The Study Of Cytotoxicity And Mechanism Of Free Dopamine On Dopaminergic Cells

It is well known that the main pathologic change of Parkinson‘s disease(PD)is the special and continuing loss of dopaminergic neuron in substantia nigra.It is also considered that the loss of dopaminergic neuron is related with the metabolism of dopamine(DA)itself.Free DA which reuptaked from extracellular through dopamine transporters or synthesized in cell is mainly transported into vesicles by vesicle transporters to avoid oxidation.However,when the intracellular free DA increased for some reasons,it is easily affected during oxidative stress and produces dopamine quinone(DAQ)that will combine with proteins of sulfhydryl,which leads to cytotoxicity and the death of dopamine neurons.In this paper,to verify the toxic effect of dopamine quinone and the cytoprotective effect of antioxidant quinone,the human neuroblastoma cell line(SH-SY5Y)was used as the model of dopamine cell to investigate the effects of increased dopamine on intracellular quinone protein formation and cell survival.On this basis,some cytoxic pathway were studied.It is also known that the sulforaphane can reduce the content of quinone compounds in cells.The protective effect of sulforaphane on the toxicity of DA and the effect on the formation of dopamine quinone protein were observed in the experiment.The experiment was divided into two parts: firstly,exogenous DA was added to SH-SY5 Y cell with different concentrations.After 24 h treatment,cell viability was determined by MTT assay,quininylated protein content determined by NBT assay,and Parkinson’s disease-related protein content was determined by Western Blotting assay.The results showed that DA had a dose-dependent toxic effect on SH-SY5 Y cells within a certain DA concentration range and it was correlated with the quininylated protein content.After 80 μM DA treatment,Parkinson’s disease-related proteins such as PINK1,Parkin and DJ-1,which are rich in sulfhydryl groups and easy to be quininylated to form dopamine quinone protein polymers,thier normal contents are significantly reduced.Secondly,SF was used as the inducer of NQO1 protein(NAD(P)H quinone oxidoreductase,NQO1)to reduce the formation of quinone compounds.The effect of SF on DA toxicity and the formation of quinone protein were verified.We pretreated with SF 1.25 μM for 12 h and then added DA to detect the content of quininylated proteins and cell survival rate.The experimental results showed that SF pretreatment reduced the content of cell quininylated protein and increased the cell survival rate,further indicating the correlation between DA toxicity and the formation of quininylated protein and the protective effect of SF on the toxicity of DA.In conclusion,the cytotoxic effect of DA may be related to the oxidative product dopamine quinone and the further formation of dopamine quinone-protein.It is well known some of the Parkinson‘s disease-related proteins such as Parkin,PINK1,DJ-1 are play an important role in the function of mitochondria and ubiquitin system.Our result shows the content of these proteins decreased obviously after treated by DA.It indicates that one way of DA‘ cytotoxicity is that DA affected the function of those Parkinson‘s disease-related proteins and eventually cause cell death.