Study On Relation Between Neurotoxicity Of Dopamine On Dopaminergic Neurons And Parkinson's Disease

ObjectiveTo study the neurotoxicity of dopamine on dopaminergic neurons and its mechanism of apoptosis in vivo and in vitro, and examine the action of Lobeline on Dopamine Uptake by Plasma Membrane dopamine transporter(DAT), and study the effect of lobeine and huperzine A in MPTP-induced Mouse Model of Parkinson's Disease. MethodsHuman neuroblastoma SK-N-SH cells, SH-SY5Y cells and Chinese hamster ovary(CHO) cells stably expressing a rat dopamine transporter (DAT) (designated D8 cells), which can take up dopamine in a selective manner, were therefore used as a model to study dopamine nerurotoxicity and analysis the mechanism of cell death by microscope, Giems staining, Hoechst33258, MTT, electron microscope, TUNEL, Flow cytometry, DNA ladder, RT-PCR in vitro. Injections of dopamine(l mol) in the rat medial forebrain bundle (MFB) directly damage the dopaminergic neurons nigrostriatal system and estimate the degrade of lesion through the number of apomorphine-induced contralateral rotations. Using Nissl staining, tyrosine hydroxylase immunohistochemical staining and TUNEL; 6-OHDA-induced hemiparkinsonism in medial forebrain bundle (MFB) evaluate effect of lobeline on nigrostriatal System from behavior in vivo; D8 cells and rat striatal synaptosomes were as one model to study effect of lobeline on DAT-mediated dopamine uptake and release, rotarod testand swim test were examined to evaluate the effect of lobeline in l-methl-4-phenyl-l,2,3,6-trtrahydropyridine(MPTP)-treated mice and Tyrosine hydroxylase(TH) immunohistochemistry and nissle's stainin were detected in substantia mgra(SN). Acetycholinesterase(AchE) was examined in apoptotic cell induced by dopamine, MPTP(MPP+) and 6-OHDA in vitro and in vivo. Percentage of cells viability was measured by MTT and FACS. Rotarod test and swim test were examined to evaluate the effect of huperzine A in MPTP-treated mice. TH immunohistochemistry and nissle's stainin were detected in substantia nigra. rotarod test and swim test were examined in l-methl-4-phenyl-l,2,3,6-trtrahydropyridine (MPTP) -treated mice and Tyrosine hydroxylase(TH) immunohistochemistry and nissle's stainin were detected in SN. ResultsWe observed the dopaminergic neurons damaged by dopamine, accompanied by retraction of processes, shrinkage, apoptosis-like atrophy, accumulation of apoptotic particle examined by microscope, Giems staining, Hoechst33258, electron microscope. DNA fragmentation and TUNEL reaction was positive and The rate of cells viability was increased by MTT and FACS in cells damaged by dopamine. The expression of caspase-3 gene only was examined and a high expression of bcl-2 gene in dopamine-treated cells by RT-PCR. Apomorphinre could induce contralateral rotations after one week with injection of dopamine in MFB. There was more than 60% reduction in the density of tyrosine hydroxylase (TH) immunoreactive cells and nissl staining cells, and positive TUNEL reaction. DNA ladder existed in SN of lesioned side respectively compared with unlesioned side. The results demonstrated lobeline(3mg/kg) could decrease the apomorphine-induced rotation rates(P<0.05) and lobeline(0.1-100M) significantly inhibited 3H-dopamine uptake into D8 cells and rat striatal synaptosomes with concentration- and time-dependent manner, but not affect DAT-mediated release in D8 cells. As an agonist at nicotine receptor on dopaminergic presynaptic terminal and dopamine reuptake inhibitor, lobeline(3mg/kg, i.p.) reversed locomotor deficits detected by rotarod test and swim test in l-methl-4-phenyl-l,2,3,6-trtrahydropyridine (MPTP)-treated mice and increased significantly Tyrosine hydroxylase(TH) positive neurons in substantia nigra (SN)and optical density of TH immunostaining in the striatum compared with only MPTP-treated mice(p<0.05). The results demonstrated that AChE participated in the apoptotic procedure induced by dopamine, MPTP(MPP+) and 6-OHDA and located in the nuclei of aopoptotic cells in vitro and vivo. Huperzine A could increase the percentage of cell viability induced by dopamine in vitro and imp...