Inhibition of calpain-regulated p35/cdk5 plays a central role in sildenafil-induced protection against chemical hypoxia produced by malonate

Phosphodiesterase 5 (PDE5) inhibitors have recently been reported to exert beneficial effects against ischemia–reperfusion injury in several organs but their neuroprotective effects in brain stroke models are scarce. The present study was undertaken to assess the effects of sildenafil against cell death caused by intrastriatal injection of malonate, an inhibitor of succinate dehydrogenase; which produces both energy depletion and lesions similar to those seen in cerebral ischemia. Our data demonstrate that sildenafil (1.5 mg/kg by mouth (p.o.)), given 30 min before malonate (1.5 μmol/2 μL), significantly decreased the lesion volume caused by this toxin. This protective effect can be probably related to the inhibition of excitotoxic pathways. Thus, malonate induced the activation of the calcium-dependent protease, calpain and the cyclin-dependent kinase 5, cdk5; which resulted in the hyperphosphorylation of tau and the cleavage of the protective transcription factor, myocyte enhancer factor 2, MEF2. All these effects were also significantly reduced by sildenafil pre-treatment, suggesting that sildenafil protects against malonate-induced cell death through the regulation of the calpain/p25/cdk5 signaling pathway. Similar findings were obtained using inhibitors of calpain or cdk5, further supporting our contention. Sildenafil also increased MEF2 phosphorylation and Bcl-2/Bax and Bcl-xL/Bax ratios, effects that might as well contribute to prevent cell death. Finally, sildenafil neuroprotection was extended not only to rat hippocampal slices subjected to oxygen and glucose deprivation when added at the time of reoxygenation, but also, in vivo when administered after malonate injection. Thus, the therapeutic window for sildenafil against malonate-induced hypoxia was set at 3 h.

► Sildenafil affords a significant protection against malonate-induced chemical hypoxia.
► The mechanism relies on the inhibition of malonate-induced calpain/p25/cdk5 signaling pathway activation.
► Sildenafil also increases the expression of the antiapoptotic proteins, Bcl-2 and Bcl-xL.
► This neuroprotection was extended to an in vitro model of oxygen glucose deprivation.
► Therapeutic window for sildenafil against malonate-induced hypoxia was set at 3 h.