The Role Of NBP On JNK Pathway In MPP~+-Induced SH-SY5Y Cell Model

Objectives To explore the role of JNK signal pathway related factors in the process of dopaminergic neuronal apoptosis involved in MPP+-induced SH-SY5 Y cell;To explore the protective effects of the NBP by modulating the JNK apoptosis pathway related factors PJNK,Fas and Fas L on PD cell model.Methods Human neuroblastoma cells SH-SY5 Y cells were inoculated in vitro cell culture flasks,with DMEM/F12=1:1 medium for culture,the medium containing 10% fetal bovine serum,penicillin 100U/m L,streptomycin 100U/m L.Experiment was divided into four groups: control group,MPP+ model group,NBP+MPP+ group,SP600125+MPP+group.Control group: SH-SY5 Y cells were treated with complete medium normal culture,the culture of the whole process without any drug intervention;MPP+ model group: added1mmol/L MPP+ in cultured cells and continued culturing for 24h;NBP+MPP+ group: the cells were cultured with 10μmol/L NBP pretreatment and added 1mmol/L MPP+ in cultured cells and continued culturing for 24h;SP600125+MPP+ group: the cells were cultured with 10μmol/L JNK inhibitor SP600125 pretreatment and added 1mmol/L MPP+in cultured cells and continued culturing for 24 h.1 Each group cells morphology was observed by inverted fluorescence microscope;2 The viability of each group cells by determined by MTT colorimetric method;3 Apoptosis rate of cells in each group carried out by Annexin-V/PI flow cytometry;4 P-JNK,Fas,Fas L protein expression in each group cells detected by western-blot;5 Fas,Fas L gene m RNA in each group cells detected by RT-PCR.Results 1 Each cell morphology observed under inverted fluorescence microscope: the control group had more adherent cell number and the morphology was substantially spindle with projections between cells significantly;The number of adherent cells in MPP+group had reduced significantly,the morphological had turned round from fusiform and the projections between cells also tuened shorter;The number of adherent cells in NBP pretreated and JNK inhibitor SP600125 pretreated increased significantly comparing with MPP+ model group.A small number of cells were round and most cell morphology was spindle shape or triangular.The morphology was closed to the control group.2 MTT assay to detect the relative cell viability: setting the control group survival rate was 100%±0.00%,compared with the control group,the relative survival rate in the MPP+ model group was49.3%±2.07%.The survival was significantly lower(P<0.05);Giving NBP pretreated and JNK inhibitor SP600125 pretreated,the cell survival rates were 71.9%±2.10% and76.4%±2.80%,the survival rate increased comparing with the MPP+ model group(P<0.05).3 Flow cytometry to detect the apoptosis rate: the control group apoptosis rate was10.63%±2.07%,compared with the control group,apoptosis in MPP+ model group was32.27%±2.26% and the apoptosis significantly increased(P<0.05);Giving NBP pretreated and JNK inhibitor SP600125 pretreated,the apoptosis rates were 21.13%±3.63% and19.15%±2.63%.Compared with the MPP+ model group,two pretreated group were significantly lower(P<0.05).4 Western blotting detection of P-JNK,Fas,Fas L protein expression: compared with the control group,three factors of MPP+ model group protein were significantly increased(P<0.05).Giving NBP and JNK inhibitor SP600125 pretreated,the protein expression of three apoptosis factors were lower(P<0.05);Control group,MPP+model group,NBP+MPP+ group,JNK inhibitor SP600125+MPP+ group P-JNK protein relative expression levels was: 0.38±0.04,0.94±0.09,0.52±0.09,0.48±0.12;Fas protein relative expression levels was: 0.48±0.09,0.93±0.08,0.61±0.10,0.61±0.12;Fas L protein relative expression levels was: 0.22±0.03,0.52±0.06,0.36±0.07,0.34±0.04.5 RT-PCR detection of Fas,Fas L m RNA gene expression: compared with the control group,two factors of MPP+ model group m RNA expression were significantly increased(P<0.05).Giving NBP and JNK inhibitor SP600125 pretreated,two factors m RNA expression were lower(P<0.05);Control group,MPP+ model group,NBP+MPP+ group,JNK inhibitor SP600125+MPP+ four groups of Fas gene m RNA expression was: 0.09±0.04,0.73±0.09,0.42±0.08,0.25±0.08;Fas L gene m RNA expression was: 0.16±0.06,0.94±0.18,0.53±0.08,0.32±0.08.Conclusions 1 The JNK pathway factor-related P-JNK,Fas,Fas L play a role in the regulation of dopaminergic neuronal apoptosis in SH-SY5 Y cell model.After giving JNK inhibitor SP600125,the expression of three kinds of factors decrease and the apoptosis also decrease;2 NBP may decrease the expression of JNK signal pathway related factors PJNK,Fas and Fas L,and these factors are the potential target points of NBP.