ИСТИНА

A key transcription factor - nuclear factor-κB (NF-κB) involved in regulation of genes encoding pro-inflammatory and pro-apoptotic factors, can be activated by a variety of stimuli, including inflammatory cytokines. Brain injury is associated with neuroinflammation, neurodegeneration, and also blood coagulation with thrombin formation and generation of activated protein C (APC). In the present study we investigated the ability of APC to modulate the production of IL-1b and IL-6 by mast cells (MC), MC survival and NF-κBp65 translocation into MC nucleus in acute inflammation in rats and at glutamate(Glu)- induced toxicity in cultural neurons. Acute inflammation was induced by intraperitoneal injection of thioglycolate broth (2ml 40% w/v) in rats. CrudeMC were isolated from the peritoneal cavity of anesthetized rats and purified in Ficoll density gradient. IL-1b, 6 in rat were analyzed using Rat IL-1β and IL-6 ELISA Kit (PeproTech, USA). We used immunoassay and immunostaining with confocal microscopy and antiNF-κBp65 antibody for analyze the translocation of NF-κBp65 to nucleus. MTT-test was used to evaluate MC viability at inflammation and neuron survival at Glu, thrombin-toxicities. Thioglycolate injection induced inflammatory response verified by IL-1β and IL-6 measurements. MC IL-6 production was 1.3, 1.8 and 1.9 fold decreased by single intraperitoneal injection of 5nMAPC at 0.5, 1.5 and 4 h of inflammation, respectively. IL-6 level in peritoneal fluid was 1.5 and 1.7 fold decreased in APC-treated rats at 1.5 and 4 h of inflammation. The enhanced production of IL-1β in MC and peritoneal cavity under action of APC was also decreased. APC protected MC from death at 30 min of acute inflammation, increasing the MC survival on 36% (to 89,6 ± 7,6%) compared to the control (53,6 ± 8,7%). The development of inflammation in rats led to activation of NF-κBp65 and its translocation into the nucleus. The maximum of nucleus NF-κBp65 was observed in 1.5 h after induction of inflammation. APC blocks the translocation of NF-κBp65 into the nucleus of MC. The maximal effect of APC was revealed in 1.5 h after induction of inflammation. We show that APC at concentrations as low as 1–2 nM inhibits translocation of NF-κB p65 into the nucleus of cultured rat hippocampal neurons, induced by 100 M Glu or 50 nM thrombin. The blocking effect of APC on NF-κB p65 translocation was observed at 1 and 4 h after treatment of neurons with Glu, when the NF-κBp 65 level in the nucleus was significantly above the basal level. The binding of APC to EPCR/PAR-1 is required to control NF-κB activation at Glu-toxicity and acute inflammation in rats. Our present data indicate that APC provides not only anti-apoptotic protection (in case of Glu-toxicity), but also antineuroinflammatory activity (in case of toxicity caused by thrombin and at acute inflammation) via decrease the nuclear level of NF-B p65 in cells.