RESUMO
The metabolic mechanisms for imidazolidine oxidation of imidacloprid (IMI) by cytochrome P450 3A4 (CYP3A4) have been investigated using quantum mechanical/molecular mechanical (QM/MM) calculations. The binding mode of CYP3A4 with IMI is examined by molecular docking in collaboration with molecular dynamics (MD) simulations. The results show that there are six amino acid residues, involving Arg192, Phe195, Ile349, Ala285, Phe284 and Phe88, closely distributed around the IMI. The binding free energy analysis exhibits that the CYP3A4-IMI binding structure is stabilized by electrostatic interaction and van der Waals interaction. Arg192 plays a major role in the binding of CYP3A4 with IMI based on its polarity and the hydrogen bond between the H atom in Arg192 side chain and the nitryl O atom of IMI. Two possible pathways, pathway 1 and pathway 2, are evaluated. Two spin states of the Fe (III) center, quartet and doublet, are considered. The free energy calculations are done using QM/MM steered molecular dynamics (SMD) simulation at the B3LYP/6-31â¯+â¯G(d):ff14SB level for two pathways. The ONIOM QM/MM single-point calculations at the B3LYP/6-311â¯+â¯G(2d,2p):ff99SB//B3LYP/6-31â¯+â¯G(d): ff14SB and M06-2X/6-311â¯+â¯G(2d,2p):ff99SB//B3LYP/6-31â¯+â¯G(d):ff14SB levels are carried out to obtain more credible energy information. The results indicate that for both pathways, the free energy barriers on the low-spin doublet state are lower than those on the high-spin quartet state. Both pathways are the stepwise processes. Pathway 1 has higher possibility to occur with the free energy barriers being lower by 10-15 kcal·mol-1 compared with pathway 2, which gives rise to trans-5'-hydroxyl-IMI as the final product. The first proton-transfer is the rate-limiting step and the calculated activation free energy is consistent with the experimental conclusion.
Assuntos
Citocromo P-450 CYP3A/química , Imidazolidinas/química , Neonicotinoides/química , Nitrocompostos/química , Relação Quantitativa Estrutura-Atividade , Sítios de Ligação , Citocromo P-450 CYP3A/metabolismo , Ligação de Hidrogênio , Imidazolidinas/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Neonicotinoides/metabolismo , Nitrocompostos/metabolismo , Oxirredução , Ligação ProteicaRESUMO
Chitosan oligosaccharides (COS) have been reported to exert many biological activities, such as antioxidant, antitumor and anti-inflammatory effects. In the present study, we examined the effect of COS on nitric oxide (NO) production in LPS induced N9 microglial cells. Pretreatment with COS (50~200 µg/ml) could markedly inhibit NO production by suppressing inducible nitric oxide synthase (iNOS) expression in activated microglial cells. Signal transduction studies showed that COS remarkably inhibited LPS-induced phosphorylation of p38 MAPK and ERK1/2. COS pretreatment could also inhibit the activation of both nuclear factor-κB (NF-κB) and activator protein-1 (AP-1). In conclusion, our results suggest that COS could suppress the production of NO in LPS-induced N9 microglial cells, mediated by p38 MAPK and ERK1/2 pathways.
Assuntos
Quitosana/farmacologia , Microglia/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Óxido Nítrico/antagonistas & inibidores , Oligossacarídeos/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Quitosana/análogos & derivados , Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Camundongos , Microglia/citologia , Microglia/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/genética , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Neuroglia/citologia , Neuroglia/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/genética , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator de Transcrição AP-1/antagonistas & inibidores , Fator de Transcrição AP-1/genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
AIM: To investigate the effect of puerarin on lipopolysaccharide (LPS)-activated N9 microglia cells. METHODS: MTT was used to detect the cytotoxic effect of puerarin and LPS on N9 microglia cells. Flow cytometry was used to determine the effect of puerarin on cellular production of nitric oxide (NO) and reactive oxygen species (ROS), apoptosis and cell cycle in LPS-activated N9 microglia cells. The amount of NO released into the supernatant was detected by Griess assay. The HE staining was used to show the morphological response of LPS-activated N9 microglia cells to puerarin. RESULTS: Morphologically, amoeba-like LPS-activated N9 cells turned to be round in quiescence after treated with puerarin (100-200 micromol/L). Puerarin (50-200 micromol/L) caused less amount of NO released into the supernatant, with 12.43+/-0.11 micromol/L of NO at a dose of 200 micromol/L puerarin compared with 23.45+/-0.19 micromol/L in untreated control (P<0.01). Likewise, puerarin (200 micromol/L) completely abolished the ROS-producing ability of LPS-activated N9 cells, with a basal ROS level comparable to the non-LPS-treated control. Puerarin also exerted an anti-apoptotic effect on the LPS-activated N9 cells and rescued the cells from G(0)/G(1) arrest. CONCLUSION: Puerarin antagonizes activation effect of LPS on N9 microglia cells and might be a useful drug for the treatment of neurodegenerative disorders.
