bay-11-7082 and Brain-Ischemia

Our previous finding suggests that p38 MAPK contributes to the GLT-1 upregulation during induction of brain ischemic tolerance by cerebral ischemic preconditioning (CIP), however, the underlying mechanism is still unclear. Here, we investigated the molecular mechanisms underlying the CIP-induced GLT-1 upregulation by using Western blotting, Co-immunoprecipitation (Co-IP), electrophoretic mobility shift assay (EMSA) and thionin staining in rat hippocampus CA1 subset. We found that application of BAY11-7082 (an inhibitor of NF-κB), or dihydrokainate (an inhibitor of GLT-1), or SB203580 (an inhibitor of p38 MAPK) could attenuate the CIP-induced neuronal protection in hippocampus CA1 region of rats. Moreover, CIP caused rapid activation of NF-κB, as evidenced by nuclear translocation of NF-κB p50 protein, which led to active p50/p65 dimer formation and increased DNA binding activity. GLT-1 was also increased after CIP. Pretreatment with BAY11-7082 blocked the CIP-induced GLT-1 upregulation. The above results suggest that NF-κB participates in GLT-1 up-regulation during the induction of brain ischemic tolerance by CIP. We also found that pretreatment with SB203580 caused significant reduction of NF-κB p50 protein in nucleus, NF-κB p50/p65 dimer nuclear translocation and DNA binding activity of NF-κB. Together, we conclude that p38 MAPK/NF-κB pathway participates in the mediation of GLT-1 up-regulation during the induction of brain ischemic tolerance induced by CIP.

Topics: Animals; Brain Ischemia; CA1 Region, Hippocampal; Excitatory Amino Acid Transporter 2; Imidazoles; Ischemic Preconditioning; Kainic Acid; Male; MAP Kinase Signaling System; Neuroprotection; NF-kappa B; NF-kappa B p50 Subunit; Nitriles; p38 Mitogen-Activated Protein Kinases; Pyridines; Rats; Rats, Wistar; Sulfones; Transcription Factor RelA

Stroke is the most common cerebrovascular disease with high morbidity and mortality around the world. However, the underlying mechanisms involved in nerve injury and cerebral ischaemia/reperfusion (I/R) during cerebrovascular disease are still not completely clear. In the present study, we investigate the role of kinesin family member 2 (KIF2) in the neuroprotection after cerebral I/R injury. KIF2 was aberrantly expressed in the cerebral tissues from middle cerebral artery occlusion (MCAO) rat model in a time dependent manner. A similar changing pattern was found in the cultured hypoxic neurons as well as SK-N-SH cells in vitro. Compared to the control, KIF2 inhibition significantly increased the level of malonic dialdehyde (MDA), and reduced the level of superoxide dismutase (SOD) as well as glutathione peroxidase (GSH-px) activity in cerebral tissues of MCAO rat model. The reactive oxygen species (ROS) level was also up-regulated after KIF2 siRNA knockdown in cultured hypoxic SK-N-SH cells. The apoptosis rates of hypoxic neurons and SK-N-SH cells as well as activated-caspase-3 level were obviously increased after KIF2 silencing. Furthermore, we found that the nuclear factor-kappa B (NF-κB) pathway was involved in KIF2-mediated neuroprotection after cerebral I/R injury, and induced apoptosis of hypoxic SK-N-SH cells by KIF2 silencing could be attenuated by the specific inhibitor BAY11-7082 of NF-κB. In conclusion, we demonstrate that KIF2 could mediate the neuroprotection in cerebral I/R injury by inhibiting activation of NF-κB pathway. This might provide a novel therapeutic target for cerebral I/R injury.

Topics: Animals; Brain Ischemia; Cell Line, Tumor; Humans; Kinesins; Male; Neuroprotection; NF-kappa B; Nitriles; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; Sulfones

Multi-protein complexes, termed "inflammasomes," are known to contribute to neuronal cell death and brain injury following ischemic stroke. Ischemic stroke increases the expression and activation of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) Pyrin domain containing 1 and 3 (NLRP1 and NLRP3) inflammasome proteins and both interleukin (IL)-1β and IL-18 in neurons. In this study, we provide evidence that activation of either the NF-κB and MAPK signaling pathways was partly responsible for inducing the expression and activation of NLRP1 and NLRP3 inflammasome proteins and that these effects can be attenuated using pharmacological inhibitors of these two pathways in neurons and brain tissue under in vitro and in vivo ischemic conditions, respectively. Moreover, these findings provided supporting evidence that treatment with intravenous immunoglobulin (IVIg) preparation can reduce activation of the NF-κB and MAPK signaling pathways resulting in decreased expression and activation of NLRP1 and NLRP3 inflammasomes, as well as increasing expression of anti-apoptotic proteins, Bcl-2 and Bcl-xL, in primary cortical neurons and/or cerebral tissue under in vitro and in vivo ischemic conditions. In summary, these results provide compelling evidence that both the NF-κB and MAPK signaling pathways play a pivotal role in regulating the expression and activation of NLRP1 and NLRP3 inflammasomes in primary cortical neurons and brain tissue under ischemic conditions. In addition, treatment with IVIg preparation decreased the activation of the NF-κB and MAPK signaling pathways, and thus attenuated the expression and activation of NLRP1 and NLRP3 inflammasomes in primary cortical neurons under ischemic conditions. Hence, these findings suggest that therapeutic interventions that target inflammasome activation in neurons may provide new opportunities in the future treatment of ischemic stroke.

Topics: Adaptor Proteins, Signal Transducing; Animals; Anthracenes; Apoptosis Regulatory Proteins; Brain; Brain Ischemia; Butadienes; Extracellular Signal-Regulated MAP Kinases; Imidazoles; Inflammasomes; Mice; Neurons; NF-kappa B; Nitriles; NLR Family, Pyrin Domain-Containing 3 Protein; Pyridines; Signal Transduction; Stroke; Sulfones

Acute ischemic stroke is one of the most important factors leading to disability and death with the characterization of accumulated neuron death and injured supportive neurovascular structures. Raf-1 kinase inhibitory protein (RKIP) is a key molecule in cell response to survival or death stimuli. However, the role of RKIP in stroke is worthy to be further studied.. We used lentivirus mediated RKIP knockdown and overexpression to investigate the effect of RKIP on animal models of focal cerebral ischemia. Cell Counting Kit-8 assay, lactate dehydrogenase release analysis, and Annexin V-APC apoptosis assay were used to detect the effect RKIP on microglial cell apoptosis and survival. Transwell migration assay was carried out to evaluate the migration of microglia cells. The releases of inflammatory cytokines were determined by ELISA. The activation of NF-kappaB signaling pathway was determined by western blot.. Overexpression of RKIP reduced focal cerebral ischemia injury. RKIP knockdown and overexpression regulated survival, activation, and motility via the NF-κB pathway. NF-κB inhibitor BAY 11-7082 blocked the changes caused by RKIP down-regulation after oxygen-glucose deprivation (OGD). RKIP overexpression inhibited the upregulation of phosphorylation of NF-κB induced by OGD and cerebral ischemia.. The present study showed that RKIP protects against ischemic stroke through inhibition of microglial excessive activation, inhibits its motility, and promotes neuronal survival partly though IKKβ-IκBα-NF-κB signaling axis and indicate that RKIP is a new target for the treatment of ischemic stroke.

Topics: Animals; Apoptosis; Brain Ischemia; Cerebral Cortex; Gene Expression Regulation; I-kappa B Kinase; Interleukin-1beta; Male; Microglia; NF-kappa B; Nitric Oxide Synthase Type II; Nitriles; Phosphatidylethanolamine Binding Protein; Primary Cell Culture; Rats; Rats, Sprague-Dawley; RNA, Small Interfering; Signal Transduction; Stroke; Sulfones; Tumor Necrosis Factor-alpha