Modulation of the ASK1–MKK3/6–p38/MAPK signalling pathway mediates sildenafil protection against chemical hypoxia caused by malonate

Abstract

PD5 inhibitors have recently been reported to exert beneficial effects against ischaemia-reperfusion injury in several organs. However, there are few studies regarding their neuroprotective effects in brain ischaemia. The present study was designed to assess the effects of sildenafil against chemical hypoxia induced by malonate. Intrastriatal injection of malonate produces energy depletion and striatal lesions similar to that seen in cerebral ischaemia through mechanisms that involve generation of reactive oxygen species (ROS). Volume lesion was analysed by cytochrome oxidase histochemistry. Generation of reactive species was determined by in situ visualization of superoxide production and nitrotyrosine measurement. Protein levels were determined by Western blot after subcellular fractionation. Sildenafil, given 30 min before malonate, significantly decreased the lesion volume in the rat. This protective effect cannot be attributed to any effect on ROS production but to the inhibition of downstream pathways. Thus, malonate induced the activation of apoptosis signal-regulating kinase-1 (ASK1) and two MAPK kinases, MKK3/6 and MKK7, which lead to an increased phosphorylation of JNK and p38 MAPK, effects that were blocked by sildenafil. Selective inhibitors of p38 and JNK (SB203580 or SP600125, respectively) were used in combination with malonate in order to evaluate the plausible implication of these pathways in the protection afforded by sildenafil. While inhibition of p38 provided a significant protection against malonate-induced neurotoxicity, inhibition of JNK did not. Sildenafil protects against the chemical hypoxia induced by malonate through the regulation of the ASK1-MKK3/6-p38/MAPK signalling pathway.