Toll-like receptors (TLRs) play a critical role in the induction of innate immune responses which have been implicated in neuronal death induced by global cerebral ischemia/reperfusion (GCI/R). The present study investigated the role and mechanisms-of-action of TLR4 signaling in ischemiainduced hippocampal neuronal death. Neuronal damage, activation of the TLR4 signaling pathway, expression of pro-inflammatory cytokines and activation of the PI3K/Akt signaling pathway in the hippocampal formation (HF) were assessed in wild type (WT) mice and TLR4 knockout mice (TLR4 -/-) mice after GCI/R. GCI/R increased expression of TLR4 protein in the hippocampal formation (HF) and other brain structures in WT mice. Phosphorylation of the inhibitor of kappa B (p-IκB) as well as activation of nuclear factor kappa B (NFκB) increased in the HF of WT mice. In contrast, there were lower levels of p-IκB and NFκB binding activity in TLR4 -/-mice subjected to GCI/R. Pro-inflammatory cytokine expression was also decreased, while phosphorylation of Akt and GSK3β were increased in the HF of TLR4 -/-mice after GCI/R. These changes correlated with decreased neuronal death/apoptosis in TLR4 -/-mice following GCI/R. These data suggest that activation of TLR4 signaling contributes to ischemia-induced hippocampal neuronal death. In addition, these data suggest that modulation of TLR4 signaling may attenuate ischemic injury in hippocampal neurons.

. Glucan phosphate attenuates cardiac dysfunction and inhibits cardiac MIF expression and apoptosis in septic mice. Am J Physiol Heart Circ Physiol 291: H1910 -H1918, 2006. First published June 9, 2006 doi:10.1152/ajpheart.01264.2005.-Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. We have previously reported that glucan phosphate (GP) significantly increased survival in a murine model of cecal ligation and puncture (CLP)-induced sepsis. In the present study, we examined the effect of GP on cardiac dysfunction in CLP-induced septic mice. GP was administered to ICR/HSD mice 1 h before induction of CLP. Sham surgically operated mice served as control. Cardiac function was significantly decreased 6 h after CLP-induced sepsis compared with sham control. In contrast, GP administration prevented CLPinduced cardiac dysfunction. Macrophage migration inhibitory factor (MIF) has been implicated as a major factor in cardiomyocyte apoptosis and cardiac dysfunction during septic shock. CLP increased myocardial MIF expression by 88.3% (P Ͻ 0.05) and cardiomyocyte apoptosis by 7.8-fold (P Ͻ 0.05) compared with sham control. GP administration, however, prevented CLP-increased MIF expression and decreased cardiomyocyte apoptosis by 51.2% (P Ͻ 0.05) compared with untreated CLP mice. GP also prevented sepsis-caused decreases in phospho-Akt, phospho-GSK-3␤, and Bcl-2 levels in the myocardium of septic mice. These data suggest that GP treatment attenuates cardiovascular dysfunction in fulminating sepsis. GP administration also activates the phosphoinositide 3-kinase/Akt pathway, decreases myocardial MIF expression, and reduces cardiomyocyte apoptosis.

phosphate attenuates myocardial HMGB1 translocation in severe sepsis through inhibiting NF-B activation. Am J Physiol Heart Circ Physiol 301: H848 -H855, 2011. First published June 3, 2011 doi:10.1152/ajpheart.01007.2010.-Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. High-mobility group box 1 (HMGB1) serves as a late mediator of lethality in sepsis. We have reported that glucan phosphate (GP) attenuates cardiac dysfunction and increases survival in cecal ligation and puncture (CLP)-induced septic mice. In the present study, we examined the effect of GP on HMGB1 translocation from the nucleus to the cytoplasm in the myocardium of septic mice. GP was administered to mice 1 h before induction of CLP. Sham-operated mice served as control. The levels of HMGB1, Toll-like receptor 4 (TLR4), and NF-B binding activity were examined. In an in vitro study, H9C2 cardiomyoblasts were treated with lipopolysaccharide (LPS) in the presence or absence of GP. H9C2 cells were also transfected with Ad5-IB␣ mutant, a super repressor of NF-B activity, before LPS stimulation. CLP significantly increased the levels of HMGB1, TLR4, and NF-B binding activity in the myocardium. In contrast, GP administration attenuated CLP-induced HMGB1 translocation from the nucleus to the cytoplasm and reduced CLP-induced increases in TLR4 and NF-B activity in the myocardium. In vitro studies showed that GP prevented LPS-induced HMGB1 translocation and NF-B binding activity. Blocking NF-B binding activity by Ad5-IB␣ attenuated LPSinduced HMGB1 translocation. GP administration also reduced the LPS-stimulated interaction of HMGB1 with TLR4. These data suggest that attenuation of HMGB1 translocation by GP is mediated through inhibition of NF-B activation in CLP-induced sepsis and that activation of NF-B is required for HMGB1 translocation.high-mobility group box 1; myocardium; nuclear factor-B HIGH-MOBILITY GROUP BOX 1 (HMGB1) is an evolutionarily conserved protein present in the nucleus of almost all eukaryotic cells where it functions to stabilize nucleosomes and acts as a transcription factor (4,14,25,28). Recent evidence suggests that HMGB1 is an important danger-associated molecular pattern (DAMP) (14, 28) and is actively secreted by activated macrophages and released from necrotic (25) and damaged tissues (4). In vivo neutralization of HMGB1 by specific antibodies protects mice against lethal endotoxemia and sepsis (28), as well as endotoxin-induced acute lung injury (29). More significantly, anti-HMGB1 antibodies rescued mice from lethal sepsis even when the first dose of antibodies was given as late as 24 h after cecal ligation and puncture (CLP) surgery (29). These data suggest that HMGB1 serves as a late mediator of lethality in sepsis (6,28). In vitro studies have demonstrated that the HMGB1-stimulated inflammatory response could be mediated through receptors for advanced glycation end products (RAGE) (27), Toll-like receptor 2 (TLR2), TLR4 (23, 24), and TLR9 (26).TLR-mediated sig...

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