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.

[6]-gingerol and [6]-shogaol, active ingredients of the traditional Japanese medicine hangeshashinto, relief oral ulcerative mucositis-induced pain via action on Na+ channels.

The traditional Japanese herbal medicine hangeshashinto (HST) has beneficial effects for the treatment of oral ulcerative mucositis (OUM) in cancer patients. However, the ingredient-based mechanism that underlies its pain-relieving activity remains unknown. In the present study, to clarify the analgesic mechanism of HST on OUM-induced pain, we investigated putative HST ingredients showing antagonistic effects on Na+ channels in vitro and in vivo. A screen of 21 major ingredients using automated patch-clamp recordings in channel-expressing cells showed that [6]-gingerol and [6]-shogaol, two components of a Processed Ginger extract, considerably inhibited voltage-activated Na+ currents. These two ingredients inhibited the stimulant-induced release of substance P and action potential generation in cultured rat sensory neurons. A submucosal injection of a mixture of [6]-gingerol and [6]-shogaol increased the mechanical withdrawal threshold in healthy rats. In a rat OUM model, OUM-induced mechanical pain was alleviated 30min after the swab application of HST despite the absence of anti-bacterial and anti-inflammatory actions in the OUM area. A swab application of a mixture of [6]-gingerol and [6]-shogaol induced sufficient analgesia of OUM-induced mechanical or spontaneous pain when co-applied with a Ginseng extract containing abundant saponin. The Ginseng extract demonstrated an acceleration of substance permeability into the oral ulcer tissue without an analgesic effect. These findings suggest that Na+ channel blockage by gingerol/shogaol plays an essential role in HST-associated analgesia of OUM-induced pain. This pharmacological mechanism provides scientific evidence supporting the use of this herbal medicine in patients suffering from OUM-induced pain.

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.

Plasma Pharmacokinetics of Polyphenols in a Traditional Japanese Medicine, Jumihaidokuto, Which Suppresses Propionibacterium acnes-Induced Dermatitis in Rats.

Most orally administered polyphenols are metabolized, with very little absorbed as aglycones and/or unchanged forms. Metabolic and pharmacokinetic studies are therefore necessary to understand the pharmacological mechanisms of polyphenols. Jumihaidokuto (JHT), a traditional Japanese medicine, has been used for treatment of skin diseases including inflammatory acne. Because JHT contains various types of bioactive polyphenols, our aim was to clarify the metabolism and pharmacokinetics of the polyphenols in JHT and identify active metabolites contributing to its antidermatitis effects. Orally administered JHT inhibited the increase in ear thickness in rats induced by intradermal injection of Propionibacterium acnes. Quantification by LC-MS/MS indicated that JHT contains various types of flavonoids and is also rich in hydrolysable tannins, such as 1,2,3,4,6-penta-O-galloyl glucose. Pharmacokinetic and antioxidant analyses showed that some flavonoid conjugates, such as genistein 7-O-glucuronide and liquiritigenin 7-O-glucuronide, appeared in rat plasma and had an activity to inhibit hydrogen peroxide-dependent oxidation. Furthermore, 4-O-methylgallic acid, a metabolite of Gallic acid, appeared in rat plasma and inhibited the nitric oxide reaction. JHT has numerous polyphenols; it inhibited dermatitis probably via the antioxidant effect of its metabolites. Our study is beneficial for understanding in vivo actions of orally administered polyphenol drugs.

Rikkunshito ameliorates bleomycin-induced acute lung injury in a ghrelin-independent manner.

Acute lung injury (ALI) is a critical syndrome consisting of acute respiratory failure associated with extensive pulmonary infiltrates. The pathological characterization of ALI includes injuries of alveolar epithelial cells (AECs), alveolar neutrophilic infiltration, and increases in proinflammatory cytokines, which cause destruction of the alveolar capillary barrier and subsequent devastating lung fibrosis. Rikkunshito (RKT), a traditional Japanese herbal medicine, is widely used for the treatment of patients with gastrointestinal symptoms and is known to stimulate ghrelin secretion. The therapeutic effects of RKT on organ inflammation and fibrosis remain unknown. We investigated the pharmacological potential of RKT in the treatment of ALI by using a bleomycin-induced ALI model in mice. RKT or distilled water (DW) was given to mice daily starting 12 h after bleomycin administration. The RKT-treated mice showed a definitively higher survival rate than the DW-treated mice after injury. They also had smaller reductions in body weight and food intake. The amelioration of neutrophil alveolar infiltration, pulmonary vascular permeability, induction of proinflammatory cytokines, activation of the NF-κB pathway, apoptosis of AECs, and subsequent lung fibrosis were notable in the RKT-treated mice. RKT administration increased the plasma ghrelin levels in wild-type mice, and it also mitigated the ALI response in both ghrelin-deficient mice and growth hormone secretagogue receptor-deficient mice after lung injury. Our results indicate that RKT administration exerts protective effects against ALI by protecting the AECs and regulating lung inflammation independently of the ghrelin system, and they highlight RKT as a promising therapeutic agent for the management of this intractable disease.

Impaired ghrelin signaling is associated with gastrointestinal dysmotility in rats with gastroesophageal reflux disease.

Gastroesophageal reflux disease (GERD) is often associated with decreased upper gastrointestinal motility, and ghrelin is an appetite-stimulating hormone known to increase gastrointestinal motility. We investigated whether ghrelin signaling is impaired in rats with GERD and studied its involvement in upper gastrointestinal motility. GERD was induced surgically in Wistar rats. Rats were injected intravenously with ghrelin (3 nmol/rat), after which gastric emptying, food intake, gastroduodenal motility, and growth hormone (GH) release were investigated. Furthermore, plasma ghrelin levels and the expression of ghrelin-related genes in the stomach and hypothalamus were examined. In addition, we administered ghrelin to GERD rats treated with rikkunshito, a Kampo medicine, and examined its effects on gastroduodenal motility. GERD rats showed a considerable decrease in gastric emptying, food intake, and antral motility. Ghrelin administration significantly increased gastric emptying, food intake, and antral and duodenal motility in sham-operated rats, but not in GERD rats. The effect of ghrelin on GH release was also attenuated in GERD rats, which had significantly increased plasma ghrelin levels and expression of orexigenic neuropeptide Y/agouti-related peptide mRNA in the hypothalamus. The number of ghrelin-positive cells in the gastric body decreased in GERD rats, but the expression of gastric preproghrelin and GH secretagogue receptor mRNA was not affected. However, when ghrelin was exogenously administered to GERD rats treated with rikkunshito, a significant increase in antral motility was observed. These results suggest that gastrointestinal dysmotility is associated with impaired ghrelin signaling in GERD rats and that rikkunshito restores gastrointestinal motility by improving the ghrelin response.

Role of transient receptor potential ankyrin 1 in gastric accommodation in conscious guinea pigs.

We report the establishment of a new model for measuring gastric tone and liquid meal-induced accommodation in conscious guinea pigs and the role played by transient receptor potential ankyrin 1 (TRPA1). An indwelling polyethylene bag was placed in proximal stomachs of 5-week-old male Hartley guinea pigs. Gastric tone was measured by distending the bag and recording changes in intrabag pressure at various volumes. Gastric accommodation was measured by administering liquid meals and recording intrabag pressure over time. N(ω)-nitro-L-arginine methyl ester hydrochloride (L-NAME) (a nitric-oxide synthase inhibitor), atropine sulfate (atropine) (a muscarinic receptor antagonist), allyl isothiocyanate (AITC) (a TRPA1 agonist), or theophylline-7-(N-4-isopropylphenyl) acetamide (HC-030031) (a selective TRPA1 antagonist) was administered 15 to 60 min before measurement. Gastric tone was increased by stepwise distension of the bag and was further significantly increased by L-NAME and significantly decreased by atropine. A liquid meal (15% w/v; 1.7 kcal) significantly decreased intrabag pressure 5 to 20 min after administration, indicating gastric accommodation; this was completely suppressed by L-NAME and further enhanced by atropine. AITC significantly increased gastric tone; this increase was decreased by HC-030031 and atropine. A combination of AITC and L-NAME significantly increased gastric tone compared with L-NAME alone. HC-030031 alone significantly decreased gastric tone. Liquid meal-induced gastric accommodation was significantly suppressed by pretreatment with AITC. We established a new model for measuring gastric tone and accommodation in conscious guinea pigs. TRPA1 activation suppresses gastric accommodation by increasing gastric tone through cholinergic neuronal pathways.

Urocortin 1 reduces food intake and ghrelin secretion via CRF(2) receptors.

Although it is known that urocortin 1 (UCN) acts on both corticotropin-releasing factor receptors (CRF(1) and CRF(2)), the mechanisms underlying UCN-induced anorexia remain unclear. In contrast, ghrelin, the endogenous ligand for the growth hormone secretagogue receptor, stimulates food intake. In the present study, we examined the effects of CRF(1) and CRF(2) receptor antagonists (CRF(1)a and CRF(2)a) on ghrelin secretion and synthesis, c-fos mRNA expression in the caudal brain stem, and food intake following intracerebroventricular administration of UCN. Eight-week-old, male Sprague-Dawley rats were used after 24-h food deprivation. Acylated and des-acylated ghrelin levels were measured by enzyme-linked immunosorbent assay. The mRNA expressions of preproghrelin and c-fos were measured by real-time RT-PCR. The present study provided the following important insights into the mechanisms underlying the anorectic effects of UCN: 1) UCN increased acylated and des-acylated ghrelin levels in the gastric body and decreased their levels in the plasma; 2) UCN decreased preproghrelin mRNA levels in the gastric body; 3) UCN-induced reduction of plasma ghrelin and food intake were restored by CRF(2)a but not CRF(1)a; 4) UCN-induced increase of c-fos mRNA levels in the caudal brain stem containing the nucleus of the solitary tract (NTS) was inhibited by CRF(2)a; and 5) UCN-induced reduction of food intake was restored by exogenous ghrelin and rikkunshito, an endogenous ghrelin secretion regulator. Thus, UCN increases neuronal activation in the caudal brain stem containing NTS via CRF(2) receptors, which may be related to UCN-induced inhibition of both ghrelin secretion and food intake.

Decreased plasma ghrelin contributes to anorexia following novelty stress.

We hypothesized that anorexia induced by novelty stress caused by exposure to a novel environment may be due to activation of corticotropin-releasing factor (CRF) and subsequently mediated by decreasing peripheral ghrelin concentration via serotonin (5-HT) and melanocortin-4 receptors (MC4R). Each mouse was transferred from group-housed cages to individual cages to establish the novelty stress. We observed the effect of changes in feeding behavior in a novel environment using the method of transferring group-housed mice to individual cages. We investigated the effect of an intracerebroventricular injection of antagonists/agonists of CRF1/2 receptors (CRF1/2Rs), 5-HT(1B)/(2C) receptors (5-HT(1B)/(2C)R), and MC4R to clarify the role of each receptor on the decrease in food intake. Plasma ghrelin levels were also measured. The novelty stress caused a reduction in food intake that was abolished by administering a CRF1R antagonist. Three hours after the novelty stress, appetite reduction was associated with reduced levels of neuropeptide Y/agouti-related peptide mRNA, increased levels of proopiomelanocortin mRNA in the hypothalamus, and a decrease in plasma ghrelin level. Administering a CRF1R antagonist, a 5-HT(1B)/(2C)R antagonist, an MC4R antagonist, exogenous ghrelin, and an enhancer of ghrelin secretion, rikkunshito, resolved the reduction in food intake 3 h after the novelty stress by enhancing circulating ghrelin concentrations. We showed that anorexia during a novelty stress is a process in which CRF1R is activated at the early stage of appetite loss and is subsequently activated by a 5-HT(1B)/(2C)R and MC4R stimulus, leading to decreased peripheral ghrelin concentrations.

10-Gingerol, a component of rikkunshito, improves cisplatin-induced anorexia by inhibiting acylated ghrelin degradation.

Rikkunshito (RKT), a Japanese traditional medicine, has been shown to stimulate food intake in rats with cisplatin-induced anorexia; however, the underlying mechanisms remain unknown. In this study, we investigated whether RKT is involved in the degradation of peripheral ghrelin. RKT inhibited decreases in plasma ghrelin level and enhanced acyl- to desacyl-ghrelin (A/D) ratio in cisplatin-treated rats. Several components of RKT demonstrated inhibitory activity against ghrelin deacylating enzymes. In addition, 10-gingerol, a component of RKT, inhibited exogenous ghrelin deacylation. Therefore, RKT may enhance plasma acyl-ghrelin level, at least in part, by inhibiting the circulating ghrelin degrading enzyme.

Decreased motility of the lower esophageal sphincter in a rat model of gastroesophageal reflux disease may be mediated by reductions of serotonin and acetylcholine signaling.

To elucidate the altered function of the lower esophageal sphincter (LES) in gastroesophageal reflux disease (GERD), we evaluated the motility proximal to LES using force transducers, contraction and relaxation responses to neurotransmitters in LES strips, and gene expression of neurotransmitter receptors in GERD rats. Force transducers were applied to the proximal LES, and contraction of the LES was monitored during free moving. In addition, LES was isolated from sham-operated and GERD rats to investigate the LES function in an organ bath, and to determine gene expression. The in vivo motility proximal to LES (% motility index) in conscious rats was decreased by atropine treatment and increased by cisapride (5-HT(4) receptor agonist) treatment. Acetylcholine- and serotonin (5-HT)-induced LES contraction and sodium nitroprusside-induced relaxation in LES strips of GERD rats markedly decreased compared to sham-operated rats. The mRNA expressions of 5-HT(4) and muscarinic acetylcholine 3 receptors were significantly reduced in esophageal LES strips of GERD rats compared with sham-operated rats. Intraperitoneal administration of cisapride improves the erosive damage in the esophagus in GERD rats. It is suggested that the reduction of 5-HT-induced contraction in LES strips in GERD rats may be partly due to the decrease in 5-HT(4)-receptor activation. The reduction of LES function may be due to the decrease in neurotransmitters signal transduction, leading to the deterioration of histopathological damage in GERD.

Effects of TJN-598, a new selective phosphodiesterase type IV inhibitor on anti-Thy1 nephritis in rats.

BACKGROUND: Phosphodiesterase type IV (PDEIV) plays an important role in the immune response and inflammation. However, it is well known that classical PDEIV inhibitors have systemic side effects, so the clinical and chronic use of these agents as therapy for glomerulonephritis is difficult. This study was performed to elucidate the anti-nephritic effects of TJN-598, a new chemical compound derived from herbal components, on experimental mesangial proliferative glomerulonephritis. METHODS: We first examined the effects of TJN-598 and captopril on mesangial expansion induced by anti-Thy1 serum in rats. Second, to investigate the effects of TJN-598 and rolipram, which are typical PDEIV inhibitors, on the production of tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-ß1, glomeruli were isolated from rats with anti-Thy1 nephritis and incubated with the test drugs in vitro for 48 h. RESULTS: Treatment with TJN-598 prevented an increase in the mesangial area/total glomerular area, in the number of cells in the glomerular cross section and matrix index. TJN-598 also inhibited the increases in the expression of α-smooth muscle actin, the TGF-ß1-positive area, in the number of ED-1 positive cells and proliferating cell nuclear antigen-positive cells in the glomeruli. Furthermore, administration of TJN-598 inhibited increases in the levels of TGF-ß1 protein derived from glomeruli with anti-Thy-1 nephritis. The addition of both TJN-598 and rolipram to the culture supernatant inhibited both increased expression of TGF-ß1 and increases in levels of TNF-α in glomeruli isolated from rats with anti-Thy1 nephritis in a dose-dependent manner. CONCLUSION: These results suggest that TJN-598, a PDEIV inhibitor, is effective against expansion of mesangial cells, via the suppression of secretion of TGF-ß1 and TNF-α from inflamed glomeruli.

Ghrelin Enhancer, the Latest Evidence of Rikkunshito.

Rikkunshito is a Japanese herbal medicine (Kampo) that has been attracting attention and researched by many researchers not only in Japan but also worldwide. There are 214 rikkunshito articles that can be searched on PubMed by August 2021. The reason why rikkunshito has attracted so much attention is due to an epoch-making report (Gastroenterology, 2008) discovered that rikkunshito promotes the secretion of the orexigenic peptide ghrelin. Since then, many researchers have discovered that rikkunshito has a direct effect on the ghrelin receptor, GHS-R1a, and an effect of enhancing the ghrelin signal to the brain. Additionally, a lot of evidence that rikkunshito is expected to be effective for various gastrointestinal diseases have also been demonstrated. Numerous basic and clinical studies have suggested that rikkunshito affects (i) various discomforts caused by anticancer drugs, gastroesophageal reflux disease, functional dyspepsia, (ii) various stress-induced anorexia, (iii) hypophagia in the elderly, and (iv) healthy lifespan. In this review, as one who discovered the ghrelin enhancer effect of rikkunshito, we will review the research of rikkunshito so far and report on the latest research results.

TJN-419 improves dextran sulfate sodium-induced colitis via inhibition of interleukin-12 release.

We investigated the association of interleukin-12 (IL-12) with development of dextran sulfate sodium (DSS)-induced colitis in mice, and examined the effects of TJN-419, a synthetic compound derived from acteoside, on this process. Enhanced IL-12 production in lipopolysaccharide (LPS)-stimulated macrophages was dose-dependently inhibited by addition of TJN-419 to culture medium, and this effect was abolished by pretreatment with PD98059, an inhibitor of extracellular-regulated kinase. We then assessed the effect of TJN-419 or a neutralizing antibody against murine IL-12 in a DSS-induced colitis model in C57 BL/6 mice. Colitis was induced by 5% DSS solution given as drinking water. Treatment with the anti-IL-12 antibody was performed intravenously and TJN-419 was administered orally. We also investigated the effect of TJN-419 on erosion in the rectum in a DSS-induced colitis model in rat. The IL-12 level in the rectum was significantly enhanced and the IL-10 level was significantly decreased in animals with DSS-induced colitis compared with untreated controls. Intravenous injection of the anti-IL-12 antibody and oral administration of TJN-419 inhibited clinical symptoms in DSS-induced colitis. TJN-419 also inhibited the increase in IL-12 and suppressed the area of erosion in the rectum in DSS-induced colitis in rats. These results indicate that IL-12 has a possible role in development of DSS-induced colitis and that TJN-419 is effective for treatment of this disease model via inhibition of IL-12 production.

TJN-331 improves anti-glomerular basement membrane nephritis by inhibiting the production of intraglomerular transforming growth factor-beta1.

Transforming growth factor-beta1 (TGF-beta1) plays an important role in the development of glomerulonephritis. The study of experimental glomerulonephritis in rats was performed to examine the antinephritic effects of TJN-331, a new herbally-derived chemical compound. To clarify the action of TJN-331 ((E)-N-(3,4-dimethoxyphenethyl)-N-methyl-3-(3-pyridyl)-2-propenamide) on TGF-beta1 production, glomeruli were isolated from rats with antiglomerular basement membrane (GBM) nephritis and incubated for 48 h with test drugs in vitro. Next, we examined the effects of TJN-331 on rat anti-GBM nephritis induced by injection with anti-GBM serum. TJN-331 dose-dependently inhibited the increase in total and mature TGF-beta1 production from nephritic glomeruli, although it did not inhibit TGF-beta1 production from normal glomeruli. Administration of TJN-331, at a dose of 2 mg/kg/d, per os (p.o.), prevented proteinuria and increased crescent formation and adhesion of capillary walls to Bowman's capsule. The increases in mature TGF-beta1 protein production and TGF-beta1 staining score in nephritic rats were reversed by TJN-331 treatment. These results suggest that TJN-331 inhibits proteinuria and histopathological changes in glomeruli via suppression of TGF-beta1 production from inflamed glomeruli.

Effect of TJN-331 on anti-Thy1 nephritis in rats via inhibition of transforming growth factor-ß1 production.

This study was performed to examine the effects of the antifibrotic agents TJN-331 and tranilast on mesangial expansion in a rat model of anti-Thy1 nephritis. We first investigated the effects of TJN-331 and tranilast on mesangial expansion induced by anti-Thy1 serum in rats, and determined the counts of glomerular cells and proliferative cell nuclear antigen (PCNA)-positive cells. The effects of TJN-331 and tranilast on production of transforming growth factor-ß1 (TGF-ß1) by isolated glomeruli incubated for 48 h were then examined. The TGF-ß1 staining score, the number of TGF-ß1-positive cells and the TGF-ß1 receptor-positive area in the anti-Thy1 nephritis model were also measured using immunohistochemistry. TJN-331 administered from day 1 (the day after anti-Thy1 serum injection) blocked an increase in mesangial matrix accumulation on days 4 and 8, compared to untreated anti-Thy1 nephritic rats. TJN-331 also inhibited both the increase in the number of glomerular cells on day 8 and the decrease in this cell count on day 2 observed in untreated nephritic rats, and TJN-331 and tranilast inhibited an increase in PCNA-positive cells in the glomerular cross section on days 4 and 8. Both TJN-331 and tranilast inhibited increases in the TGF-ß1 protein content from nephritic glomeruli, the TGF-ß1-positive area, and the number of TGF-ß1-positive cells/cross section in anti-Thy1 nephritic glomeruli. These results suggest that TJN-331 and tranilast prevent expansion of the mesangial area by suppression of TGF-ß1 secretion from inflamed glomeruli.

(E)-N-[(3,4-dimethoxyphenethyl)]-N-methyl-3-(3-pyridyl)-2-propenamide (TJN-331) inhibits mesangial expansion in experimental IgA nephropathy in ddY mice.

BACKGROUND: TJN-331 is an inhibitor of transforming growth factor ß1 (TGF-ß1) production that has similar structural features to the natural product acteoside. This study was performed to examine the antinephritic effects of TJN-331 in a mouse model of experimental IgA nephropathy. MATERIALS AND METHODS: IgA nephropathy was induced in ddY mice by oral administration of bovine γ globulin, followed by reticuloendothelial blocking by colloidal carbon injection and heminephrectomy. Effects of TJN-331 were examined over oral administration periods from 10 to 15 weeks after the third colloidal carbon injection. Intravenous administration of a TGF-ß1-neutralizing antibody was used to investigate the role of TGF-ß1 in IgA nephropathy. RESULTS: Administration of TJN-331 or captopril prevented elevation of serum creatinine. Histopathological examination after both experimental periods showed that TJN-331 inhibited increases in the mesangial matrix index and the number of nuclei per glomerular cross-section, compared with in untreated ddY mice with IgA nephropathy. TJN-331 prevented increase in glomerular TGF-ß1 staining without affecting IgA. In the in vitro study, TJN-331 prevented total TGF-ß1 production from splenocytes stimulated with concanavalin A. A neutralizing antibody against TGF-ß1 prevented increase in the mesangial matrix index and the number of glomerular cells per cross-sectional area. CONCLUSION: These results suggest that TJN-331 is effective against IgA nephropathy in ddY mice and acts via suppression of TGF-ß1 production in glomeruli.

Vulnerability to psychological stress-induced anorexia in female mice depends on blockade of ghrelin signal in nucleus tractus solitarius.

BACKGROUND AND PURPOSE: Women have a higher incidence of eating disorders than men. We investigated whether the effects of ghrelin on feeding are affected by sex and stress, and to elucidate the mechanisms that may cause sex differences in stress-mediated anorexia, focusing on ghrelin. EXPERIMENTAL APPROACH: Acylated ghrelin was administered to naïve and psychologically stressed male and female C57BL/6J mice, followed by measurements of food intake and plasma hormone levels. Ovariectomy was performed to determine the effects of ovary-derived oestrogen on stress-induced eating disorders in female mice. The numbers of Agrp or c-Fos mRNA-positive cells and estrogen receptor α/c-Fos protein-double-positive cells were assessed. KEY RESULTS: Ghrelin administration to naïve female mice caused a higher increase in food intake, growth hormone secretion, Agrp mRNA expression in the arcuate nucleus and c-Fos expression in the nucleus tractus solitarius (NTS) than in male mice. In contrast, psychological stress caused a more sustained reduction in food intake in females than males. The high sensitivity of naïve females to exogenous ghrelin was attenuated by stress exposure. The stress-induced decline in food intake was not abolished by ovariectomy. Estrogen receptor-α but not -ß antagonism prevented the decrease in food intake under stress. Estrogen receptor-α/c-Fos-double-positive cells in the NTS were significantly increased by stress only in females. CONCLUSION AND IMPLICATIONS: Stress-mediated eating disorders in females may be due to blockade of ghrelin signalling via estrogen receptor-α activation in the NTS. Targeting the ghrelin signal in the brain could be a new treatment strategy to prevent these disorders.

Rikkunshito, an herbal medicine, suppresses cisplatin-induced anorexia in rats via 5-HT2 receptor antagonism.

BACKGROUND & AIMS: Chemotherapy with an anticancer agent generally causes gastrointestinal tract disorders such as vomiting and anorexia, but the mechanism remains unclear. Rikkunshito, a kampo preparation, is known to alleviate such adverse reactions. In this study, we attempted to clarify the mechanism. METHODS: We investigated the decreases of plasma acylated-ghrelin level and food intake caused by cisplatin, serotonin (5-HT), 5-HT agonists, and vagotomy as well as the decrease-suppressing effects of rikkunshito and 5-HT antagonists. In addition, binding affinities of rikkunshito components were determined in receptor-binding assays using 5-HT2B and 5-HT2C receptors. RESULTS: Cisplatin, 5-HT, BW723C86 (5-HT2B-receptor agonist), and m-chlorophenylpiperazine HCl (5-HT2C agonist) markedly decreased plasma acylated-ghrelin levels, although 5-HT3 and 5-HT4 agonists had no effect. In contrast, 5-HT2B and 5-HT2C antagonists suppressed the cisplatin-induced decrease of plasma acylated-ghrelin level and food intake. Administration of rat ghrelin improved the cisplatin-induced decrease in food intake. Vagotomy decreased the plasma acylated-ghrelin level, which was decreased further by cisplatin. Rikkunshito suppressed such cisplatin-induced decreases of plasma acylated-ghrelin level and food intake. The suppressive effect of rikkunshito was blocked by a ghrelin antagonist. Components of rikkunshito, 3,3',4',5,6,7,8-heptamethoxyflavone, hesperidin, and iso-liquiritigenin showed a 5-HT2B-antagonistic effect in vitro, and oral administration of rikkunshito suppressed the cisplatin-induced decrease in the plasma acylated-ghrelin level. CONCLUSIONS: The cisplatin-induced decreases of the plasma acylated-ghrelin level and food intake are mediated by 5-HT2B/2C receptors and suppressed by flavonoids in rikkunshito.