| Aims1.To study neuroprotective effect of Eerdun Wurile extracts and their gene regulation;2.To study the promotion of neurite outgrowth effect of Eerdun Wurile extracts and related gene expressionMethods1.Extraction and analysis: The powder of Eerdun Wurile(0.5 g)was extract using water,ethanol,n-butanol,ethyl acetate and petroleum ether,respectively.0.5 g powder was dispersed in the above mentioned solvent and heat at 37 degree for 8 h,while stirring at 210 rpm/min.Then,after the insoluble parts was removed by filtration,the filtrate was concentrated and dried under reduced pressure before submitted for UPLC-q TOF MS analysis.2.Cytotoxicity: The cytotoxicity of all extracts was assessed by MTT method.PC-12 cells were seeded in 96-well plate(1x104cells/well)and were incubated at 37 degree under 5% CO2 for 24 h.Then,extract with different concentration were added to the cells and the cell viability was measure according to the MTT protocol.3.Neuroprotective effect of Eerdun Wurile extracts:3.1.MTT method: PC-12 cells were seeded in 96-well plate(1x104cells/well)and were incubated at 37 degree under 5% CO2 for 24 h.Then,Quercetin(positive control,50-80 ug/m L)as well as Eerdun Wurile extracts(50-100 ug/m L)were added to the cells,respectively.After 12 h incubation,the cell were treated with H2O2(250 u M)for 4 h,followed by assessment of cell viability according to MTT protocol.3.2.Anti-oxidant effect: The anti-oxidant effect of Eerdun Wurile extracts was measure using ROS kit,SOD kit and CAT kit,according to the manufactures' instructions.3.3.Live/dead cell staining method: H2O2 treated PC-12 cells were further treated3.4.with various Eerdun Wurile extracts.Then,cells stained with red and green were observed under microscope.3.5.RT-q PCR method: The expression levels of neuroprotection and apoptosis related genes(Bax,NF-k B,Caspase 9,Bcl-2,PARP,p38,Jnk and Akt)were analyzed by RT-q PCR.4.The promotion of neurite outgrowth effect of Eerdun Wurile extracts4.1.The promotion of neurite outgrowth effect of 5 Eerdun Wurile extracts;4.2.RT-q PCR analysis: The expression of neurite outgrowth related genes(Nefh,Sym1,Dlg4)in Eerdun Wurile extracts treated PC-12 cells were analyzed by RT-q PCR.Results1.Analysis of Eerdun Wurile extracts by UPLC-q TOF MS identified 16 neuroactive compounds.2.Cytotoxicity: MTT assay indicated that water extract of Eerdun Wurile shows minor toxicity at the concentration of 150-200 ug/m L.All other extracts show cytotoxicity at the concentration of 150 ug/m L.Therefore,a concentration range of 50-100ug/m L was used for the further experiments.3.Neuroprotective effects: MTT assay indicated that treatment of PC-12 cells with H2O2 decreased cell viability by 60%.Treatment with Eerdun Wurile extract significantly increases cell viability.The ROS content,which increases upon H2O2 treatment,was decreased by Eerdun Wurile extracts;SOD content,as well as CAT activity of PC-12 cells was elevated upon treatment with Eerdun Wurile extracts.The extracts of Eerdun Wurile also enhanced cell viability after H2O2 treatment,while efficiently regulating the expression of neuroprotective related genes including Bax,NF-k B,Caspase 9,Bcl-2,PARP,p38,Jnk and Akt.4.The promotion of neurite outgrowth effect of Eerdun Wurile extracts: The Eerdun Wurile extract efficiently promoted neurite outgrowth of PC-12 cells.The mechanism of such neurite outgrowth effect may be the up-regulation of genes such as Nefh,Sym1,Dlg4.Conclusion1.Eerdun Wurile contains at least 16 neuroactive compounds;2.These compounds exhibit neuroprotective effects;3.The active molecules also promotes neurite outgrowth. |
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