The role of inflammation in ischemic brain damage has been reported in human and in different animal models of stroke. Since statins (3-hydroxy-3-methylglutaril (HMG)–coenzymeA (CoA) reductase inhibitors), besides their lipid lowering action, exert also anti-inflammatory activity we sought to investigate the effect of simvastatin treatment on the expression of interleukin-1beta (IL-1beta), monocyte chemoattractant protein-1 (MCP-1), NF-kB activity and on signaling pathways related with NF-kB activation in a rat model of permanent middle cerebral artery occlusion (pMCAO). Unilateral pMCAO was carried out in anesthetized Sprague-Dawley rats using a microbipolar coagulator to permanently occlude the right middle cerebral artery. Expression of IL-1beta and MCP-1 was determined in cerebral cortex using RT-PCR. NF-kB DNA binding activity was assessed with Trans-AM NF-kB p65 transcription factor assay kit using cortical nuclear extracts. Binding of NF-kB to DNA was visualized by anti-p65 antibody that specifically recognizes activated NF-kB. MAP kinases were revealed by immunoblotting. Intravenous administration of a specific MEK (MAPK/ERK kinase) inhibitor, U0126 (150 mg/kg), was used to evaluate the causal relationship between anti-inflammatory actions and the neuroprotective activity. Expression of lL-1b and MCP-1 was enhanced by pMCAO and this effect was inhibited by administration of simvastatin before ischemia. We then investigated the ability of NF-kB p65 subunit to bind DNA at different time after pMCAO. The cerebral cortex ipsilateral to the occlusion displayed an increase in binding activity which reached its peak 16 hours after the ischemic insult. In order to determine whether simvastatin could interfere with pMCAO-induced activation of NF-kB, animals were treated for 3 days with 20 mg/kg of statin before permanent occlusion of the artery or 2 hours after pMCAO. Pre-treatment with simvastatin abolished the activation of NF-kB observed in vehicle-treated animals. In contrast, an acute administration of the drug, after induction of permanent ischemia, did not show any inhibitory effect on NF-kB activation. The effect of simvastatin was specific for NF-kB since other transcription factors such as Jun, Fos and NF-YA were not affected by the treatments. Following the same schedule of treatment, we have also evaluated the modulation of different signal transduction pathways such as ERK 1/2, SAPK/JNK 46/54 and p38. Under our experimental conditions, only the expression of ERK1/2 was enhanced by ischemia and this activation was prevented by prophylactic, but not post-ischemic, administration of simvastatin. Administration of U0126, after pMCAO, reduced the ischemic damage, inhibited NF-kB activation and reduced the activation of inflammatory markers. These results provide evidence for the role of simvastatin in the protection of ischemic brain damage and suggests that this effect is mediated by inhibition of ERK1/2 pathway.
