Effect of 6-hydroxydopamine on the levels of phosphorylation of signal transduction and transcription factor 1 and 3: experiment with dopaminergic neurons

Abstract

To observe the effects of 6-hydroxydopamine (6-OHDA) on the levels of phosphorylation of signal transducer and activator of transcription (STAT) 1 and STAT3, and the role of STASD in the neurotoxicity of 6-OHDA on dopaminergic neurons so as to help study the treatment of Parkinson's disease. Dopaminergic neurons, human neuroblastoma cells of the line SH-SY5Y were cultured and were treated with 6-OHDA of the concentrations of 50, 100, 200, and 400 micromol/L respectively. SH-SY5Y cells not treated with 6-OHDA were used as control group. MTT method was used to observe the cell viability rate 24, 48, and 72 h later. 3H-thymidine deoxyribose (TdR) was added into the culture fluid 12, 36, or 60 h after the treatment of 6-OHDA for 12 h, then the cell viability rate was observed. Western blotting was used to detect the levels of STAT1 and STAT3 phosphorylation. Twenty-four hours after the treatment of 50 - 400 micromol/l 6-OHDA the cell viability rates decreased to 8.1% - 81.9% that of the control cells (all P < 0.05 or P < 0.01), e.g., 100 micromol/L 6-OHDA decreased the cell viability rate to 50% that of the control cells (P < 0.01). 3H-TdR proliferation assay showed that the cell viability rate decreased with the increase of the 6-OHDA concentration and time of treatment too. Western blotting showed that 0.5 - 24 h after the treatment with 100 micromol/L 6-OHDA, the STAT3 phosphorylation level was decreased obviously with the comparative band density of 0.747 +/- 0.011 - 0.238 +/- 0.007, and 2 hours after the treatment with 50 - 200 micromol/L 6-OHDA the comparative band density of STAT3 phosphorylation was 0.895 +/- 0.003 - 0.633 +/- 0.002 (all P < 0.05). However, the level of STAT1 phosphorylation was not influenced by the treatment of 6-OHDA of the concentration of 100 mol/L at different time points. Decreased STAT3 phosphorylation may be involved in the neurotoxicity mechanisms of 6-OHDA on dopaminergic neurons.