ABSTRACT
PURPOSE: Plant-derived extracellular vesicles (EVs) have been reported to exert biological activity on intestinal tissues by delivering their contents into intestinal cells. We previously reported that ASBT/SLC10A2 mRNA was downregulated by apple-derived extracellular vesicles (APEVs). ASBT downregulation is effective in the treatment of cholestasis and chronic constipation, similar to the beneficial effects of apples. Therefore, this study aimed to establish the mechanism of ASBT downregulation by APEVs, focusing on microRNAs present in APEVs. RESULTS: APEVs downregulated the expression of ASBT, but no significant effect on SLC10A2-3'UTR was observed. Proteomics revealed that APEVs decreased the expression of RARα/NR1B1. The binding of RARα to SLC10A2 promoter was also decreased by APEVs. The stability of NR1B1 mRNA was attenuated by APEVs and its 3'UTR was found to be a target for APEVs. Apple microRNAs that were predicted to interact with NR1B1-3'UTR were present in APEVs, and their mimics suppressed NR1B1 mRNA expression. CONCLUSIONS: Suppression of ASBT by APEVs was indirectly mediated by the downregulation of RARα, and its stability was lowered by microRNAs present in APEVs. This study suggested that macromolecules in food directly affect intestinal function by means of EVs that stabilize them and facilitate their cellular uptake.
Subject(s)
Extracellular Vesicles , Malus , MicroRNAs , Symporters , Humans , Transcription Factors/genetics , Transcription Factors/metabolism , Down-Regulation , Malus/genetics , Malus/metabolism , 3' Untranslated Regions , Bile Acids and Salts , MicroRNAs/genetics , Extracellular Vesicles/genetics , Extracellular Vesicles/metabolism , Symporters/genetics , Symporters/metabolism , Organic Anion Transporters, Sodium-Dependent/genetics , Organic Anion Transporters, Sodium-Dependent/metabolismABSTRACT
PURPOSE: Dry eye syndrome (DES) is characterized by an increase in tear osmolarity and induction of the expression and nuclear localization of an osmoprotective transcription factor (nuclear factor of activated T-cells 5 [NFAT5]) that plays an important role in providing protection against hyperosmotic tears. In this study, we screened medicines already in clinical use with a view of finding compounds that protect cultured human corneal epithelial cells against hyperosmolarity-induced cell damage. METHODS: Viable cell number was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and cellular NFAT5 level was measured by immunoblotting. The rat model for DES was developed by removal of the lacrimal glands, with an assessment of corneal surface damage based on levels of fluorescein staining and epithelial apoptosis. RESULTS: Some nonsteroidal anti-inflammatory drugs (NSAIDs), including diclofenac sodium (diclofenac), were identified during the screening procedure. These NSAIDs were able to suppress hyperosmolarity-induced apoptosis and cell growth arrest. In contrast, other NSAIDs, including bromfenac sodium (bromfenac), did not exert such a protective action. Treatment of cells with diclofenac, but not bromfenac, stimulated both the nuclear localization and expression of NFAT5 under hyperosmotic conditions. In the rat model for DES, topical administration of diclofenac (but not bromfenac) to eyes reduced corneal surface damage without affecting the volume of tear fluid. CONCLUSIONS: Diclofenac appears to protect cells against hyperosmolarity-induced cell damage and NFAT5 would play an important role in this protective action. The findings reported here may also indicate that the topical administration of diclofenac to eyes may be therapeutically beneficial for DES patients.
Subject(s)
Diclofenac/therapeutic use , Dry Eye Syndromes/drug therapy , Epithelium, Corneal/drug effects , Animals , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Cells, Cultured , Disease Models, Animal , Dry Eye Syndromes/metabolism , Dry Eye Syndromes/pathology , Epithelium, Corneal/metabolism , Epithelium, Corneal/pathology , Male , Osmotic Pressure/drug effects , Rats , Rats, Sprague-Dawley , Transcription Factors/metabolism , Treatment OutcomeABSTRACT
The gene of a novel O-methyltransferase was isolated from tea cultivars (Camellia sinensis L.). Using the recombinant enzyme, O-methylated (-)-epigallocatechin-3-O-gallate (EGCG) in all cases were synthesized. EGCG and the synthesized O-methylated EGCGs including (-)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG3''Me), (-)-epigallocatechin-3-O- (4-O-methyl)-gallate(EGCG4''Me), (-)-epigallocatechin-3-O-(3,5-O-dimethyl)-gallate (EGCG3'',5''diMe), and (-)-3-O-methyl-epigallocatechin-3-O-(3,5-O-dimethyl)-gallate (EGCG3',3'',5''triMe) were assayed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay and antibacterial activity. EGCG was the most effective of the O-methylated EGCGs. The antiallergic effects of EGCG and the other O-methylated EGCGs were measured by conducting histamine release assays using bone marrow-derived mouse mast cells, and the order of potency was EGCG3',3'',5''triMe = EGCG3'',5''diMe > EGCG3''Me > EGCG. These results indicated that reducing the number of hydroxyl groups decreases the effectiveness of DPPH radical scavenging and antibacterial activity. In contrast, the inhibition of histamine release was potentiated by an increase in the number of methyl groups in EGCG, especially in the galloyl moiety.
Subject(s)
Camellia sinensis/enzymology , Catechin/analogs & derivatives , Cloning, Molecular , Plant Proteins/genetics , Protein O-Methyltransferase/genetics , Amino Acid Sequence , Animals , Anti-Allergic Agents/chemistry , Anti-Allergic Agents/pharmacology , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bone Marrow Cells/drug effects , Bone Marrow Cells/immunology , Camellia sinensis/genetics , Catechin/chemistry , Catechin/pharmacology , Free Radical Scavengers/chemistry , Free Radical Scavengers/pharmacology , Histamine Release/drug effects , Mice , Molecular Sequence Data , Plant Proteins/metabolism , Protein O-Methyltransferase/metabolism , Sequence AlignmentSubject(s)
Benzene Derivatives/chemical synthesis , Copper/chemistry , Organometallic Compounds/chemical synthesis , Piperidines/chemical synthesis , Benzene Derivatives/chemistry , Crystallography, X-Ray , Molecular Structure , Organometallic Compounds/chemistry , Piperidines/chemistry , Quantum Theory , StereoisomerismABSTRACT
To find novel PPAR ligands, we prepared several 3-{3 or 4-[2-(nonylpyridin-2-ylamino)ethoxy]phenyl}propanoic acid derivatives which were designed based on the structure of our previous PPARgamma ligand 1. In PPAR binding affinity assays, compound 4, which had an ethoxy group at the C-2 position of the propanoic acid of 1, showed selective binding affinity for PPARgamma. Compound 3, with an ethyl group at the C-2 position, was found to be a PPARalpha/gamma dual ligand. Compound 6, the meta isomer of 1, has been shown to be a PPARalpha ligand. The introduction of methyl (7) and ethyl (8) groups to the C-2 position of the propanoic acid of 6 further improved PPARalpha-binding potency. In cell-based transactivation assay, compounds 3 and 4 showed dual-agonist activity toward PPARalpha and PPARgamma. Compound 6 was found to be a triple agonist and compound 8 proved to be a selective PPARalpha agonist. In the human hypodermic preadipocyte differentiation test, it was demonstrated that the maximal activity of compounds 3 and 4 was higher than that of rosiglitazone.
Subject(s)
Aminopyridines/chemistry , Aminopyridines/chemical synthesis , Peroxisome Proliferator-Activated Receptors/agonists , Adipocytes/cytology , Adipocytes/drug effects , Aminopyridines/pharmacology , Cell Differentiation/drug effects , Cell Line , Cells, Cultured , Drug Evaluation, Preclinical , Humans , Ligands , Molecular Structure , Rosiglitazone , Thiazolidinediones/pharmacologyABSTRACT
To find novel PPARdelta-selective agonists, we designed and synthesized phenylpropanoic acid derivatives bearing 6-substituted benzothiazoles. Optimization of this series led to the identification of a potent and selective PPARdelta agonist 17. Molecular modeling suggested that compound 17 occupies the Y-shaped pocket of PPARdelta appropriately.
Subject(s)
Benzothiazoles/chemistry , PPAR delta/agonists , Phenylpropionates/pharmacology , Humans , Models, Molecular , Pyrimidines/pharmacologyABSTRACT
To develop novel PPARalpha ligands, we designed and synthesized several 3-{3-[2-(nonylpyridin-2-ylamino)ethoxy]phenyl}propanoic acid derivatives. Compound 10, the meta isomer of a PPARgamma agonist 1, has been identified as a PPARalpha ligand. The introduction of methyl and ethyl groups at the C-2 position of the propanoic acid of 10 further improved the PPARalpha-binding potency.
Subject(s)
PPAR alpha/chemistry , PPAR alpha/metabolism , PPAR gamma/chemistry , PPAR gamma/metabolism , Ligands , Models, Molecular , Molecular Structure , Structure-Activity RelationshipABSTRACT
To develop novel PPARgamma ligands, we synthesized thirteen 3-{4-(2-aminoethoxy)phenyl}propanoic acid derivatives, which are designed based on the structures of rosiglitazone and 15d-PGJ2. Among these compounds, compound 9 was found to be as potent as rosiglitazone in a binding assay and a preadipocyte differentiation test. Molecular modeling suggested that the nonyl group of 9 interacted with hydrophobic amino acid residues constructing the hydrophobic region of PPARgamma protein where the alkyl chain of 15d-PGJ2 is expected to be located.
