Background and Purpose— The natural product baicalein is a specific inhibitor of 12/15-lipoxygenase, but it also has antioxidant properties. The current study was designed to test if the neuroprotective properties of baicalein are related to its lipoxygenase inhibition. Methods— The presence of 12/15-lipoxygenase in the ischemic mouse brain was demonstrated by immunohistochemistry. A mouse model of transient middle cerebral artery occlusion was used to study lipoxygenase-dependent protection of the ischemic brain by baicalein. Rat primary neurons were subjected to oxidative stress in the presence or absence of baicalein. Results— In a mouse model of transient middle cerebral artery occlusion, 12/15-lipoxygenase is increased in the peri-infarct area surrounding the primary infarction, predominantly in neurons. Oxidative toxicity in primary rat neurons is reduced by baicalein. C57Bl6J mice are protected against transient focal ischemia by intraperitoneal injection of baicalein, and a similar degree of protection is seen in 12/15-lipoxygenase knockout mice compared with wild-type mice. In contrast, the 12/15-LOX knockout mice are not further protected by baicalein. Conclusion— Baicalein protects against ischemia/reperfusion injury by inhibiting the 12/15-lipoxygenase pathway to neuronal cell death.

Background and Purpose— The concept of the neurovascular unit suggests that effects on brain vasculature must be considered if neuroprotection is to be achieved in stroke. We previously reported that 12/15-lipoxygenase (12/15-LOX) is upregulated in the peri-infarct area after middle cerebral artery occlusion in mice, and 12/15-LOX contributes to brain damage after ischemia–reperfusion. The current study was designed to investigate 12/15-LOX involvement in vascular injury in the ischemic brain. Methods— In cell culture, a human brain microvascular endothelial cell line was subjected to either hypoxia or H ₂ O ₂ -induced oxidative stress with or without lipoxygenase inhibitors. For in vivo studies, mice were subjected to 90 minutes middle cerebral artery occlusion, and the effects of either 12/15-LOX gene knockout or treatment with lipoxygenase inhibitors were compared. Expression of 12/15-LOX and claudin-5 as well as extravasation of immunoglobulin G were detected by immunohistochemistry. Edema was measured as water content of brain hemispheres according to the wet–dry weight method. Results— Brain endothelial cells were protected against hypoxia and H ₂ O ₂ by the lipoxygenase inhibitor baicalein. After focal ischemia, 12/15-LOX was increased in neurons and endothelial cells. The vascular tight junction protein claudin-5 underwent extensive degradation in the peri-infarct area, which was partially prevented by the lipoxygenase inhibitor baicalein. Leakage of immunoglobulin G into the brain parenchyma was significantly reduced in 12/15-LOX knockout mice as well as wild-type mice treated with baicalein. Likewise, brain edema was significantly ameliorated. Conclusion— 12/15-LOX may contribute to ischemic brain damage not just by causing neuronal cell death, but also by detrimental effects on the brain microvasculature. 12/15-LOX inhibitors may thus be effective as both neuroprotectants and vasculoprotectants.

12/15‐Lipoxygenase (12/15‐LOX) is an important mediator of brain injury following experimental stroke in rodents. It contributes to neuronal death, but the underlying mechanism remains unclear. We demonstrate here that in neuronal HT22 cells subjected to glutamate‐induced oxidative stress, 12/15‐LOX damages mitochondria, and this represents the committed step that condemns the cell to die. Importantly these events, including breakdown of the mitochondrial membrane potential, the production of reactive oxygen species, and cytochrome c release, can all be replicated by incubation of 12/15‐LOX with mitochondria in vitro, without the need to add other cytosolic factors. Proteasome activity is required downstream of mitochondrial damage to complete the cell death cascade, but proteasome inhibition is only partially protective. These findings position 12/15‐LOX as the central executioner in an oxidative stress‐related neuronal death program.

A key challenge facing drug discovery today is variability of the drug target between species, such as with 12/15-lipoxygenase (12/15-LOX), which contributes to ischemic brain injury, but its human and rodent isozymes have different inhibitor specificities. In the current work, we have utilized a quantitative high-throughput (qHTS) screen to identify compound 1 (ML351), a novel chemotype for 12/15-LOX inhibition that has nanomolar potency (IC₅₀ = 200 nM) against human 12/15-LOX and is protective against oxidative glutamate toxicity in mouse neuronal HT22 cells. In addition, it exhibited greater than 250-fold selectivity versus related LOX isozymes, was a mixed inhibitor, and did not reduce the active-site ferric ion. Lastly, 1 significantly reduced infarct size following permanent focal ischemia in a mouse model of ischemic stroke. As such, this represents the first report of a selective inhibitor of human 12/15-LOX with demonstrated in vivo activity in proof-of-concept mouse models of stroke.