Efeito protetor da enzima Fosfatidilinositol 3-QUINASE (PI3K) em excitotoxicidade e seu envolvimento no mecanismo de ação anticonvulsivante e neuroprotetor do Canabidiol

Abstract

Introduction: The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt) /mammalian target of rapamycin (mTOR) signaling pathway has been associated with several pathologies in the central nervous system (CNS), including in epilepsy. However, the specific role of PI3K in modulating seizures and post-ictal events is poorly understood. In addition, a phytocannabinoid extracted from Cannabis sativa leaves, cannabidiol (CBD), exhibit a powerful anticonvulsant effect in animal models and clinical studies. However, the anticonvulsant and neuroprotective mechanisms of action of CBD have not yet been elucidated. Aims: To test the hypothesis that PI3K reduces excitotoxicity and its consequences, as well as its signaling pathway mediates the anticonvulsant and neuroprotective effects of CBD. Methods: In vitro and in vivo experiments were performed on C57Bl/6 (WT) and PI3K-/- mice. Status epilepticus (SE) was induced by bilateral intra-hippocampal pilocarpine (PILO) microinjection. After 8 h, the animals had their hippocampus removed, for further analysis of the neurotrophic factors BDNF, NGF and GDNF levels. After 24 h, animals were perfused and their brains removed and processed, for further histological techniques to evaluate neuronal death, microglia and astrocyte staining. Primary cultures of hippocampal neurons were used for glutamate induced cell death assay, as well as hippocampal synaptosomal preparations were used for analysis of glutamate release and cytosolic calcium content. Finally, pentylenetetrazole (PTZ)induced seizures was performed by intraperitoneal route, for further behavioral and/or electroencephalographic analysis. Results: PILO-induced SE promoted some post-ictal changes in WT and PI3K-/- animals, such as increased neuronal death and microglial and astrocytic activation. Pharmacological inhibition of PI3K promoted time and dose-dependent changes in the glutamate-induced cell death assay, and the genetic or pharmacological inhibition of PI3K increased glutamate release and cytosolic calcium content induced by KCl in preparations of hippocampal synaptosomes. In addition, behavioral and electroencephalographic alterations were observed between the WT and PI3K-/- animals, submitted to the PTZ model of seizures. Subsequently, a potent anticonvulsant effect of CBD was observed in the PILO model, followed by hippocampal reduction of neuronal death and microglial and astrocytic activation. Pretreatment with the CB1 receptor antagonist, AM251, with the mTOR inhibitor, rapamycin, or in PI3K-/- animals, reduced the anticonvulsant effect of CBD. In addition, it was observed a reduction in in vivo and in vitro neuroprotective effects of CBD, through genetic and pharmacological PI3K inhibition, as well as alterations in the hippocampal levels of BDNF, NGF and GDNF. The anticonvulsant effect of CBD was also demonstrated in PTZ model, and this effect was also reduced by genetic deletion of PI3K as well as by pharmacological inhibition of PI3K/Akt/mTOR signaling pathway. Conclusion: According to the results, we suggest that the enzyme PI3K has a protective role against excitotoxicity. In addition, the modulation of PI3K/Akt/mTOR signaling pathway is involved in the anticonvulsive and neuroprotective effects of CBD.