| Microglial cells are resident macrophage-like immune cells in the brain, activated microglial cells produce large amount of biologically active substances, Activated microglial cells play important role in modulating oxidative stress, inflammatory response and immune response in central nervous system diseases. Therefore, a better understanding of the responses of microglial cells to certain kinds of cytokines may provide a valuable clue for the treatment of such central nervous system diseases.Microglia-derived reactive species such as hydrogen peroxide (H2O2) and nitric oxide (NO) are thought to play a defensive role in pathological conditions. However, the reactive species may in turn harm microglial cells. For self-protection against oxidative damage, therefore, microglial cells must be equipped with antioxidative defense mechanisms. In this study, we investigated the regulation of interferon-γ(IFN-γ) on oxidative injury in microglial cells as well as the mechanisms involved in the regulation effect. Firstly, the regulation effects of IFN-γand IL-1βon H2O2 toxicity in microglial cells were assessed by morphological examination and LDH measurement. We found that pretreatment with IFN-γfor 20 h, but not IFN-γcotreatment and IL-1βpretreatment, protected microglial cells from the H2O2-evoked toxicity. Pretreatment of IFN-γalso protected microglial cells from the toxicity of various reactive species such as superoxide anion (O2-), peroxynitrite (ONOO-),4-Hydroxynonenal (HNE) and rotenone. In addition, IFN-γdid not protect astrocytes or macrophages from H2O2 toxicity. IFN-γprotected microglial cells in a time-dependent manner when the pretreatment time increased from 6h to 24 h. In addition, the cytoprotective effect of IFN-γpretreatment was abolished by the protein synthesis inhibitor cycloheximide. These results imply that protein synthesis is required for the protection by IFN-γ.In order to investigate the effect of IFN-γon microglial antioxidant enzyme system, the expression and activity of antioxidant enzymes were determined with western blot analysis and activity assay kits respectively. Among various antioxidant enzymes such as manganese or copper/zinc superoxide dismutase (Mn-SOD or Cu/Zn-SOD), catalase, glutathione peroxidase (GPx), peroxiredoxin (Prx) and sulfiredoxin (Srx), only Mn-SOD and Srx was augmented in IFN-γ-pretreated microglial cells. Furthermore, the expression of Prx-SO2 induced by H2O2 was attenuated by IFN-y pretreatment. Whereas the activities of Mn-SOD and catalase were up-regulated by IFN-y pretreatment, those of Cu/Zn-SOD and GPx were not. Transfection with siRNA of Mn-SOD abolished both up-regulation of Mn-SOD expression and protection of IFN-y pretreatment on H2O2 toxicity. Application of catalase inhibitor partially abolished the protective effect of IFN-y pretreatment. Moreover, increased level of Srx could enhance the reduction of Prx-SO2 under oxidative stress, which is proposed to keep the activity of Prx. These results indicate that IFN-y pretreatment protects microglial cells from oxidative stress via selective up-regulation of the level of Mn-SOD and Srx and the activities of Mn-SOD and catalase.Nitric oxide, which was identified have dual effect on oxidative stress, was produced by IFN-y-treated microglial cells. In this study, iNOS inhibitor inhibited the NO production induced by IFN-y and partially blocked the protective effect of IFN-y pretreatment. Interestingly, NO donor DEA/NO showed similar protective effect on H2O2-induced microglial cell death. The expression of Srx and the reduction of Prx-SO2 were increased by DEA/NO. These results indicate that NO mediates, at least in part, the protection of IFN-y pretreatment.Mitogen-activated protein kinases (MAPKs) pathways are well known to be involved in the oxidative stress responses. In order to further investigate the pathways mediating the protection of IFN-y pretreatment on H2O2 toxicity, its effects on H2O2-induced MAPKs phosphorylation were examined. The results showed that the MAPKs including p38, c-jun NH2-terminal kinase (JNK) and extracellular signal-regulated protein kinases (ERK1/2) were activated by H2O2 treatment. And H2O2-induced cell death in microglial cells was markedly blocked by the p38 inhibitor SB203580 or the JNK inhibitor SP600125, but not the ERK inhibitor PD98059, indicating the importance of p38 and JNK MAPK in microglial cell death induced by H2O2. Pretreatment with IFN-y for 20h significantly attenuated phosphorylation of p38 and JNK evoked by H2O2. The regulation of IFN-y pretreatment on the expression of MAPK phosphatase-1 (MKP-1) was also studied here. The expression of MKP-1 of microglial cells was up-regulated time-dependently by pretreatment of IFN-y for 1-24h. These result suggest that besides antioxidant enzyme enhancement, IFN-y pretreatment protects microglial cells from H2O2 toxicity via up-regulation of MKP-1 and subsequent dephosphorylation of p38 and JNK MAPK.In conclusion, the results demonstrate that IFN-y pretreatment protects microglial cells from oxidative stress through augmenting the function of antioxidant enzyme system and inhibiting the phosphorylation of MAKPs induced by H2O2.
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