Although experiments on animals and cells indicate that anti

Although experiments on animals and cells indicate that antioxidant and free radical scavenging agents have protective effects against brain damage, the results of preclinical trials are not as encouraging. Edaravone is a type of free radical scavenging drug that was first used as a neuroprotective drug in 2001 in Japan (Amemiya et al., 2005), and it is also the most effective drug used in clinical study. However, its new mechanism via the 5-lipoxygenase (5-LOX) pathway is inexplicitly and need to be further investigated. Once brain injury occurred, damaged blood-brain barrier and neuronal cells oedema exhibits, accompanied by declined activity of Na+/K+-ATPase enzymes on the cell membrane and reabsorption blockage, all of these changes can cause inflammatory mediators to be released into the injured parts of brain. High level of arachidonic SB505124 metabolites is one of the most intensively studied mediator; leukotriene, generated by arachidonic acid metabolites, is neurotoxic to neurons (Cho et al., 2011, Tomimoto et al., 2002) and often triggers brain oedema at later periods of injury and delayed neuronal apoptosis. It is widely accepted that three main enzymes participate in lipoxygenase metabolism of arachidonic acid: 5-lipoxygenase (5-LOX), 12-lipoxygenase (12-LOX) and 15-lipoxygenase (15-LOX), which pathology and physiological characteristics are understood clearly. Watanabe’s (Watanabe and Egawa, 1994) experiment suggests that edaravone can significantly reduce leukotriene level after cerebral ischemia, which can ease cerebral oedema in late-period ischemia. However, it has not yet been reported how edaravone regulates the signalling pathways of arachidonic metabolites. In the current study, middle cerebral artery occlusion (MCAO) model was used to determine whether neuroprotective effect of edaravone, a free radical scavenging agent, is correlated with 5-LOX. In vitro, we used an OGD injury model on PC12 cells to further validate its effect, which include observing 5-LOX activation induced by OGD injury and investigating whether the inhibitory effect of edaravone was dependent on 5-LOX signalling pathways. Finally, we evaluated the new mechanism of edaravone and comprehensively discussed the relationship between the 5-LOX pathway and mitochondria.
Results
Discussion At present, MCAO model is a world-acclaimed method for ischemic stroke research. For better performance, MCAO for 90 min with 24 h reperfusion has been chosen for the models. Comparing with MCAO group, rats in edaravone (ED) and caffeic acid (CA) group have a lower neurological deficit score, a slighter infract areas in TTC staining, which indicate edaravone and caffeic acid can remarkably ameliorates ischemia symptom pathologically. All the results above elucidated that edaravone is an effective agent for treatment of ischemia stroke in vivo. Interestingly, we found the expression of 5-LOX on the nuclear membrane after ED and CA administration was dramatically diminished when compared with MCAO group and triflingly expressed in sham group. To uncover the relationship between the neuroprotective effect of edaravone and 5-LOX signalling pathway in-depth, PC12 cells were utilized for the following study. In vitro experiment, cell culture without oxygen and glucose (OGD) is the most classic model of cerebral ischemia. Our data showed that OGD damages PC12 cells in a time-dependent manner. Because the morphology of the remaining cells was not completely changed at OGD 6 h, it was easy to observe the subcellular distribution of 5-LOX. As such, we chose OGD 6 h as our acute damage injury model and OGD 6 h with 24 h reperfusion as our late injury model (OGD-R). Edaravone (0.01, 0.1, 1 μmol/L) had the protective effects on OGD/OGD-R injury in a concentration-dependent manner. Hoechst 33258/PI double staining, TUNEL and electron microscopy of PC12 cells showed not only necrosis, but also apoptosis in our OGD-R injury model. However, the number of apoptotic cells was obviously decreased when edaravone was administered while OGD. Taken together, edaravone can apparently protect both SD rats and PC12 cells from ischemic injury.