NADPH Inhibits MPTP-induced Neuroinflammation And Neurotoxicity

Aim:To observe the effect of nicotinamide adenine dinucleotide phosphate(NADPH)on neuroinflammation and neurotoxicity in 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-and MPP~+-induced model of Parkinson disease(PD)and study the mechanisms.Methods:In vivo PD model was developed with intraperitoneal injection of MPTP to C57BL/6 mice and in vitro PD model was produced with MPP~+(active metabolite of MPTP)in microglia cell line BV-2 cells.The levels of NADPH and GSH in midbrain were analyzed by using the enzychrom NADPH/NADP~+assay kit and the GSH assay kit.The levels of reactive oxygen species(ROS)in the SNpc and BV-2 cells were determined with DHE staining.Western blot analysis and immunofluorescence analysis were performed to assess the protein levels of GFAP,Iba-1,p-IKK?,I?B?,p-p38 MAPK,COX2,inflammasome components and THase in SNpc or BV-2 cells.Immunofluorescence analysis and Western blot assay were used to detect the protein levels of nuclear factor-?B(NF-?B)in the nucleus and cytoplasm in SNpc.ELISA assay was employed to determine the levels of IL-1?and TNF-?in SNpc or co-culture model of microglial and neuronal cells.In vitro,the nuclear staining with Hoechst 33342 and PI was used to determine the apoptosis of neuronal cells in co-culture model and behavioral experiments were carried out in vivo.Results:MPTP/MPP~+increased levels of ROS,activated glial cells,activation of NF-?B,phosphorylation of p38 MAPK,and inflammasome proteins in SNpc.These responses were inhibited by supplementation of exogenous NADPH.NADPH effectively decreased MPP~+-induced excessive production of ROS,p38 phosphorylation and inflammatory protein of cyclooxygenase2(COX2)in cultured microglial BV-2 cells in vivo studies.Similarly,the p38 MAPK inhibitor SB203580 suppressed the upregulation of p38 phosphorylation and COX2 protein levels induced by MPP~+.Co-culture of neuronal cells with MPP~+-primed BV-2 cells increased the levels of TNF-?and induced cell death.These effects were diminished by TNF-?neutralizing antibody and NADPH.NADPH reduced motor dysfunction and the loss of dopaminergic(DA)cell induced by MPTP.Conclusion:NADPH protects DA neurons by inhibiting oxidative stress and glia-mediated neuroinflammation both in vitro and in vivo,thus suggesting a potential of clinical application for PD and other neurodegenerative diseases.