Study On The Protective Effects And Pathogenesis Of 1,5-diCQA Preconditioning On Astrocytes Induced By OGD/Reperfusion

Objective To investigate the expression and the effect of NF-E2-related factor2 on oxidative stress in astrocytes induced by ischemia/reperfusion.Methods Oxygen-glucose-deprivation and (OGD)/reperfusion to induce the injury of primary cultured rat astrocytes, and ROS level, cellular GSH content, changes of survival rate and the expression of Nrf2 in cytoplasm and nucleus were observed respectively.Results With the prolongation of OGD/reperfusion, the cellular ROS level was gradually increased, GSH content was delption and the cell viability was decreased. However, at the early stage of injury (reperfusion 0.5h), the expression of Nrf2 was decreased in cytoplasm but increased in nuclear. With the injury continuing, Nrf2 in both cytoplasm and nuclear were decreased. After 12h reperfusion, the expression of Nrf2 showed a tendency toward stabilization.Conclusion The Nrf2 nuclear translocation possessed correlation with the oxidative stress in astrocytes induced by OGD/reperfusion.Objective To identify the effect of Nrf2 siRNA transfaction in astrocytes for the silenceof target gene.Methods The small interfering RNA specific targeted to Nrf2 gene was constructed by chemical synthesis and was transfected into primary culture astrocytes using lipofectamine2000. Transfection efficiency was detected using fluorescence microscopy ans flow cytometry. The expression of Nrf2 mRNA and protein in the tansfected astrocytes was identified by RT-PCR and western blot analysis respectively.Results The eukaryotic expression vector of Nrf2 was constructed and tansfected primary culture astrocytes. The transfection efficiency was 52.19%,59.72%,61.43%, 70.61% and 62.16% respectively in 24,48,72 and 96h. RT-PCR and western blot analysis showed that Nrf2 siRNA can efficiently suppress Nrf2 mRNA and protein expression (P＜0.05).Conclusions Transfect Nrf2 siRNA into astrocytes can effectively inhibite Nrf2 mRNA and protein expression, prepare a condition for subsequent research of medical intervention in ischemia model of astrocytes.Objective To investigate the protective effects of 1,5-diCQA preconditioning on the injury of astrocytes induced by OGD/reperfusion and the related mechanisms.Methods Oxygen-glucose-deprivation and (OGD)/reperfusion to induce the injury of primary cultured rat astrocytes. The cells were divided into:the control group, OGD/reperfusion group,1,5-diCQA+OGD/reperfusion group. The release of LDH, ROS level, cellular GSH content, changes of survival rate and the expression of Nrf2 in cytoplasm and nucleus, glutamate-cysteine ligase (GCL) activity were observed respectively. The indicators mentioned above were also detected in Nrf2 siRNA tansfected astrocytes to verificated whether the protective effects of 1,5-diCQA depend on the activation of Nrf2/ARE pathway.Results 1,5-diCQA pretreatment significantly suppressed cell death, reduced the production of reactive oxygen species, prevented glutathione (GSH) depletion, increased the activity of glutamate-cysteine ligase (GCL) and triggered Nrf2 nuclear translocation in astrocytes induced by 4 h OGD and 24 h reperfusion. Interestingly, these protective effects were greatly attenuated in Nrf2 siRNA-transfected cells.Conclusions 1,5-diCQA has antioxidant signaling properties that upregulate GSH synthesis by stimulating the Nrf2 pathway in astrocytes and protects them from cell death in an in vitro model of ischemia/reperfusion.Objective To study the effect of persistent oxygen-glucose-deprivation (OGD) on the death pathway of primary culture rat astrocytes and the relevant mechanisms.Methods The primary culture rat astrocytes were divided into three groups:①the control group:cells maintained in high glucose DMEM supplemented with 20% fetal calf serum;②the cells treated with OGD;③pretreatment with PD 150606 (100μmol/L) before OGD. the changes of the cellular morphology, the energy metabolism of astrocytes, and the percentages of apoptosis or oncosis of the astrocytes induced by OGD were observed respectively, and Western blot was adoped to assay the protein expression of several cytoskeletal proteins, including paxillin, vinculin, vimentin and GFAP.Results Electron microscopy revealed the coexistence of ultrastructural features of both apoptosis and oncosis in individual cells. The cellular ATP content was gradually decreased (P＜0.05) and the percentages of apoptotic and oncotic cells were increased over the OGD time(P＜0.05). After 4 h OGD, the ATP depletion below 35% of control and oncosis became the main pathway of astrocytes death. OGD led to a significant decrease in paxillin, vinculin, vimentin protein levels in a time-dependent manner (P<0.05) while GFAP appeared markedly more than the control at 0.5 h OGD treatment (P＜0.05), then decreased later. Pre-treatment with 100μmol/L Calpain inhibitor PD150606 led to decrease in the loss of cytoskeleton-associated proteins, and delayed the LDH release of astrocytes associated OGD damage.Conclusion Astrocytes induced by persistent OGD would be go though apoptosis and oncosis, and there was a narrow range of ATP threshold (<35%of the control) that determines astrocytes oncotic death induced by persistent OGD; calpain-mediated hydrolysis of the cytoskeleton-associated proteins may contribute to astrocytes oncosis.