Abstract: Objective To investigate the possible correlation between the expression of Nurr1 gene, a midbrain transcription factor, and selective apoptotic cell death induced by neurotoxin 6-OHDA. Methods Firstly, the expression of Nurr1 was detected by immunohistochemistry and RT-PCR in transfected SK-N-SH/Nurr1 cells. Secondly, following treatment with 50~100 μmol/L 6-OHDA on both SK-N-SH and SK-N-SH/Nurr1 cells for 6 and 12 h, the cell loss was detected to discriminate between apoptosis and necrosis by using flow cytometry(FCM) and Annexin V/PI double staining. Thirdly, the ultrastructural changes of both cells were observed by transmission electronic microscopy(TEM) after treatment with 75 μmol/L 6-OHDA for 12 h. Finally, the level of apoptosis effector Caspase-3 of both cells were detected after exposure to 75 μmol/L 6-OHDA for 6 and 12 h by Western blotting analysis. Results Firstly, immunohistochemical staining and RT-PCR analysis showed that the over expression of Nurr1 in transfeced SK-N-SH cells. Secondly, Annexin V/PI double staining with FCM showed treatment with 75 μmol/L 6-OHDA for 12 h induced a significant apoptotic phase with Annexin V+/PI-only in SK-N-SH/Nurr1 cells(P=0.000), while both cells showed increased necrotic phase with Annexin V+/PI+ after exposure to 100 μmol/L 6-OHDA for 6 and 12 h. Thirdly, the result of TEM showed that treatment with 75 μmol/L 6-OHDA for 12 h induced typical apoptosis in SK-N-SH/Nurr1 cells. In contrast, no morphological characteristics of apoptosis in SK-N-SH cells were observed. Finally, Western blot analysis showed that treatment with 75 μmol/L 6-OHDA for 6 and 12 h decreased the level of pro-Caspase-3 in SK-N-SH/Nurrl cells but not in SK-N-SH cells(P=0.000). Conclusion Nurr1-overexpression stimulated apoptotic pathway induced by 6-OHDA,specifically in a dose-dependence manner.

Key words: nuclear receptor related factor1, overexpression, 6-hydroxydopamine, apoptosis, flow cytometry