| Parkinson's disease (PD) characterised pathologically by the selective loss of dopaminergic neurons and the presence of Lewy bodies (LB) in the substantia nigra is the second most common neurodegenerative disorder after Alzheimer's disease, affecting people in mid and late life. Lots of reports suggest that apoptosis is concerned with the process of Parkinson's disease, but the mechanisms remain unknown. It has been suggested that mitochondrial dysfimction and oxidative stress might be involved. Symptomatic treatments are efective in the early stage of PD, however, most patients may develop motor complications. So those neuroprotective therapies that can slow disease progression shall be instituted.1-methyl-4-phenylpyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-2, 3, 6-tetrahydropyridine (MPTP), can enter the mitochondria to induce oxidative stress and impair energy metabolism by inhibiting mitochondrial complexâ… , inducing a syndrome closely resembling PD. The neurotoxicity of exogenous dopamine (DA) has been described in in vivo, primary cultures and several cell lines. In dopaminergic neurons, DA is oxidized easily in vitro and in vivo to a variety of neurotoxic metabolites such as highly cytotoxic quinine molecules, which participate in the generation of reactive oxygen species (ROS). Many studies indicate that an imbalance between cytoplasmic and vesicular DA may lead to oxidative stress and cell degeneration. DA may cause apoptosis, but most studies on DA toxicity agree that non-apoptofic mechanisms are also involved in DA-induced cell death. model. SH-SY5Y neuroblastoma possesses many of the qualities of human neurons, and moreover, its membrane receptors and transmitters are similar to dopaminergic neuron. So SH-SY5Y cells have been widely and extensively used for the model of PD and for assessment of neurotoxicity and neuroprotection.The present research includes two parts. In the first part, a cell-based model ofα-synucleinopathy was established by Stable transfection of SH-SY5Y with mutant G209A humanα-synuclein for screening compounds with therapeutic potential of Parkinson's disease and for PD-related research. In the second part, 140 compounds or extracturns were screened by MTT method to investigated which one could decrease DA-induced cytotoxicity on cell viability, and then three compounds extracted from Fraxinus sielboldiana blume belonging to the Oleaceae family were studied on their neuroprotection.In the first part of this research, we successfully developed a cell-based model by stable transfection of SH-SYSY with mutant G209A humanα-synuclein, recapitulating neurotoxicity ofα-synuclein overexpression. The cDNA encoding humanα-synuclein was obtained by RT-PCR from SH-SY5Y cells. The mutant G209Aα-synuclein gene was procured by mega primer and gene splicing and overlap extension (SOE) on site-directed mutagenesis of wild-type humanα-synuclein. The mutant G209Aα-synuclein genes were cloned into pcDNA3.1(+) vector and identified by restriction enzyme analysis and DNA sequencing. The overexpression of mutantα-synuclein was analyzed by Western blotting, immunocytochemistry, and RT-PCR. One of the stably transfected clone overexpressed exogenous gene on both protein level and transcriptive level. Cell viability was processed when treated with different concentrations of MPP+ and DA for 24, 48 and 72h by MTT assay. Significant difference in cytotoxicity was found between pcDNA3.1(+) group and pcDNA3.1(+)-hmα-synuclein group in the presence of same concentration of MPP+ and DA within same incubation time. Early apoptosis, late apoptosis/necrosis were analyzed by flow cytometry using Annexin V-FITC and propidium iodide (PI) double staining, respectively. The level of either early apoptosis or late apoptosis/necrosis was remarkably increased in transfected cells compared with control after treated with 100μmol/L MPP+ for 24h. In addition, the presence of typical DNA "ladder" was observed in pcDNA3.1(+)-hmα-synuclein group when treated with 200μmol/L MPP+ for 48h. In this part, it was indentified that cells overexpressing G209A mutantα-synuclein were significantly vulnerable against MPP+ or dopamine exposures and this cell-based model is useful for studying the function ofα-synuclein in PD and screening compounds with therapeutic potential. In the second study, we examined the effect of the three compouds on DA-induced cytotoxicity and the underlying mechanism in human neuroblastoma SH-SY5Y cells. All groups of cells were treated with a range of concentrations (10-8, 10-7, 10-6, 10-5, and10-4 mol/L) of liriodendrin for 24h and cell viability was determined by MTT assay. It was demonstrated that the three compounds do not induce cytotoxicity to SH-SY5Y cells. Our results suggest that the protective effects of them (10-7, 10-6 and 10-5mol/L) on DA-induced cytotoxicity may be ascribed to their anti-oxidative properties by reducing ROS level, anti-apoptotic effect via protection of mitochondrion membrane potential (MMP,ΔΨm), enhancement of the activity of superoxide dismutaee (SOD) and the level of reduced glutathione (GSH), and regulation of P53, Bax and Bcl-2 expression. In addition, they inhibited the release of cytochrome c (Cyt c) and apoptosis-inducing factor(AIF), and protein expression of activated caspase 3. These data indicate that the three compounds may provide a useful therapeutic strategy for the treatment of progressive neurodegenerative diseases such as PD.
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