The Study Of Dopamine-quinone Toxic Effect And Elimination In PD Model Of Brain Slice

Parkinson’s disease (PD) is a neurodegenerative disease commonly seen in aged people, characterized by the loss of dopaminergic neurons especially in the Substantia Nigra, whose pathogenesis is complicated and unclear, The effect of actual therapy is not ideal.Objective:To establish brain slice culture system in vitro, and evaluate PD models on brain slice. To investigate the molecular mechanism of dopamine and L-DOPA induced dopaminergic neuron oxidatvie injury and the effect of quinone oxidoreductase, NQO1, in preventing cytotoxicity of dopamine-quinone. To study the PD pathogenesis and explore potential pathway in therapeutic strategy.Methods:Establish brain slice models and evaluate by immunofluorescence and Western Blot to detect the expression of dopaminergic cell specific protein Tyrosine Hydroxylase (TH). NBT/Glycinate assay is applied to detect the content of quinone protein to determine the relevance of formation of quinone and dopamine induced toxicity. Inhibit the function of Vesicular monamine transporter2(VMAT2) to explore the dependency of dissociative dopamine on oxidative dysfunction. Pre-incubate Sulforaphane (SF) to induce the expression of endogenous phase Ⅱ enzymes and estimate the elimination and detoxification effect on dopamine induced cellular damage.Results:In vitro brain slice culture methods are established to investigate pathogenesis of dopamine-quinone PD models. Significant decreases in viability of DA cell can be observed in DA600μM condition while the content of quinone raises. The livability of DA neurons reduces with the suppression of the activity of VMAT2, accompanied by the increase of quinone content. Marked uprise of survival cells and diminishment of quinone protein occurred after the incubation of SF due to the induced NQO1. Application of L-DOPA to DA cells and5-HT cells lead to dose-dependent manner and a majority of cells disappear under600μM. What’s more, the inhibition of VMAT2by RS results in further cellular damage.Conclusion:High level of dopamine is responsible for the toxicity effect on dopamine neurons and the formation of dopamine-quinone, which suggest that the pathogenesis of PD result from dopamine metabolism and generation of quinone. Besides, side-effects of L-DOPA therapy in PD may also relate to dopamine-quinone and quinone protein, whose toxicity accumulates in dopamine neurons and5-HT neurons. Highly expressed NQO1protect cells against dopamine induced cellular damage on brain slice level, which indicates potential virtual measure to prevent PD and provides new extends in practice.