The Effects Of TRAF2 On Stroke Outcomes

Objective: Ischemic stroke cleads to brain tissue damage and neurological deficits. Tumor necrosis factor receptor-associated factors(TRAFs) are a type of adapter proteins that are important for various signal cascades via its interaction with signal molecule and cell surface receptors. TRAF2 is a member of TRAFs family, which is a ubiquitin ligase with a RING domain. It interacts with sphingosine kinase 1(SphK1) to mediate TNF-α and NF-κB-mediated cascades. Our previous studies have shown that Sph K1 plays an important role in cerebral ischemia. Thus, we investigated in this study the role of TRAF2 in ischemic brain damage.Methods: Cerebral ischemia was induced in adult male ICR mice by middle cerebral artery occlusion(MCAO) using the filament technique. To mimic in vivo cerebral ischemia, primary neurons were subjected to oxygen glucose deprivation(OGD) or primary microglia were treated with conditioned medium collected from neurons subjected to OGD. Real-time quantitative PCR(Quantitative real-time PCR, q-PCR) and Western blot were performed to assess the TRAF2 m RNA and protein levels in the ischemic brain following MCAO and primary neurons or microglia following OGD treatment; immunofluorescence(immunofluorescence staining) was performed to examine the types of cells expressing TRAF2 in the ischemic brain. Then, to investigate the role of TRAF2 in cerebral ischemia, si RNA against TRAF2 was administrated into the lateral ventricle via stereotactic injection. The effects of TRAF2 siRNA on acute infarction was assessed with 2, 3, 5-tripheyltetrazoliumchloride(TTC) histology at 24 and 96 hrs following MCAO, and the effects of TRAF2 siRNA on post-ischemic neuroinflammation was assessed by measuring the m RNA levels of inflammatory cytokines in the ischemic brain at 24 hrs following MCAO. Then, to investigate the role of TRAF2 in primary microglia, lentivirus against TRAF2 was transfected into the cells. After that, the cells were treated with OGD Neuron CM and the effects of TRAF2 lentivirus on inflammation was assessed by measuring the m RNA levels of inflammatory cytokines in the cells. Primary microglia Necroptosis model was built with OGD Neuron CM and ZVAD, and the Necroptosis extent was assessed by Calcein-AM/ Propidium bromide(Calcein-AM/PI) staining.Results: 1) The expression levels of TRAF2 mRNA and protein were significantly enhanced in the cortical penumbra following MCAO;2) The TRAF2 m RNA levels were induced in primary neurons subjected to OGD or in primary microglia stimulated with conditioned medium collected from OGD neurons. Immunofluorescence showed that TRAF2 was co-localization with microglial marker(Iba 1) and neuronal marker(NeuN);3) TRAF2 si RNA-injected mice displayed remarkably reduced infarct volumes at 24 and 96 hrs after MCAO compared to NC si RNA-injected or sham treated mice;4) The expression levels of inflammatory cytokines in mouse cortical penumbra was significantly lower than in TRAF2 siRNA-injected group than in sham mice and in NC siRNA-injected mice;5) Lentivirus against TRAF2 had no effects for the expression levels of inflammatory cytokines in microglia stimulated with OGD Neuron CM;6) The mortality of primary microglial stimulated with OGD Neuron CM and ZVAD was increased compared to the control group’s;7) Necroptosis occured in primary microglial when the cells were stimulated with OGD Neuron CM and ZVAD for 24hrs;8) Necroptosis of primary microglial stimulated with OGD Neuron CM and ZVAD could be enhanced with lentivirus against TRAF2.Conclusion: The expression of TRAF2 was significantly induced following cerebral ischemia, and blocking post-ischemic induction of TRAF2 with siRNA reduces acute infarct damage and post-ischemic neuroinflammation. Blocking induction of TRAF2 with lentivirus enhances the extend of microglia Necroptosis may be one of the mechanisms of inflammation decline. Our study suggested that TRAF2 contributed to stroke pathology and TRAF2 was a potential therapeutic target for ischemic stroke.