The HMnSOD-transfected PC12 Cells And Its Anti-oxidative Stress

Increasing evidence implicates oxidative stress in the neurodegenerative diseases,such as Alzheimer's disease(AD), Parkinson~ s diease, aging, ischemia and reperfusion. Hydrogen peroxide is an important precursor of highly reactive free radicals. Hydrogen peroxide can induced many cell lines apoptosis, one of the mechanism of which is by reactive oxygen species from mitochondria or via changing the mitochondrial membrane potential. Manganese superoxide dismutase (MnSOD) is strictly a mitochondrial enzyme located in the inner membrane and encoded by nuclear gene. It protects mitochondria from oxidative damage. So this study to explore that MnSOD modulates hydrogen peroxide induced apoptosis in the transformed PC12 cells. The sense and antisense human MnSOD cDNA under the transcriptional control of a human P -actin promoter were introduced into PC12 cells by Iipofectin transfection with recombinant plasmid containing neomycin selectable marker. MnSOD activity increased about two-fold in the cells transfected with the sense MnSOD cDNA, and decreased to about 33% in the cells transfected with the antisense MnSOD cDNA. When PC12 cells were exposed to the low concentration (50-lOOjtM) of hydrogen peroxide, the direct consequence of oxidative damage is apoptosis. The cytotoxicity was assayed by MTT assay, flow cytometry was used to analyze DNA fragmentation quantitatively and Hoechst 33258 stain revealed the morphological changes of nucleus. But the cells transfected sense MnSOD gene can significantly prevent cells from the insult of hydrogen peroxide; on the other hand, the cells transfected with antisense MnSOD gene were more sensitive to hydrogen peroxide cytotoxicity than parent PCI2 cells. The expression of APP gene didn't alter in neither of the cells transfected with sense MnSOD eDNA or the cells transfected with antisense MnSOD cDNA. So the results show that the cells overexpressing MnSOD confer protection against H202-induced apoptosis on PC12 cells. And the results suggest the damage of mitochondria may be involved in the cytotoxicity of H202 on PC12 cells, which induces imbalance of cellular redox state and apoptosis.