RESUMO
Vizantin has immunostimulating properties and anticancer activity. In this study, we investigated the molecular mechanism of immune activation by vizantin. THP-1 cells treated with small interfering RNA for TLR-4 abolished vizantin-induced macrophage activation processes such as chemokine release. In addition, compared with wild-type mice, the release of MIP-1ß induced by vizantin in vivo was significantly decreased in TLR-4 knockout mice, but not in TLR-2 knockout mice. Vizantin induced the release of IL-8 when HEK293T cells were transiently cotransfected with TLR-4 and MD-2, but not when they were transfected with TLR-4 or MD-2 alone or with TLR-2 or TLR-2/MD-2. A dipyrromethene boron difluoride-conjugated vizantin colocalized with TLR-4/MD-2, but not with TLR-4 or MD-2 alone. A pull-down assay with vizantin-coated magnetic beads showed that vizantin bound to TLR-4/MD-2 in extracts from HEK293T cells expressing both TLR-4 and MD-2. Furthermore, vizantin blocked the LPS-induced release of TNF-α and IL-1ß and inhibited death in mice. We also performed in silico docking simulation analysis of vizantin and MD-2 based on the structure of MD-2 complexed with the LPS antagonist E5564; the results suggested that vizantin could bind to the active pocket of MD-2. Our observations show that vizantin specifically binds to the TLR-4/MD-2 complex and that the vizantin receptor is identical to the LPS receptor. We conclude that vizantin could be an effective adjuvant and a therapeutic agent in the treatment of infectious diseases and the endotoxin shock caused by LPS.
Assuntos
Endotoxinas/imunologia , Glicolipídeos/farmacologia , Imunidade/efeitos dos fármacos , Antígeno 96 de Linfócito/metabolismo , Trealose/análogos & derivados , Animais , Quimiocina CCL4/biossíntese , Citocinas/biossíntese , Expressão Gênica , Glicolipídeos/metabolismo , Células HEK293 , Humanos , Imunidade/genética , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Antígeno 96 de Linfócito/química , Antígeno 96 de Linfócito/genética , Macrófagos/química , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Transporte Proteico , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Trealose/metabolismo , Trealose/farmacologiaRESUMO
A simple reaction procedure for chemiluminescence of firefly luciferin (D-luc) using n-propylphosphonic anhydride (T3P) is reported. A luminescent photon is produced as a result of one-pot reaction, only requiring mixing with the substrate carboxylic acid and T3P in the presence of a mild organic base.