| 【Introduction】Parkinson's disease(PD) is a neurological disorder characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta.Although the etiology of PD remains unclear,several risk factors have been associated with the disease,including age,genetic and environmental fators.1-methyl-4-phenyl-1,2,3,6-tetrahy-dropyridine(MPTP) is a neurotoxin which can lead to parkinsonism in animals and humans. Its active metabolite is 1-methyl-phenyl pyridinium cation(MPP~+) which can hurt the DA neurons in the substantia nigra corpara striata.2,3,5,4-tetrahydroxystilbene-2-o-β-D-glucoside(TSG) is the main ingredient found in Polygonum multiflorum, can anti-oxidation, scavenge free radicals and improve the memory.Recently ,the neuroprotective properties of TSG are rapidly becoming focused.Resesrch indicated that TSG can improve the learning and memory capability of senile dementia mice. However,it is still unknown that whether TSG could provide neuroprotective effects aginst the injury which induced by MPP~+ in dopaminergic neurons or not.The central hypothesis guiding this study is that TSG may play a protective role in MPP~+ induced injury of dopaminergic neurons.PC12 cells retaining dopaminergic characteristic,was used as an in vitro model.The possible effect of TSG was investigated and the related mechanism was proved.【Objective】1. To set up Parkinson's disease model using MPP~+ on PC12 cells,seive the effective concentrations of MPP~+ inducing injury and the protective concentrations of TSG against the injury in PC12 cells.2. To explore the protective effect of TSG against the apoptosis induced by MPP~+.3. To explore the mechanisms of the neuroprotective effects of TSG on MPP~+-induced apoptosis in PC12 cells.【Methods】1. Setting control group and 200,300,500,700,800,1000,1200,1500 mol/L MPP~+ groups on PC12 cells for 24h;To choose the protective concentrations of TSG, control group, MPP~+ group (500 mol/L) and the different concentrations of TSG(1,5,10,50,100 mol/L) protevtive groups were set.The cell viability was measured by MTT assay.2. Set normal control group,MPP~+group and the different concentrations TSG pretreatment(1,5,10 mol/L)groups, cell viability was measured by MTT assay,the cells and the changes in nuclear morphology of cells are observed by labeling the cells with the nuclear stain Hoechst 33258,apoptosis rate was measured by flow cytometry,determination of the DNA fragmentation was measured by terminal deoxynucleotidyl transferase( TdT)-mediated dUTP biotinnickend- labeling(TUNEL)staining cells.3. The expression of caspase-3,p38MAPK ,p53 in PC12 cells was observed by Western blotting.【Results】1.MTT assay showed that cell viability is 48.3±3.6%(P<0.001)after using 500 mol/L concentration of MPP+ treatment for 24 h,compared with the control. While TSG pretreated for 24h followed by MPP(+500 mol/L)treated for 24h,cell viability in the 1,5,10 mol/L TSG pretreatment groups respectively increased to 57.8±1.2%(P<0.05),74.3±2.7%(P<0.05),86.8±2.0%(P<0.05)respectively, but the high concentration of TSG(50,100 mol/L)show no protection effect on the cells.2.MTT assay showed that TSG(1,5,10 mol/L)could inhibit MPP+-induced cell viability which decreased in PC12 cells. The cell viability in the 1,5,10 mol/L TSG pretreatment groups increased to 57.8±1.2% ( P<0.05 ) ,74.3±2.7%(P<0.05),86.8±2.0%(P<0.05)respectively, comparaed with the group of MPP+ treated (48.3±3.6%). Hoechst stain showed that normal cell nucleus were blue, smooth and integrity in the edge,could see uniformly hypochromic staining. While the MPP+ group show cyto-chromatin concentration,cellular presentation pykno-strong staining,showing stronger fluorescence,even observed that the clearage of cellular,appearance pyknicly granula-like fluorescence. The rate of apoptosis after useing TSG pretreatment decreased to 31.6±2.3% (P<0.05),22.4±1.8% (P<0.05) and 13.4±1.1% (P<0.05), respectively,and nuclear morphous improved ,fluorescence intensity weaken;the results of FCM show that the rate of apoptic cells in the normal cell group,MPP+ cell group,TSG( 1,5,10 mol/L)groups were 1.5%,35.6%,27.2%,19.1% and 13.3%, respectively. TUNEL staining showed that the TUNEL positive cells in the MPP+ group increased to 35.3±1.2%(P<0.01),while the TUNEL positive cells in the TSG groups dramatically decreased to 25.3±1.2% (P<0.01),17.3±0.9% (P<0.01) and 11.7±0.9%(P<0.01), respectively,which all comparaed with the normal control group( 4.0±0.6%).3. Results of Western blotting assay showed that the expression of caspase-3 , p38MAPK and p53 in the TSG groups is evidently weaker than that in the MPP~+ group.【Conclusion】1.TSG can inhibit MPP~+-induced apoptosis in PC12 cell,and have dose-dependent feature in the low concentration range.2.The mechanisms of TSG protective effects on MPP~+-induced apoptosis in PC12 cell may be concerned with TSG inhibiting the activation of caspase-3, lowing regulation the expression of p38MAPK and p53. |
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