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.

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.

Lymphocytic infundibuloneurohypophysitis with positive anti-rabphilin-3A antibodies nine years post-onset of central diabetes insipidus.

Childhood-onset lymphocytic infundibuloneurohypophysitis (LINH) due to infiltration of autoimmune lymphocyte in the neurohypophysis is rarely reported. Its definitive diagnosis requires a pituitary biopsy, which is an invasive procedure. Recently, anti-rabphilin-3A antibody has been reported as a potential diagnostic marker for LINH in adults; however, only a few cases have been reported in children. Here, we present a case of childhood-onset LINH in a 10-yr-old boy identified as anti-rabphilin-3A antibody positive during chronic phase, 9 yr post-onset of central diabetes insipidus (CDI). T1-weighted magnetic resonance imaging (MRI) revealed pituitary stalk thickening and absence of posterior pituitary bright signal spot, and the hormonal responses of the adenohypophysis to GHRH, TRH, CRH, and LHRH revealed no abnormalities during the first admission. MRI at 5 mo post-onset indicated reduced stalk swelling; however, replacement treatment with intranasal desmopressin was continued to counter unimproved CDI. Additionally, GH replacement therapy was also initiated to counter its deficiency. Pituitary re-enlargement was not observed in the subsequent routine MRI, and no increase was observed in the levels of tumor markers during follow-up, which was considered clinically consistent with LINH. Our case study suggests that anti-rabphilin-3A antibody may be considered as a useful diagnostic marker for LINH in children.

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.

Preventive effect of oral goshajinkigan on chronic oxaliplatin-induced hypoesthesia in rats.

Oxaliplatin, a widely used chemotherapeutic agent, induces peripheral neuropathy that manifests itself as two distinct phases: acute cold hyperesthesia and chronic peripheral hypoesthesia/dysesthesia. The latter is a serious dose-limiting side effect that can often lead to withdrawal of treatment. We have developed a rat model expressing both phases and used the model to investigate the action of goshajinkigan (GJG), a traditional Japanese herbal medicine, which was reported to ameliorate oxaliplatin-induced neuropathy in a placebo-controlled double-blind randomized phase II study. In this study, neuropathy was induced by injection of oxaliplatin twice weekly for 8 wks. The maximum level of cold hyperesthesia was observed at 4 wks with heat hypoesthesia developing later. Microscopy studies revealed atrophy of axons of myelinated sciatic nerve fibers in oxaliplatin-treated rats at 8 wks. Co-administration of GJG ameliorated both abnormal sensations as well as histological damage to the sciatic nerve. A pharmacokinetic study revealed numerous neuroprotective components of GJG that are rapidly absorbed into the blood. GJG and some of its components attenuated the generation of oxaliplatin-induced reactive oxygen species, which is a possible mechanism of oxaliplatin-induced neurotoxicity. The present study provides a useful animal model for oxaliplatin-induced neurotoxicity as well as a promising prophylactic agent.

Focal bacterial nephritis without pyuria in a boy presenting with high urinary ß2-MG and NAG levels.

Acute focal bacterial nephritis (AFBN) is a localized bacterial infection of the kidney presenting as an inflammatory mass without frank abscess formation. We report a case of acute focal bacterial nephritis without pyuria in a five-month-old boy presenting with high urinary ß2-microglobulin (ß2-MG) and N-Acetyl-ß-(D)-Glucosaminidase (NAG) levels. The infant initially presented with high-grade fever, and plain computed tomography (CT) showed a nearly isodense mass, and contrast-enhanced abdominal CT showed a wedge-shaped hypodense lesion. Enterococcus was detected in the subsequent urine culture. A diagnosis of AFBN was made on the basis of his high inflammatory reaction, contrast-enhanced abdominal CT findings and high urinary ß2-MG and NAG levels. He was treated with Ceftriaxone and subsequent improvement in inflammatory reaction and contrast-enhanced CT findings were observed. Voiding cystourethrogram (VCUG) showed a grade V right VUR. This case suggests that urinary ß2-MG and NAG levels may be useful additional markers for the diagnosis of AFBN without pyuria.

Assembly of self-assembled monolayer-coated Al2O3 on TiO2 thin films for the fabrication of renewable superhydrophobic-superhydrophilic structures.

A renewable superhydrophobic-superhydrophilic pattern with a minimum dimension of 50 microm is prepared from octadecyltrimethoxysilane self-assembled monolayer-covered superhydrophobic Al2O3 overlayers on a superhydrophilic TiO2 surface via self-assembly and calcination of boehmite (AlOOH.nH2O) particles. The resulting Al2O3 layer plays dual roles as a superhydrophobic layer and as a UV-blocking layer for the underlying TiO2.