HMGB1-binding heptamer suppresses the synergistic effect of HMGB1 and LPS by interacting directly with HMGB1.

High mobility group box 1 (HMGB1) is an endogenous danger signal molecule. In the postischemic brain, HMGB1 is massively released during acute damaging process and triggers inflammatory processes. Moreover, it has been reported HMGB1 augments the proinflammatory effect of LPS by direct interaction. In previous studies, the authors showed intranasally delivered a HMGB1 binding heptamer peptide (HBHP; HMSKPVQ) has robust neuroprotective effects in the ischemic brain after middle cerebral artery occlusion and that it exerts an anti-inflammatory effect. In the present study, the authors investigated whether HBHP suppresses the augmentation of the proinflammatory effect of LPS by HMGB1. In primary microglial cultures, low doses of LPS (5 ng/ml) and recombinant HMGB1 (rHMGB1, 20 ng/ml) synergistically activated microglial cells, and HMGB1-LPS binding was detected. In addition, synergistic NO accumulation along with direct HMGB1-LPS binding was also observed when primary microglial cultures were treated with LPS (5 ng/ml) and HMGB1 accumulated in NMDA-conditioned medium (NCM). Co-treatment of microglial cells with HBHP and LPS or rHMGB1 (NCM), or treatment with rHMGB1 or NCM and LPS after pre-incubating rHMGB1 (or NCM) with HBHP markedly suppressed their synergistic activation. Furthermore, interactions between rHMGB1 and LPS or between HMGB1 in NCM and LPS were suppressed dose-dependently by HBHP, indicating that HBHP suppressed the synergism between HMGB1 and LPS and the underlying mechanism involved inhibition of HMGB1-LPS binding. Together these results show HBHP has anti-inflammatory effects, and that it inhibits synergism caused by the binding of HMGB1 and LPS.

Ethyl pyruvate inhibits HMGB1 phosphorylation and secretion in activated microglia and in the postischemic brain.

Ethyl pyruvate (EP) has been shown to have anti-inflammatory effects and confer protective effects in various pathological conditions. For example, EP inhibits secretion of high mobility group box 1 (HMGB1), which is known to be released from activated or dying cells and aggravate inflammatory pathways. In the present study, we investigated whether EP reduces HMGB1 phosphorylation and release in ischemic brain and in cultured microglia. In the postischemic brains (60 min middle cerebral artery occlusion (MCAO)), HMGB1 was released extracellularly, generating dual peaks in cerebrospinal fluid (CSF) around 1 and 7 days after ischemic insult, which were probably generated from damaged neurons and activated inflammatory cells, respectively. We showed that treatment with EP 30 min post-MCAO (5 mg/kg, i.v.), which has been shown to confer a robust neuroprotective effect in the postischemic brain, reduced both peaks. In addition, delayed EP treatment from 4 days post-MCAO reduced HMGB1 accumulation in CSF at 7 day post-MCAO in the absence of accompanying amelioration of ischemic brain damage, indicating that the suppression of HMGB1 release is a direct effect. We also found that EP markedly suppressed the LPS-induced nuclear translocations of protein kinase C alpha and calcium/calmodulin-dependent protein kinase IV, HMGB1 phosphorylation, and subsequent secretion of HMGB1 induced by LPS in BV2 cells and EP-mediated above-mentioned effects were also independent of cell death or survival. These results indicate that EP inhibits HMGB1 phosphorylation and release in activated microglia, which might be responsible for EP-mediated suppression of HMGB1 release in the postischemic brain.