Cellular Toxicity Of Dopamine Quinone And Prevention Study

AIM:To study the influence of dopamine (DA) on proliferation and survival of dopaminergic cells and determine whether Quinone oxidoreductase (NQO1) protects dopaminergic cells against dopamine-induced cytotoxicity; to investigate the pathogenic mechanism of Parkinson's disease and explore the potential therapeutic application of phase Ⅱ inducers in the treatment of Parkinson's disease.METHODS:The immunohistochemistry and immunofluorescence method were used to detetct the nerve cell's and dopamine neuron's special antigen Microtubule-associated protein2(MAP2) and Tyrosine hydroxylase (TH) in cell lines and primary cells. MTT assay (mono-nuclear cell direct cytotoxicity assay) was hered to determine the cells survival rate after different treatments.Westem blot was applied to investigate the expression of proteins such as endogenous phase Ⅱ enzymes. The content of quinone protein was determined by the NBT/glycinate assay.. Adenovirus was used to transfer NQO1genes by infect various cell lines and primary cells and NQO1expression was detected by immuno fluorescence staining and Western blot..Immunohistochemistry and Western blot was also used to detect NQO1expression in brain region of animal model after intraperitoneal injection of SF.RESULTS:SH-SY5Y cells show more differentiated neuron phenotype and express TH more; Original generation cells as a model of DAergic neurons for PD research successively obtained alternatively after one week culture in DMEM/F12+10%FBS and one week in Neurobasal+Diff.Dopamine reduces the viability of SH-SY5Y and primary midbrain cells in a dose-dependant manner.Dopamine also reduces the TH positive cells of primary cultured midbrain cells.SF was effective phase Ⅱ enzyme inducer and protected cells against DA-induced cytotoxicity. SF increases NQO1expression and alleviates DA-induced toxicity in primary neurons. Increased expression of NQO1in primary neurons by infection of an NQOl-adenovirus alleviates DA-caused cytotoxicity, indicating that the SF protective effect was at least partly due to the higher levels of NQO1expression. The intracellular content of quinone protein correlated with cell viability, which was reduced by SF pre-incubation. In primary neuronal cultures, DA-induced toxicity was also reduced by SF pre-incubation.The primary midbrain cells expressed NQO1in microglia cells; SF can induce astrocyte and oligodendrocyte to express NQO1; all the neuron and glia can express NQO1after infected by NQO1-adeno virus. After intraperitoneal injection of SF, NQO1expression was increased in the midbrain.CONCLUSIONS:Dopamine reduces cell proliferation in SH-SY5Y cells in a dose-dependant manner.. Expression of NQO1can be increased via SF treatment and adenovirus infection. Overexpression of NQO1in primary neurons protects cells against cytotoxicity caused by dopamine. High levels of NQO1expression also protect TH-positive neurons against DA-induced toxicity. The amount of intracellular quinone protein correlates with cell viability, indicating that the formation of quinone protein contributes to the toxicity in dopaminergic neurons. A pilot study indicated that an intraperitoneal SF injection enhances NQO1expression in the midbrain, supporting the potentially application of phase Ⅱ inducers like SF in the treatment of Parkinson's disease.