TUMOR NECROSIS FACTOR-α CONFERS CYTOTOXICITY IN ASTROCYTES UNDER OXIDATIVE STRESS VIA INHIBITION OF NF-κB SIGNALING

Oxidative stress and inflammation together recognized as central feature of stroke and other neurodisorders. In acute ischemic stroke, formation of H2O2 causes brain injury, which appear to be exacerbated by inflammation such as IL-1β or TNF-α produced after reperfusion. However, evidences also show that TNF-α helps in recovery and repair. Therefore, role of TNF-α is unclear. Further, it is unknown how our brain cells i.e. astrocytes are affected when oxidative stress and inflammation coexist. Here we examined the effects of H2O2 on cell survival in cultured human astrocytes co-stimulated with TNF-α or IL-1β. Data showed H2O2-treatment significantly increased astrocytes death; however, IL-1β or TNF-α-alone did not. Interestingly, co-treatment of TNF-α, but not IL-1β with non-toxic dose of H2O2 significantly increased astrocyte cell death. The toxicity of co-treatment of TNF-α and H2O2 was significantly higher than respective dose of H2O2-alone. Investigations of mechanisms revealed that H2O2 inhibited TNF-α-induced translocation of a transcription factor NF-κB to the nucleus in astrocytes thereby inhibiting cellular defense and/or survival pathways. H2O2 also decreased other TNF-a receptor associated proteins, RIP1, IκB kinases activation, thereby inhibited IκB-α degradation and NF-κB nuclear translocation. This supports the evidence of H2O2 as a modulator of pro-inflammatory signaling and explains the increased sensitivity of astrocytes during brain injury. These data also signify need to design strategy to combat oxidative stress during neuroinflammation and repair. Purpose (a): Oxidative stress and inflammation together recognized as central feature of both acute and chronic neurological disorders. In acute ischemic stroke formation of H2O2 causes brain injury, which appear to be exacerbated by IL-1β or TNF-α produced after reperfusion. However, evidences also show that TNF-α helps in recovery and repair. Therefore, role of TNF-α is unclear. Further, it is unknown how astrocytes are affected when oxidative stress and inflammation coexist. Methods (b): Here we examined the effects of H2O2 on cell survival and NF-kB dynamics in cultured human astrocytes co-stimulated with TNF-α or IL-1β. Results (c): Data showed H2O2-treatment significantly increased apoptosis in astrocytes in dose-dependent manner; however, IL-1β or TNF-α-alone did not. Interestingly, co-treatment of TNF-α, but not IL-1β with non-toxic dose of H2O2 significantly increased apoptosis in astrocytes. The toxicity of co-treatment of TNF-α and H2O2 was significantly higher than respective dose of H2O2-alone. Investigations of mechanisms revealed that H2O2 inhibited TNF-α-induced translocation of NF-kB to the nucleus in astrocytes thereby inhibiting cellular defense and/or survival pathways. H2O2 decreased TNFR1 associated protein, RIP1 level, necessary for IkB kinases activation, thereby inhibited IkB-a degradation and NF-kB nuclear translocation. The real time PCR analysis of oxidative stress pathway showed H2O2 decrease of antioxidant machinery of astrocytes. Investigation of apoptosis pathway showed that H2O2 increased the expression of TRAILR1/R2, Fas and FADD, which lead TNF-α-induced caspase-dependent apoptosis. Conclusions (d): This study supports the evidence of H2O2 as a modulator of pro-inflammatory signaling and explains the increased sensitivity of astrocytes during brain injury. These data also signify need to design strategy to combat oxidative stress during neuroinflammation and repair.