Brain microglia are a major source of inflammatory cytokines, such as tumor necrosis factor‐α (TNF‐α), which have been implicated in the progression of neurodegenerative diseases. Recently, microglia were revealed to be highly responsive to ATP, which is released from nerve terminals, activated immune cells, or damaged cells. It is not clear, however, whether released ATP can regulate TNF‐α secretion from microglia. Here we demonstrate that ATP potently stimulates TNF‐α release, resulting from TNF‐α mRNA expression in rat cultured brain microglia. The TNF‐α release was maximally elicited by 1 mM ATP and also induced by a P2X₇ receptor‐selective agonist, 2′‐ and 3′‐O‐(4‐benzoylbenzoyl)adenosine 5′‐triphosphate, suggesting the involvement of P2X₇ receptor. ATP‐induced TNF‐α release was Ca²⁺‐dependent, and a sustained Ca²⁺ influx correlated with the TNF‐α release in ATP‐stimulated microglia. ATP‐induced TNF‐α release was inhibited by PD 098059, an inhibitor of extracellular signal‐regulated protein kinase (ERK) kinase 1 (MEK1), which activates ERK, and also by SB 203580, an inhibitor of p38 mitogen‐activated protein kinase. ATP rapidly activated both ERK and p38 even in the absence of extracellular Ca²⁺. These results indicate that extracellular ATP triggers TNF‐α release in rat microglia via a P2 receptor, likely to be the P2X₇ subtype, by a mechanism that is dependent on both the sustained Ca²⁺ influx and ERK/p38 cascade, regulated independently of Ca²⁺ influx.

Stroke is the second leading cause of death worldwide. Treatment options for ischemic stroke are limited, and the development of new therapeutic agents or combined therapies is imperative. Growing evidence suggests that metformin treatment, due to its anti-inflammatory action, exerts a neuroprotective effect against ischemia/reperfusion-induced brain damage. Experimental assessment has typically been performed in models of cerebral transient ischemia followed by long-term reperfusion. The aim of this study was to evaluate the neuroprotective effect of metformin treatment after permanent middle cerebral artery occlusion (pMCAO) without reperfusion in rats. Neurological deficits were assessed using the Longa scale, which offers a graded scale on body movement following pMCAO. Both infarct size and brain oedema area were measured by staining with 2,3,5-triphenyltetrazolium chloride. The number of neurons and total and activated microglia, as well as interleukin 10 (IL-10) production, in brain sections were evaluated by immunohistochemical staining. Our results show that metformin treatment improves the neurological state and reduces infarct size after 120 h of pMCAO. Metformin also prevents neuronal loss in the ischemic cortex but not in the striatum after 48 h of pMCAO. Moreover, post-stroke treatment with metformin significantly decreases the number of total and activated microglia at 48 h. The anti-inflammatory effect of metformin is associated with increased IL-10 production at 48 h after pMCAO. The results of the present study suggest that post-stroke treatment with metformin exerts anti-inflammatory and neuroprotective effects in a pMCAO model.

Comprehensive LC-MS and MS/MS analysis of the crude venom extract from the solitary eumenine wasp Eumenes micado revealed the component profile of this venom mostly consisted of small peptides. The major peptide components, eumenine mastoparan-EM1 (EMP-EM1: LKLMGIVKKVLGAL-NH₂) and eumenine mastoparan-EM2 (EMP-EM2: LKLLGIVKKVLGAI-NH₂), were purified and characterized by the conventional method. The sequences of these new peptides are homologous to mastoparans, the mast cell degranulating peptides from social wasp venoms; they are 14 amino acid residues in length, rich in hydrophobic and basic amino acids, and C-terminal amidated. Accordingly, these new peptides can belong to mastoparan peptides (in other words, linear cationic α-helical peptides). Indeed, the CD spectra of these new peptides showed predominantly α-helix conformation in TFE and SDS. In biological evaluation, both peptides exhibited potent antibacterial activity, moderate degranulation activity from rat peritoneal mast cells, and significant leishmanicidal activity, while they showed virtually no hemolytic activity on human or mouse erythrocytes. These results indicated that EMP-EM peptides rather strongly associated with bacterial cell membranes rather than mammalian cell membranes.