis factor-alpha, interleukin-1, interleukin-6 and interferon-gamma have been implicated in the induction of cancer-related muscle wasting. Cytokine-induced skeletal muscle wasting is probably a multifactorial process, which involves a depression in protein synthesis, an increase in protein degradation or a combination of both. Cancer patients suffer from the reduction in physical function, tolerance to anti-cancer therapy and survival, while many effective chemotherapeutic agents for cancer are burdened by toxicities that can reduce patient's quality of life or hinder their effective use. Herbal medicines have been widely used to help improve such conditions. Recent studies have shown that herbal medicines such as rikkunshito enhance ghrelin signaling and consequently improve nausea, appetite loss and cachexia associated with cancer or cancer chemotherapy, which worsens the quality of life and life expectancy of the patients. The multicomponent herbal medicines capable of targeting multiple sites could be useful for future drug discovery. Mechanistic studies and identification of active compounds could lead to new discoveries in biological and biomedical sciences.

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.

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.

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.

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.

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".

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.

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.

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.

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.

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.