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1.
J Pharmacol Exp Ther ; 369(3): 466-472, 2019 06.
Article in English | MEDLINE | ID: mdl-30967403

ABSTRACT

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.


Subject(s)
Gastric Mucosa/metabolism , Receptors, G-Protein-Coupled/metabolism , Stomach/physiology , Animals , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/pharmacology , Flavonoids/chemistry , Guinea Pigs , Male , Stomach/drug effects
2.
Int J Mol Sci ; 19(2)2018 Jan 23.
Article in English | MEDLINE | ID: mdl-29360768

ABSTRACT

Recent studies have demonstrated that flavonoid glucuronides can be deconjugated to the active form aglycone by ß-glucuronidase-expressing macrophages. Keigairengyoto (KRT) is a flavonoid-rich traditional Japanese medicine reported to enhance bacterial clearance through immune modulation. Our aims are to examine the pharmacokinetics of KRT flavonoids and to identify active flavonoids contributing to the adjuvant effects of KRT. KRT was evaluated at pharmacokinetic analysis to quantify absorbed flavonoids, and cutaneous infection assay induced in mice by inoculation of Staphylococcus aureus. Preventive or therapeutic KRT administration reduced the number of bacteria in the infection site as well as macroscopic and microscopic lesion scores with efficacies similar to antibiotics. Pharmacokinetic study revealed low plasma levels of flavonoid aglycones after KRT administration; however, plasma concentrations were enhanced markedly by ß-glucuronidase treatment, with baicalein the most abundant (Cmax, 1.32 µg/mL). In random screening assays, flavonoids such as bacalein, genistein, and apigenin enhanced bacteria phagocytosis by macrophages. Glucuronide bacalin was converted to aglycone baicalein by incubation with living macrophages, macrophage lysate, or skin homogenate. Taken together, the adjuvant effect of KRT may be due to some blood-absorbed flavonoids which enhance macrophage functions in host defense. Flavonoid-rich KRT may be a beneficial treatment for infectious skin inflammation.


Subject(s)
Anti-Bacterial Agents/pharmacokinetics , Flavonoids/pharmacokinetics , Plant Extracts/pharmacokinetics , Staphylococcus aureus/drug effects , Animals , Anti-Bacterial Agents/chemistry , Chromatography, High Pressure Liquid , Flavonoids/chemistry , Glucuronides/chemistry , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Medicine, Traditional , Molecular Structure , Phagocytosis/drug effects , Phagocytosis/immunology , Phytochemicals/analysis , Phytochemicals/chemistry , Plant Extracts/chemistry , Rats , Staphylococcal Infections/drug therapy , Staphylococcal Infections/microbiology , Staphylococcal Skin Infections/drug therapy , Staphylococcal Skin Infections/microbiology
3.
Molecules ; 22(11)2017 Oct 27.
Article in English | MEDLINE | ID: mdl-29077044

ABSTRACT

Stratum corneum tryptic enzyme kallikrein 5 (KLK5) is a serine protease that is involved in the cell renewal and maintenance of the skin barrier function. The excessive activation of KLK5 causes an exacerbation of dermatoses, such as rosacea and atopic dermatitis. Some triterpenoids are reported to suppress the serine proteases. We aimed to investigate whether bioactive triterpenoids modulate the KLK5 protease. Nineteen triterpenoids occurring in medicinal crude drugs were evaluated using an enzymatic assay to measure the anti-KLK5 activity. The KLK5-dependent cathelicidin peptide LL-37 production in human keratinocytes was examined using immunoprecipitation and Western blotting. Screening assays for evaluating the anti-KLK5 activity revealed that ursolic acid, oleanolic acid, saikosaponin b1, tumulosic acid and pachymic acid suppressed the KLK5 protease activity, although critical molecular moieties contributing to anti-KLK5 activity were unclarified. Ursolic acid and tumulosic acid suppressed the proteolytic processing of LL-37 in keratinocytes at ≤10 µM; no cytotoxicity was observed. Both triterpenoids were detected in the plasma of rats administered orally with triterpenoid-rich crude drug Jumihaidokuto. Our study reveals that triterpenoids, such as ursolic acid and tumulosic acid, modulate the KLK5 protease activity and cathelicidin peptide production. Triterpenoids may affect the skin barrier function via the regulation of proteases.


Subject(s)
Biological Products/chemistry , Kallikreins/chemistry , Triterpenes/chemistry , Animals , Biological Products/administration & dosage , Biological Products/pharmacokinetics , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Humans , Kallikreins/antagonists & inhibitors , Keratinocytes/drug effects , Keratinocytes/metabolism , Molecular Structure , Rats , Triterpenes/administration & dosage , Triterpenes/pharmacokinetics
4.
Immun Inflamm Dis ; 5(3): 265-279, 2017 09.
Article in English | MEDLINE | ID: mdl-28480538

ABSTRACT

INTRODUCTION: Flavonoids are converted to inactive metabolites like glucuronides in the gut, and circulate mainly as glucuronides in blood stream, resulting in low concentrations of active aglycones in plasma. It is therefore unclear how oral flavonoids exert their effects in tissues. We recently reported the plasma pharmacokinetics of some flavonoids and suggested the possibility that the absorbed flavonoids modified macrophage functions leading to enhance bacterial clearance. We aimed to confirm their pharmacological profiles focusing on tissue macrophages. METHODS: Pseudoinfection was induced by intradermal injection of FITC-conjugated and killed Staphylococcus aureus into the ears of mice treated with or without genistein 7-O-glucuronide (GEN7G, 1 mg/kg, i.v.). FACS analysis was performed on single cell suspensions dispersed enzymatically from the skin lesions at 6 h post pseudoinfection to evaluate phagocytic activities of monocytes/macrophages (CD11b+ Ly6G- ) and neutrophils (CD11b+ Ly6G+ ). Phagocytosis of the FITC-conjugated bacteria by four glucuronides including GEN7G was evaluated in cultures of mouse macrophages. RESULTS: After GEN7G injection, genistein was identified in the inflamed ears as well as GEN7G, and the phagocytic activity of CD11b+ Ly6G- cells was increased. GEN7G was converted to genistein by incubation with macrophage-related ß-glucuronidase. Macrophage culture assays revealed that GEN7G increased phagocytosis, and the action was dampened by a ß-glucuronidase inhibitor. Binding of aglycones to estrogen receptors (ERs), putative receptors of flavonoid aglycones, correlated to biological activities, and glucuronidation reduced the binding to ERs. An ER antagonist suppressed the increase of macrophage function by GEN7G, whereas estradiol enhanced phagocytosis as well. CONCLUSIONS: This study suggests a molecular mechanism by which oral flavonoids are carried as glucuronides and activated to aglycones by ß-glucuronidase in tissue macrophages, and contributes to the pharmacological study of glucuronides.


Subject(s)
Flavonoids/metabolism , Glucuronidase/metabolism , Glucuronides/metabolism , Macrophages/metabolism , Phytoestrogens/metabolism , Staphylococcal Skin Infections/metabolism , Staphylococcus aureus , Animals , Macrophages/pathology , Mice , Mice, Inbred ICR , Staphylococcal Skin Infections/pathology
5.
Molecules ; 22(2)2017 Feb 08.
Article in English | MEDLINE | ID: mdl-28208738

ABSTRACT

Insufficient detoxification and/or overproduction of reactive oxygen species (ROS) induce cellular and tissue damage, and generated reactive oxygen metabolites become exacerbating factors of dermatitis. Keishibukuryogan-ka-yokuinin (KBGY) is a traditional Japanese medicine prescribed to treat dermatitis such as acne vulgaris. Our aim was to verify the antioxidant properties of KBGY, and identify its active constituents by blood pharmacokinetic techniques. Chemical constituents were quantified in extracts of KBGY, crude components, and the plasma of rats treated with a single oral administration of KBGY. Twenty-three KBGY compounds were detected in plasma, including gallic acid, prunasin, paeoniflorin, and azelaic acid, which have been reported to be effective for inflammation. KBGY decreased level of the diacron-reactive oxygen metabolites (d-ROMs) in plasma. ROS-scavenging and lipid hydroperoxide (LPO) generation assays revealed that gallic acid, 3-O-methylgallic acid, (+)-catechin, and lariciresinol possess strong antioxidant activities. Gallic acid was active at a similar concentration to the maximum plasma concentration, therefore, our findings indicate that gallic acid is an important active constituent contributing to the antioxidant effects of KBGY. KBGY and its active constituents may improve redox imbalances induced by oxidative stress as an optional treatment for skin diseases.


Subject(s)
Antioxidants/administration & dosage , Drugs, Chinese Herbal/administration & dosage , Medicine, East Asian Traditional , Reactive Oxygen Species/blood , Administration, Oral , Animals , Antioxidants/chemistry , Antioxidants/pharmacokinetics , Chromatography, Liquid , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/pharmacokinetics , Gas Chromatography-Mass Spectrometry , Male , Oxidative Stress/drug effects , Phytochemicals/chemistry , Plant Extracts/administration & dosage , Plant Extracts/chemistry , Plant Extracts/pharmacokinetics , Rats , Tandem Mass Spectrometry
6.
Article in English | MEDLINE | ID: mdl-26495013

ABSTRACT

Purpose. Macrophages serve as sweepers of microbes and inflammation-derived wastes and regulators of inflammation. Some traditional Japanese medicines are reported to have adjuvant effects by modifying macrophages. Our aim was to characterize the actions of jumihaidokuto (JHT) for treatment of skin inflammations including acne vulgaris, in which Propionibacterium acnes has pathogenic roles. Methods. Dermatitis was induced in rat ears by intradermal injection of P. acnes. JHT or prednisolone (PDN) was given orally, and ear thickness and histology were evaluated. The effects of constituents and metabolites of JHT on monocytes were tested by cell-based assays using the human monocytic THP-1 cell. Results. JHT and PDN suppressed the ear thickness induced by P. acnes injection. Histological examinations revealed that JHT, but not PDN, promoted macrophage accumulation at 24 h after the injection. PDN suppressed the macrophage chemokine MCP-1 in the inflamed ears, while JHT did not affect it. The JHT constituents liquiritigenin and isoliquiritin increased expression of CD86 (type-1 macrophage marker) and CD192 (MCP-1 receptor) and enhanced phagocytosis by THP-1. Conclusions. JHT suppressed dermatitis, probably by enhancing type-1 macrophage functions, with an action different from PDN. JHT may be a beneficial drug in treatment of skin inflammation induced by P. acnes.

7.
Molecules ; 20(10): 18031-46, 2015 Sep 30.
Article in English | MEDLINE | ID: mdl-26437394

ABSTRACT

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.


Subject(s)
Anti-Inflammatory Agents/pharmacokinetics , Antioxidants/pharmacokinetics , Dermatitis/drug therapy , Plant Extracts/pharmacokinetics , Polyphenols/pharmacokinetics , Propionibacterium acnes/immunology , Administration, Oral , Animals , Anti-Inflammatory Agents/administration & dosage , Anti-Inflammatory Agents/blood , Antioxidants/administration & dosage , Dermatitis/microbiology , Flavanones/blood , Flavanones/pharmacokinetics , Genistein/blood , Genistein/pharmacokinetics , Male , Medicine, Traditional , Plant Extracts/administration & dosage , Plant Extracts/blood , Polyphenols/administration & dosage , Polyphenols/blood , Rats , Rats, Sprague-Dawley
8.
Article in English | MEDLINE | ID: mdl-25709710

ABSTRACT

Objective. Bokusoku (BK) is an extract from the Quercus cortex used in folk medicine for treatment of skin disorders and convergence, and is present in jumihaidokuto, a traditional Japanese medicine that is prescribed for purulent skin diseases like acne vulgaris. The excess of sebum production induced by androgen is involved in the development of acne. Our aim is to examine whether BK and its constituents inhibit testosterone metabolism and testosterone-induced sebum synthesis. Methods. Measurements of 5α-reductase activity and lipogenesis were performed using rat liver microsomes and hamster sebocytes, respectively. Results. BK dose-dependently reduced the conversion of testosterone to a more active androgen, dihydrotestosterone in a 5α-reductase enzymatic reaction. Twenty polyphenols in BK categorized as gallotannin, ellagitannin, and flavonoid were identified by LC-MS/MS. Nine polyphenols with gallate group, tetragalloyl glucose, pentagalloyl glucose, eugeniin, 1-desgalloyl eugeniin, casuarinin, castalagin, stenophyllanin C, (-)-epicatechin gallate, and (-)-epigallocatechin gallate, inhibited testosterone metabolism. In particular, pentagalloyl glucose showed the strongest activity. BK and pentagalloyl glucose suppressed testosterone-induced lipogenesis, whereas they weakly inhibited the lipogenic action of insulin. Conclusions. BK inhibited androgen-related pathogenesis of acne, testosterone conversion, and sebum synthesis, partially through 5α-reductase inhibition, and has potential to be a useful agent in the therapeutic strategy of acne.

9.
J Pharmacol Exp Ther ; 341(1): 205-12, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22262922

ABSTRACT

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.


Subject(s)
Calcium Channels/physiology , Muscle, Smooth/physiology , Nerve Tissue Proteins/physiology , Stomach/physiology , Transient Receptor Potential Channels/physiology , Wakefulness/physiology , Animals , Cholinergic Neurons/physiology , Gastric Emptying/physiology , Gastric Mucosa/metabolism , Guinea Pigs , Male , Muscle Relaxation/physiology , Muscle, Smooth/cytology , Signal Transduction/physiology , Stomach/cytology , TRPA1 Cation Channel
10.
Lab Invest ; 92(1): 22-31, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21912379

ABSTRACT

Acid is a major cause of gastro-esophageal reflux disease. However, the influence of acid on the esophageal stratified epithelial barrier function and tight junction (TJ) proteins is not fully understood. Here, we explore the influence of acid on barrier function and TJ proteins using a newly developed model of the esophageal-like squamous epithelial cell layers that employs an air-liquid interface (ALI) system. Barrier function was determined by measuring trans-epithelial electrical resistance (TEER) and diffusion of paracellular tracers. TJ-related protein (claudin-1, claudin-4, occludin and ZO-1) expression and localization was examined by immunofluorescent staining, and by western blotting of 1% NP-40 soluble and insoluble fractions. We also examined the influence of acid (pH 2-4) on the barrier created by these cells. The in vitro ALI culture system showed a tight barrier (1500-2500 Ω·cm(2)) with the expression of claudin-1, claudin-4, occludin and ZO-1 in the superficial layers. Claudin-1, claudin-4, occludin and ZO-1 were detected as dots and whisker-like lines in the superficial layers, and as a broad line in the suprabasal layers. These localization patterns are similar to those in the human esophagus. On day 7 under ALI culture, TJ proteins were detected in the superficial layers with functional properties, including decreased permeability and increased TEER. Dilated intercellular spaces were detected at the suprabasal cell layers even under the control conditions of ALI cells. pH 2 acid on the apical side significantly reduced the TEER in ALI-cultured cells. This decrease in TEER by the acid was in parallel with the decreased amount of detergent-insoluble claudin-4. Claudin-4 delocalization was confirmed by immunofluorescent staining. In conclusion, TJs are located in the superficial layers of the esophagus, and acid stimulation disrupts barrier function, at least in part by modulating the amount and localization of claudin-4 in the superficial layers.


Subject(s)
Claudins/analysis , Esophagus/metabolism , Gastric Acid/physiology , Membrane Proteins/analysis , Cells, Cultured , Claudin-1 , Claudin-4 , Electric Impedance , Epithelium/chemistry , Epithelium/metabolism , Esophagus/chemistry , Fluoresceins/metabolism , Humans , Occludin , Permeability , Phosphoproteins/analysis , Zonula Occludens-1 Protein
11.
Am J Physiol Cell Physiol ; 300(6): C1422-9, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21307344

ABSTRACT

Current experimental models of esophageal epithelium in vitro suffer from either poor differentiation or complicated culture systems. We have established a model to study stratified squamous epithelium in vitro, which is very similar to esophageal epithelium in vivo. A stratified squamous multilayer epithelium was formed by seeding primary normal human bronchial epithelial (NHBE) cells onto collagen- and fibronectin-coated trans-well inserts and then cultivating the cells under air-liquid interface (ALI) conditions in the presence of growth factors and low levels of all-trans-retinoic acid. Trans-epithelial electrical resistance (TEER) measurements revealed the presence of a tight barrier, previously only achievable with esophageal biopsies mounted in Ussing chambers. Molecular markers for desmosomes, cornified envelope, tight junctions, and mature esophageal epithelium were upregulated in the differentiating culture in parallel with functional properties, such as decreased permeability and acid resistance and restoration. Acid exposure resulted in a decrease in TEER, but following 1-h recovery the TEER values were fully restored. Treatment with all-trans-retinoic acid decreased TEER and inhibited the recovery after acid challenge. PPAR-delta agonist treatment increased TEER, and this temporary increase in TEER was consistent with an increase in involucrin mRNA. Global gene expression analysis showed that ALI-differentiated NHBE cells had expression profiles more similar to epithelial biopsies from the esophageal tissue of healthy volunteers than to any other cell line. With respect to morphology, molecular markers, barrier properties, and acid resistance, this model presents a new way to investigate barrier properties and the possible effects of different agents on human esophagus-like epithelium.


Subject(s)
Bronchi/cytology , Cell Culture Techniques , Epithelial Cells/cytology , Epithelium/anatomy & histology , Esophagus/cytology , Models, Biological , Respiratory Mucosa/cytology , Animals , Biomarkers/metabolism , Cell Differentiation/drug effects , Cell Line , Epithelial Cells/drug effects , Epithelial Cells/physiology , Gene Expression Profiling , Humans , Keratolytic Agents/pharmacology , Membrane Proteins/genetics , Membrane Proteins/metabolism , PPAR delta/agonists , Tight Junctions/chemistry , Tretinoin/pharmacology
12.
Clin Exp Nephrol ; 15(1): 14-24, 2011 Feb.
Article in English | MEDLINE | ID: mdl-20820840

ABSTRACT

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.


Subject(s)
Acrylamides/therapeutic use , Cyclic Nucleotide Phosphodiesterases, Type 4 , Glomerulonephritis/drug therapy , Glomerulonephritis/immunology , Isoantibodies/immunology , Phosphodiesterase Inhibitors/therapeutic use , Pyridines/therapeutic use , Acrylamides/chemistry , Animals , Disease Models, Animal , Humans , Male , Molecular Structure , Phosphodiesterase Inhibitors/chemistry , Plant Preparations/therapeutic use , Pyridines/chemistry , Rats , Rats, Wistar , Thy-1 Antigens/immunology , Transforming Growth Factor beta1/metabolism , Tumor Necrosis Factor-alpha/metabolism
13.
Biol Pharm Bull ; 33(10): 1710-5, 2010.
Article in English | MEDLINE | ID: mdl-20930380

ABSTRACT

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.


Subject(s)
Acrylamides/pharmacology , Glomerular Mesangium/drug effects , Glomerulonephritis/drug therapy , Proliferating Cell Nuclear Antigen/metabolism , Pyridines/pharmacology , Renal Agents/pharmacology , Transforming Growth Factor beta1/metabolism , ortho-Aminobenzoates/pharmacology , Acrylamides/therapeutic use , Animals , Cell Count , Glomerular Mesangium/cytology , Glomerular Mesangium/physiology , Glomerulonephritis/chemically induced , Male , Pyridines/therapeutic use , Rats , Rats, Wistar , Renal Agents/therapeutic use , Thy-1 Antigens , ortho-Aminobenzoates/therapeutic use
14.
Clin Exp Nephrol ; 14(6): 528-35, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20814808

ABSTRACT

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.


Subject(s)
Acrylamides/pharmacology , Glomerulonephritis, IGA/pathology , Pyridines/pharmacology , Animals , Concanavalin A/pharmacology , Creatinine/blood , Disease Models, Animal , Glomerular Mesangium/drug effects , Glomerular Mesangium/pathology , Kidney Glomerulus/pathology , Mice , Mice, Inbred Strains , Nephrectomy , Spleen/cytology , Transforming Growth Factor beta1/antagonists & inhibitors , Transforming Growth Factor beta1/biosynthesis
15.
Biol Pharm Bull ; 33(8): 1349-54, 2010.
Article in English | MEDLINE | ID: mdl-20686230

ABSTRACT

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.


Subject(s)
Acrylamides/therapeutic use , Anti-Glomerular Basement Membrane Disease/drug therapy , Pyridines/therapeutic use , Transforming Growth Factor beta1/antagonists & inhibitors , Acrylamides/administration & dosage , Actins/metabolism , Animals , Anti-Glomerular Basement Membrane Disease/blood , Anti-Glomerular Basement Membrane Disease/metabolism , Anti-Glomerular Basement Membrane Disease/urine , Creatinine/blood , Disease Models, Animal , Gene Expression/drug effects , Immunohistochemistry , Kidney Glomerulus/drug effects , Kidney Glomerulus/metabolism , Kidney Glomerulus/pathology , Male , Molecular Structure , Proteinuria/metabolism , Proteinuria/pathology , Proteinuria/prevention & control , Proteinuria/urine , Pyridines/administration & dosage , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , Transforming Growth Factor beta1/biosynthesis , Transforming Growth Factor beta1/genetics
16.
Biol Pharm Bull ; 33(1): 84-90, 2010.
Article in English | MEDLINE | ID: mdl-20045941

ABSTRACT

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.


Subject(s)
Acetates/therapeutic use , Anti-Inflammatory Agents/therapeutic use , Cinnamates/therapeutic use , Colitis/drug therapy , Glucosides/metabolism , Interleukin-12/antagonists & inhibitors , Macrophages/drug effects , Phenols/metabolism , Plant Extracts/metabolism , Rectum/drug effects , Acetates/administration & dosage , Acetates/pharmacology , Animals , Anti-Inflammatory Agents/administration & dosage , Anti-Inflammatory Agents/pharmacology , Antibodies, Neutralizing , Cinnamates/administration & dosage , Cinnamates/pharmacology , Colitis/chemically induced , Colitis/metabolism , Dextran Sulfate , Dose-Response Relationship, Drug , Flavonoids , Lipopolysaccharides , Lippia/chemistry , Male , Mice , Mice, Inbred C57BL , Models, Animal , Rats , Rats, Sprague-Dawley , Rectum/pathology
17.
J Gastroenterol ; 45(5): 478-87, 2010 May.
Article in English | MEDLINE | ID: mdl-20016992

ABSTRACT

BACKGROUND: A traditional Japanese medicine, rikkunshito, has been reported to relieve dyspepsia symptoms. We investigated the effect of rikkunshito on RE-induced abdominal dyspepsia, and performed experiments to elucidate the mechanism of that effect. METHODS: RE model rats were prepared using 8-week-old male Wistar rats, and rikkunshito was administered in drinking water. Voluntary movement was used as an index of RE-induced abdominal dyspepsia, which was monitored by an infrared sensor. On the tenth day after surgery, the total area of esophageal erosion was measured, and samples of nonerosive mucosa were collected. Using those samples, intercellular spaces of epithelial mucosa were examined by transmission electron microscopy, and the NP-40-soluble and -insoluble levels of the tight junction proteins claudin-1, -3 and -4 and their mRNAs were determined. RESULTS: Rikkunshito did not reduce the average total area of erosive lesions in the esophageal mucosa of RE model rats. On day 10, voluntary movement was significantly decreased in the RE model rats and rikkunshito significantly increased it. Nonerosive esophageal mucosa from RE rats showed dilation of intercellular spaces in epithelium, and significantly decreased claudin-3 mRNA and protein levels. Rikkunshito significantly suppressed intercellular space dilation and significantly increased the level of NP-40-insoluble claudin-3, but it did not affect the mRNA level, suggesting that it promoted tight junction formation by facilitating the translocation of proteins. CONCLUSION: Rikkunshito increased voluntary movement in RE model rats. This may have been because rikkunshito ameliorated the symptoms of RE by improving the barrier function of esophageal mucosa.


Subject(s)
Drugs, Chinese Herbal/pharmacology , Epithelial Cells/drug effects , Esophagitis, Peptic/pathology , Tight Junctions/drug effects , Animals , Behavior, Animal/drug effects , Claudin-3 , Disease Models, Animal , Drugs, Chinese Herbal/therapeutic use , Epithelial Cells/metabolism , Epithelial Cells/ultrastructure , Esophagitis, Peptic/drug therapy , Esophagitis, Peptic/metabolism , Extracellular Space/drug effects , Extracellular Space/metabolism , Japan , Male , Membrane Proteins/metabolism , Motor Activity/drug effects , Mucous Membrane/drug effects , Mucous Membrane/metabolism , Mucous Membrane/pathology , Rats , Rats, Wistar , Tight Junctions/metabolism , Tight Junctions/ultrastructure
18.
Biol Pharm Bull ; 32(10): 1728-33, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19801835

ABSTRACT

TJN-259 is a chemical substance based on the structural features of the botanically derived ingredient acteoside. This study was performed in order to elucidate the antinephritic effects of TJN-259 in experimental immunoglobulin A (IgA) nephropathy. In this study, 28-week-old ddY mice were used as a spontaneous model of IgA nephropathy. With regard to spontaneous IgA nephropathy, we investigated the effects of TJN-259 administered from 28 to 40 weeks. In addition, an accelerated model of IgA nephropathy was experimentally induced in ddY mice by oral administration of bovine serum albumin, followed by reticuloendothelial blocking by colloidal carbon injection and heminephrectomy. At 10 weeks after the 3rd carbon injection, we also examined the effects of TJN-259 on accelerated IgA nephropathy. To investigate the effects of TJN-259 on transforming growth factor (TGF)-beta1 production in accelerated IgA nephropathy, kidneys were isolated and measured TGF-beta1 by the enzyme-linked immunosorbent assay (ELISA) method. The administration of TJN-259 to mice with spontaneous IgA nephropathy decreased the incidence of mesangial expansion as well as the number of nuclei per glomerular cross-section in comparison with that of non-treated mice. In addition, TJN-259 treatment prevented the increase in the incidence of mesangial expansion, crescent formation, and segmental sclerosis in glomeruli in accelerated IgA nephropathy. TJN-259 also inhibited the increased immunostaining score of collagen type IV and TGF-beta1 in glomeruli of accelerated IgA nephropathy. Treatment with TJN-259 inhibited the increases in renal total and mature TGF-beta1 protein levels in accelerated type IgA nephropathy. TJN-259 failed to inhibit the increase in serum IgA levels in both models. These results suggest that TJN-259 was an effective treatment against IgA nephropathy in ddY mice, acting via the suppression of TGF-beta1 production in glomeruli.


Subject(s)
Anti-Inflammatory Agents/therapeutic use , Glomerular Mesangium/drug effects , Glomerulonephritis, IGA/drug therapy , Immunoglobulin A/blood , Kidney/drug effects , Pyridines/therapeutic use , Transforming Growth Factor beta1/biosynthesis , Animals , Anti-Inflammatory Agents/chemical synthesis , Anti-Inflammatory Agents/pharmacology , Cattle , Collagen Type IV/metabolism , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Glomerular Mesangium/metabolism , Glomerular Mesangium/pathology , Glomerulonephritis, IGA/chemically induced , Glomerulonephritis, IGA/pathology , Glucosides , Kidney/metabolism , Kidney/pathology , Male , Mice , Mice, Inbred Strains , Phenols , Plant Extracts , Pyridines/chemical synthesis , Pyridines/pharmacology , Sclerosis/drug therapy , Serum Albumin , Stachys
19.
Clin Exp Nephrol ; 11(4): 275-282, 2007 Dec.
Article in English | MEDLINE | ID: mdl-18085387

ABSTRACT

BACKGROUND: In this study, we administered saireito to high serum IgA (HIGA) mice and investigated its inhibitory effect on platelet-derived growth factor (PDGF) receptor tyrosine kinase (which causes mesangial proliferation) as one of the possible antinephritic mechanisms of saireito. METHODS: Female HIGA/NscSlc mice, aged 10 weeks, were divided into five groups (each, n = 12; a control group, three saireito-mixed feed groups, and a captopril-mixed feed group) so that the plasma IgA levels were comparable among the groups. After the grouping, the animals were administered the saireito or captopril, mixed in the feed, until the age of 45 weeks. RESULTS: At the age of 45 weeks, the glomerular cell number was 47.8 +/- 3.9 / cross section in the HIGA mice in the control group, but 41.6 +/- 2.3 / cross section in the 1.3% saireito-mixed feed group and 38.7 +/- 3.5 / cross section in the captopril-mixed feed group, being significantly lower in both these treatment groups than in the control group. At the age of 45 weeks, the sclerosis score in the HIGA mice in the control group was 0.92 +/- 0.23. However, the sclerosis scores in the 0.26% (0.59 +/- 0.26) and 1.3% (0.58 +/- 0.16) saireito-mixed feed groups were significantly lower than that in the control group. In the captopril-mixed feed group, the sclerosis score was 0.64 +/- 0.34, significantly lower than that in the control group. It was clarified that saireito suppressed mesangial cell proliferation without showing any cytotoxicity. Furthermore, as a result of investigating the mesangial cell proliferation-suppressing effect similarly with the 23 substances constituting saireito, a proliferation-suppressing effect was recognized with isoliquiritigenin (a component of Glycyrrhizae Radix) and oroxylin A (a component of Scutellariae Radix). Oroxylin A and isoliquiritigenin showed an inhibitory effect on PDGF receptor tyrosine kinase. Furthermore, the inhibitory effects of oroxylin A and isoliquiritigenin on tyrosine kinase were found to be specific to the PDGF receptor, and showed no influence on the tyrosine kinase activities of other growth-factor receptors examined. CONCLUSION: These results suggest that the antinephritic effects of saireito in HIGA mice may be partly due to the inhibiton of PDGF tyrosine kinase by oroxylin A and isoliquiritigenin, components of saireito.


Subject(s)
Cell Proliferation/drug effects , Drugs, Chinese Herbal/pharmacology , Glomerulonephritis, IGA/drug therapy , Immunoglobulin A/blood , Mesangial Cells/drug effects , Platelet-Derived Growth Factor/metabolism , Protein Kinase Inhibitors/pharmacology , Receptors, Platelet-Derived Growth Factor/antagonists & inhibitors , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Animals , Becaplermin , Captopril/pharmacology , Cell Survival/drug effects , Cells, Cultured , Chalcones/isolation & purification , Chalcones/pharmacology , Disease Models, Animal , Dose-Response Relationship, Drug , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/therapeutic use , Female , Flavonoids/isolation & purification , Flavonoids/pharmacology , Glomerulonephritis, IGA/enzymology , Glomerulonephritis, IGA/metabolism , Humans , Mesangial Cells/enzymology , Mesangial Cells/metabolism , Mice , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins c-sis , Receptors, Platelet-Derived Growth Factor/metabolism , Up-Regulation
20.
Chem Pharm Bull (Tokyo) ; 55(2): 255-67, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17268099

ABSTRACT

We found previously that 7-[3-(cyclohexylmethyl)ureido]-3-{1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl}quinoxalin-2(1H)-one (7d-6) has considerable potency as a PDGF inhibitor. This compound showed potent inhibitory activity in a PDGF-induced CPA (Cell Proliferation Assay) and APA (Auto-Phosphorylation Assay) (IC50 = 0.05 micromol/l in CPA, 0.03 micromol/l in APA). Therefore, we tried to develop a novel and effective PDGF-betaR inhibitor by optimizing a series of its derivatives. We found that trifluoroacetic acid (TFA)-catalyzed coupling of pyrrolo[2,3-b]pyridines with quinoxalin-2-ones proceeded efficiently under mild oxidation condition with manganese(IV) oxide (MnO2) in situ, so this method was applied to prepare a series of derivatives. Results of in vitro screening of newly synthesized derivatives identified compound 7d-9 as having potent (IC50 = 0.014 micromol/l in CPA, 0.007 micromol/l in APA) and selective [IC50 values against vascular endothelial growth factor receptor 2 (VEGFR2, kinase domain region, KDR), epidermal growth factor receptor (EGFR), c-Met (hepatocyte growth factor receptor) and insulin growth factor I receptor (IGF-IR)/IC50 against PDGFR were each >1000] inhibitory activity. Moreover, in this series of derivatives, 7b-2 showed potent inhibitory activity toward both PDGF- and VEGF-induced signaling (PDGFR: IC50 = 0.004 micromol/l in CPA, 0.0008 micromol/l in APA, KDR: IC50 = 0.008 micromol/l in APA). Herein we report a new and convenient synthetic method for this series of derivatives and its SAR study.


Subject(s)
Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Quinoxalines/chemical synthesis , Quinoxalines/pharmacology , Receptor, Platelet-Derived Growth Factor beta/antagonists & inhibitors , Catalysis , ErbB Receptors/metabolism , Hepatocyte Growth Factor/metabolism , Humans , Inhibitory Concentration 50 , Manganese Compounds/chemistry , Oxidation-Reduction , Oxides/chemistry , Receptor, IGF Type 1/metabolism , Structure-Activity Relationship , Trifluoroacetic Acid/chemistry
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