Protection Of TAT-SOD Fusion Protein Against Neuronal Cells Apoptosis After Cerebral Ischemia And Reperfusion

【Background】The reactive-oxygen species are inevitably formed as byproducts of various, normal cellular processes involving interactions with oxygen. These reactive- oxygen species damage macromolecules in the cells, and therefore, sometimes make significant contributions to the several pathological processes of ischemia and reperfusion injury.Indeed, numerous studies have demonstrated that antioxidant enzymes can provide substantial protection against oxidative stresses. Also, recombinant enzymes such as SOD have been used to protect against oxidative stresses. These enzymes were modified to obtain long circulating half-lives and to target specificity by conjugation with polyethylene glycol, albumin, antibody, or encapsidation with liposome. Although a variety of modified recombinant enzymes are available to protect against oxidative stresses, one of the major limitations in using these enzymes is the lack of their efficient transduction ability into cells, thus resulting in an inability to detoxify intracellular ROS.In the present study, we describe the transduction of fulllength TAT-SOD fusion protein into astrocytes and whether this transduced TAT-SOD has a protective effect against oxidative stress in the cells.【Objectives】1. Expression and purification of TAT-SOD; 2. Transduction of TAT-SOD into astrocytes; 3. Induction of transient forebrain ischemia, discover the effect of transduced TAT-SOD on neuronal viability after ischemic insult.【Methods】1. Product the TAT-SOD fusion proteins, which were confirmed by SDS-PAGE and western blot analysis.2. Analyz the effect of transduced Tat-SOD on the viability of astrocytes.3.Found models of cerebral ischemia and reperfusion, and calculate the MDA and SOD of the models.4. Analyz the effect of transduced Tat-SOD on neuronal viability afterischemic insult.【Results】1.TAT-SOD and SOD proteins were highly expressed and soluble in E. coli. The identities of the overexpressed proteins were confirmed by SDS-PAGE and Western blot analysis with monoclonal antibody.2. To evaluate the transducing ability of TAT-SOD, we analyzed the kinetics of its incorporation, by adding it to the astrocyte culture medium and measuring the level of the transduced protein by Western blotting.The activity of transduced SOD increased in a time-and dose-dependent manner.3. We found models of cerebral ischemia and reperfusion, which were evaluated successfully in behavior.4. Calculate the MDA and SOD after cerebral ischemia and reperfusion, MDA increased and SOD decreased respectively.5. We tested the effect of transduced Tat-SOD on neuronal cell viability after administering transient forebrain ischemia by Caspase3 and FCM. The observations show that Tat-SOD passes efficiently across the blood-brain barrier and protect cells against cell death. 【Conclusions】1. The SOD gene was fused with a gene fragment encoding the TAT protein transduction domain of HIV-1 in a bacterial expression vector to produce genetic inframe TAT-SOD fusion proteins.2. We found that TAT-SOD fusion protein is efficiently transduced into astrocytes and that when done so it retains its enzymatic and biologic activities in vitro. The activity of transduced SOD increased in a time-and dose-dependent manner.3. The models of cerebral ischemia and reperfusion were evaluated successfully in behavior. 4.TAT-SOD fusion protein is efficiently taken up into neuronal cells,which resulted in MDA increasing and SOD decreasing slowly respectively by eliminate ROS.5. TAT-SOD prevented neuronal cell death in the hippocampus in response to transient forebrain ischemia. These results suggest that Tat-SOD provides a strategy for therapeutic delivery in various human diseases.