The Influence Of Rotenone On The Dopaminergic SH-SY5Y Cells In Vitro

[Objective] The purpose of present study is to determine the mechanism of rotenone neurotoxicity for dopaminergic cells, and the role of mitochondrial dysfunction in the process of a-synuclein aggregate.[Methods] 1. The α-synuclein-pEGFP, a recombinant gene, were transfected into SH-SY5Y cells with lipofectamine. The expression of a-synuclein was determined by EGFP fluorescence, immunocytochemistry. 2. Cell viability was measured using MTT assay after cells were exposed to rotenone. The changes in cytoplasm were identified with HE staining and immunohistochemistry. The ultrastructural alterations of cells treated with rotenone were observed with electron microscopy. Oxidative stress was determined by DCF assay. The activity of mitochondrial complex I, the degree of the mitochondria swelling and the content of O2- in mitochondria were also detected.[Results] 1. The results of present study showed that the expression of EGFP protein with green fluorescence was observed throughout the cell body, neuritis and nucleus with fluorescent microscopy. The anti-a-synuclein immunostaining with red fluorescence was observed in cytoplasm. This result indicated that a-synuclein expressed in transfected humandopaminergic neuroblastoma cells. 2. The grown rate of cells was inhibited by exposed to rotenone (p <0.01). But. the decreased degree of the viability in transfected cells was less than that of untransfected cells (p <0.05). The α-synuclein protein was induced to aggregate and form the eosinophilic intracytoplasmic inclusions, which were immunoreactive to a-synuclein. The cytoplasmic inclusions with an electron-dense core, abnormality of the ultrastructure of the mitochondria, increment of the number of autophgosome in a-synuclein transfected cells were seen with electronic microscope. Oxidative stress was observed with enhanced intensity of DCF fluorescence in rotenone treated cells (p <0.01), while, the intensity of DCF fluorescence in transfected cells was lower than in untransfected cells (p <0.01). The mitochondrial complex I activity was decreased in rotenone treated cells and mitochondria swell was observed in these cells (p <0.01). [Conclusion] 1. The results of this present study indicated that the recombinant vector, α-synuclein-pEGFP could express both EGFP reporter gene and a-synuclein in human dopaminergic neuroblastoma cells, SH-SY5Y. 2. Rotenone could inhibit the viability of cells and induce the aggregation of a-synuclein protein to form the eosinophilic intracytoplasmic inclusions. Mitochondrial function was impaired by rotenone. The results suggested that rotenone might be a risk for PD. 3. There was no significantly neurotoxicity lesion in cells transfected with a-synuclein gene. 4. Mitochondrial dysfunction might play an important role of in the process of a-synuclein aggregates.