Establishment And Application Of A Hepatocellar Cell Model With High Expression Of Mn-SOD

Reactive oxygen species (ROS) are the set of certain metabolites of oxygen and products of oxygen reaction, which were closely related to apoptosis, cell cycle, signal transduction and cell differentiation, aging, and many other physiological phenomenons. ROS are well recognized for playing a dual role as deleterious and beneficial species. Excessive oxidative stress will lead to cell necrosis, however, an appropriate degree of oxidative stress will induce the proliferation. A dynamic equilibrium of ROS is necessary in the exercise of its normal function. Superoxide (O2.-) is an important source of intracellular oxidation pressure and can be converted to other types of ROS.As a scavenger of Reactive oxygen species, superoxide dismutase can catalysis superoxide O2.- to H₂O₂ and water, play an important role in reduce intracellular O2.- level .and, especially the Mn-SOD, which was located in the mitochondria, was first defense is to eliminate ROS. Mn-SOD are significantly lower in almost any cancer, research shows that Mn-SOD may be increased the tolerance of oxidative stress.In this study, we have constructed a hepatocellular cell model with high levels of Mn-SOD expression by transfecting cells with eukaryotic expression plasmids containing sense human Mn-SOD cDNA. Additionally, the protective effect of Mn-SOD on mitochondrial activity against the cytotoxicity of oxidant H2O2 was evaluated. Cells with elevated Mn-SOD levels were more resistant to the cytotoxic effects of H₂O₂ than Mn-SOD-deficient cells.In addition, ROS level was detected by flow cytometer; Akt phosphorylation was analyzed by Western blotting and transcriptional factors expression level was measured by RT-PCR to find the specific signal pathway. These findings shows that Overexpression of Mn-SOD selectively inhibited the Akt phosphorylation and decrease redox-sensitive transcriptional factors expression such as c-Fos /c-Jun and p65. Overall, these results indicate that Mn-SOD regulates both cellular redox status and selectively modulates Akt signaling, thereby delaying transcriptional factors activation and inhibiting cell necrosis, resulting in increase of oxidative stress resistance.