【作者】 陈侠；

【导师】 吴春福； 金源起；

【作者基本信息】 沈阳药科大学 ， 药理学， 2010， 博士

【摘要】 小胶质细胞是脑内固有的巨噬细胞样免疫效应细胞,活化的小胶质细胞释放多种生物活性物质,对中枢神经系统疾病中组织细胞的氧化应激状态、炎症进程以及免疫反应都起到重要的调节作用。充分了解小胶质细胞对体内各种活性物质包括细胞因子的反应,能够为治疗中枢神经系统疾病提供理论依据及线索。小胶质细胞释放的自由基,如过氧化氢(H2O2)和一氧化氮(NO),在病理状态下可加强细胞的防御功能,但它们同时也会攻击小胶质细胞。作为一种自我保护机制,小胶质细胞需配备完善的抗氧化防御系统,以抵御自由基诱导的细胞损伤。本论文着重考察干扰素-γ(Interferon-γ, IFN-γ)对小胶质细胞抗氧化防御系统的调节作用及作用机理。我们首先以细胞形态学改变及LDH释放量为指标,比较IFN-γ及白细胞介素-1β(Interleukin-1β,IL-1β)对H2O2诱导的小胶质细胞死亡的影响。结果显示,IFN-γ预处理能够显著抑制H2O2诱导的小胶质细胞死亡,而IFN-γ共处理以及IL-1β预处理对H2O2毒性均未显示明显的保护作用。IFN-γ预处理还能够显著抑制超氧阴离子(O2-)、过氧亚硝酸盐(ONOO-)、HNE和rotenone诱导的小胶质细胞的损伤及死亡。细胞选择性考察结果显示,IFN-γ预处理对H202诱导的星形胶质细胞和腹腔巨噬细胞损伤未显示保护作用。进一步的研究结果显示,IFN-γ预处理小胶质细胞6-24h期间,IFN-γ对H2O2毒性的抑制作用随着预处理时间的延长而增强,而且IFN-γ预处理对小胶质细胞的保护作用可被蛋白合成抑制剂(Cycloheximide)阻断。上述实验结果提示,小胶质细胞在IFN-γ预处理作用下生成的新蛋白质是IFN-γ发挥小胶质细胞保护作用的必要条件。为了研究IFN-γ预处理对小胶质细胞抗氧化酶系统的调节作用,我们利用Western blot及抗氧化酶活性测定试剂盒分别检测小胶质细胞抗氧化酶的蛋白表达及活性。在多种抗氧化酶如铜锌超氧化物歧化酶(Cu/Zn-SOD)、锰超氧化物歧化酶(Mn-SOD)、过氧化氢酶(Catalase)、谷胱甘肽过氧化物酶(Gpx)、过氧化还原酶(PrxⅠand PrxⅢ)及硫过氧化物还原因子(Srx)中,IFN-γ预处理可增强小胶质细胞Mn-SOD及Srx的表达,并且能够抑制H2O2引起的Prx过氧化产物(Prx-SO2)的表达。抗氧化酶活性分析结果则显示,IFN-γ提高小胶质细胞Mn-SOD及catalase活性,对Cu/Zn-SOD及Gpx活性无明显影响。通过siRNA转染下调小胶质细胞Mn-SOD的表达,Mn-SOD siRNA转染可部分阻断IFN-γ对Mn-SOD表达的增强作用及其对H2O2毒性的抑制作用。而Catalase抑制剂(3-AT)可部分阻断IFN-γ对小胶质细胞氧化损伤的保护作用。以上实验结果提不,IFN-y通过增强线粒体Mn-SOD的表达及Mn-SOD、catalase的活性,发挥细胞保护作用。此外,IFN-y还可增强小胶质细胞Srx的表达,并且促进氧化应激状态下Prx-SO2的还原,维持细胞内Prx的活性,从而加强小胶质细胞的抗氧化能力。IFN-γ预处理6-24h可逐渐增加小胶质细胞NO释放量,而NO对于细胞的氧化应激反应具有双向调节作用。本实验中,诱导型一氧化氮合酶(iNOS)抑制剂可部分阻断IFN-y预处理的细胞保护作用,而NO供体(DEA/NO)能够显著抑制H2O2诱导的小胶质细胞死亡。DEA/NO能够增强小胶质细胞Srx的表达,加快Prx-SO2的还原速率。以上结果提示,NO是介导IFN-y的小胶质细胞保护作用的重要活性分子。丝裂原活化蛋白激酶(mitogen-activated protein kinases, MAPKs)的磷酸化在细胞氧化应激反应中起着重要的作用。为了进一步探讨参与IFN-y预处理保护作用的信号通路,我们利用western blot检测了IFN-y对H2O2诱导的MAPKs磷酸化的影响。结果显示,H2O2可显著提高小胶质细胞MAPKs (p38, JNK, ERK)的磷酸化水平,p38和JNK抑制剂可明显抑制H2O2毒性。因此,p38和JNK MAPK在H2O2诱导的小胶质细胞死亡中起关键作用。IFN-y预处理可显著降低H2O2诱导的p38和JNK MAPK的磷酸化水平。本实验还考察了IFN-y处理1-24h对小胶质细胞丝裂原活化蛋白激酶磷酸酶-1(MAPK phosphatases-1, MKP-1)表达的影响。结果显示,IFN-y预处理1-24h可逐步增强小胶质细胞MKP-1的表达。这些结果提示,除了能够增强小胶质细胞的抗氧化酶系统的功能,上调小胶质细胞MKP-1的表达从而抑制H2O2诱导的p38/JNK MAPK磷酸化是IFN-y的小胶质细胞保护作用的又一作用机制。综上所述,IFN-y预处理20h可增强小胶质细胞抗氧化酶系统功能,并且抑制H2O2诱导的小胶质细胞MAPKs磷酸化,从而增加小胶质细胞对氧化应激损伤的抵抗能力。更多还原

【Abstract】 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.更多还原