ABSTRACT
To investigate the compounds present in wasabi leaves (Wasabia japonica Matsumura) that inhibit the adipocyte differentiation, activity-guided fractionation was performed on these leaves. 5-Hydroxyferulic acid methyl ester (1: 5-HFA ester), one of the phenylpropanoids, was isolated from wasabi leaves as a compound that inhibits the adipocyte differentiation. Compound 1 suppressed the intracellular lipid accumulation of 3T3-L1 cells without significant cytotoxicity. Gene expression analysis revealed that 1 suppressed the mRNA expression of 2 master regulators of adipocyte differentiation, PPARγ and C/EBPα. Furthermore, 1 downregulated the expression of adipogenesis-related genes, GLUT4, LPL, SREBP-1c, ACC, and FAS. Protein expression analysis revealed that 1 suppressed PPARγ protein expression. Moreover, to investigate the relationship between the structure and activity of inhibiting the adipocyte differentiation, we synthesized 12 kinds of phenylpropanoid analog. Comparison of the activity among 1 and its analogs suggested that the compound containing the substructure that possess a common functional group at the ortho position such as a catechol group exhibits the activity of inhibiting the adipocyte differentiation. Taken together, our findings suggest that 1 from wasabi leaves inhibits adipocyte differentiation via the downregulation of PPARγ.
Subject(s)
Adipocytes/drug effects , Adipogenesis/drug effects , Cell Differentiation/drug effects , Coumaric Acids/isolation & purification , Coumaric Acids/pharmacology , Esters/pharmacology , Plant Leaves/chemistry , Wasabia/chemistry , 3T3-L1 Cells , Adipocytes/physiology , Adipogenesis/genetics , Animals , CCAAT-Enhancer-Binding Protein-alpha/genetics , CCAAT-Enhancer-Binding Protein-alpha/metabolism , Cell Differentiation/genetics , Coumaric Acids/chemistry , Down-Regulation/drug effects , Down-Regulation/genetics , Esters/chemistry , Esters/isolation & purification , Mice , PPAR gamma/genetics , PPAR gamma/metabolism , Sterol Regulatory Element Binding Protein 1/genetics , Sterol Regulatory Element Binding Protein 1/metabolismABSTRACT
Wasabi is a plant of Japanese origin. It belongs to the family Brassicaceae and produces various isothiocyanates (ITCs). To clarify the type I allergies inhibited by wasabi ITCs, we investigated the inhibitory effect on chemical mediator release from dinitrophenylated bovine serum albumin (DNP-BSA)-stimulated RBL-2H3 rat basophilic leukemia cells. Allyl ITC (AITC), sec-butyl ITC (s-BuITC), and 3-butenyl ITC (3-BuITC), which have 3 or 4 carbon chains, inhibited histamine release but did not inhibit the release of leukotriene B4 (LTB4) or cysteinyl LTs (CysLTs). 4-Pentenyl ITC (4-PeITC) and 5-hexenyl ITC (5-HeITC), which have 5 or 6 carbon chains and an unsaturated bond at the end, inhibited LTB4 release but did not inhibit the release of histamine or CysLTs. 6-Methylthiohexyl ITC (6-MTITC), 6-methylsulfinylhexyl ITC (6-MSITC), and 6-methylsulfonylhexyl ITC (6-MSFITC), which have a sulfur atom inserted at the end of a 6-carbon chain, inhibited the release of histamine, LTB4, and CysLTs and the elevation in intracellular Ca(2+). These results suggest that wasabi ITCs inhibited type I allergies by inhibiting chemical mediator release and that the inhibitory effects on each chemical mediator were due to differences in the side chain structure of the wasabi ITCs.
Subject(s)
Isothiocyanates/pharmacology , Plant Extracts/pharmacology , Wasabia/chemistry , Animals , Cattle , Cell Line, Tumor , Dinitrophenols/metabolism , Histamine Release/drug effects , Leukemia, Basophilic, Acute/pathology , Leukotriene B4/antagonists & inhibitors , Leukotriene B4/metabolism , Rats , Serum Albumin, Bovine/metabolismABSTRACT
6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC) is a bioactive ingredient present in wasabi, a popular pungent spice in Japan. Previous studies have revealed the cytoprotective and cancer chemopreventive effects of 6-MSITC. This study aims to clarify the molecular mechanisms by investigating the action of 6-MSITC on the Nrf2/Keap1 system. 6-MSITC up-regulated the expression of NAD(P)H:quinone oxidoreductase 1 (NQO1) by increasing the Nrf2 level. Treatment with 6-MSITC extended the half-life (t(1/2)) of Nrf2 protein from 11.5 to 35.2 min, approximately three times longer. Moreover, 6-MSITC suppressed the ubiquitination of Nrf2 but not Keap1. Alternatively, a modified Keap1 was observed in 6-MSITC-treated cells accompanying reduction of normal Keap1 protein. The results from cellular fractionation and immunocytochemistry assay revealed that Nrf2 was primarily accumulated in nucleus. EMSA and the reporter gene assay further demonstrated that 6-MSITC augmented Nrf2-ARE binding and transcription activity. Silencing Nrf2 using Nrf2 siRNA markedly reduced the Nrf2 level and ARE-driven activity under both baseline and 6-MSITC-induced conditions. Our data revealed that 6-MSITC enhanced Nrf2/ARE-driven NQO1 expression by stabilizing Nrf2 that was accomplished by modifying Keap1 with consequent inhibition of the ubiquitination and proteasomal turnover of Nrf2. The findings provide an insight into the mechanisms underlying 6-MSITC in cytoprotection and cancer chemoprevention.