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Aim To study the protective effects of Paeoniflorin (PF) on dopaminergic neurons in brain slice of substantia nigra treated with MPP~+ and to investigate the transcription of alpha-synuclei (α-syn) mRNA.Methods The organatypic brain slice culture of substantia nigra prepared from neonatal SD rats was placed on Millicell-CM porous membranes and cultured to day-10.Then the cultrues of slice were treated with different concentrations (0.1,0.5,1.0 mmol·L~(-1)) of MPP~+ for 24 h.Some of the cultrues treated with 0.5 mmol·L~(-1) MPP~+ also received PF (1 or 10 μmol·L~(-1)).Slices cultured in normal medium were used as vehicle control.The tyrosine hydroxylase (TH) immunohistochemical staining with the cell counting was used to determine the dopaminergic neruons.The transcription of α-syn mRNA was examined by real-time quantitative RT-PCR.Results MPP~+(0.1,0.5,1.0 mmol·L~(-1)) exposure markedly decreased the number of TH~+ cells in a dose-dependent manner (P<0.05 or P<0.01) and sharply induced the transcription of α-syn mRNA (P<0.01) in slices treated with 0.5 mmol·L~(-1) MPP~+.The addition of PF (10 μmol·L~(-1)) to MPP~+-treated slices significantly increased dopaminergic neurons survival (P<0.01) and downregulated the transcription of α-syn mRNA significantly (P<0.05).Conclusion PF can effectively inhibit the injury of dopaminergic neurons induced by MPP~+ on brain slice of substantia nigra and downregulate the transcription of alpha-synuclein.
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Objective To explore the specific role of autophagy and ubiquitin-proteasome pathway in apoptosis, specific protease inhibitor and (or) macroautophagy inhibitors.Methods The stimulators were selected to work on the pheochromocytoma (PC12) cell lines transfected with human mutant α-synuclein (A53T).Cell activity and apeptosis rate were detected by MTT law and flow cytometry.NO energy, heat shock protein 70 (Hsp70) and Caspase-3 expression were determined in cell culture.Results A53T cell survival rate significantly decreased 24 hours after handling with the protease inhibitor (100 nmol/L) and (or) autophagy inhibitors 3-MA (10 mmol/L, A =0.23±0.01,0.19±0.01 and 0.17±0.01 respectively; P <0.05) compared with the control group (A =0.32±0.06).Cell survival rate was significantly higher than the other drug group after 24 hours handling with autophagy stimulators (A =0.44±0.08).Compared with the control group or autophagy stimulator of rapamycin (0.2 μg/ml) group (1.55%±1.15%), A53T cells apeptosis percentage rate was significantly higher after treated with proteasome inhibitor and macroautophagy inhibitors 24 hours (4.74%±0.91%, 4.59%±1.18% and 5.40%±1.75%respectively, P <0.05); and a slight decrease with stimulators.Protein Hsp70 and NO were significantly higher in proteasome inhibitor groups than the control group.But in antophagy inhibitor and stimulator group, NO and Hsp70 protein was similar to the control group.Conclusion The inhibition of macroautophagy and proteasome can promote apoptosis.Inhibiting or stimulating autophagy has less impact on Hsp70 and NO than proteasome pathway.