Oxygen‐glucose deprivation activates 5‐lipoxygenase mediated by oxidative stress through the p38 mitogen‐activated protein kinase pathway in PC12 cells

Abstract

5-Lipoxygenase (5-LOX) is a key enzyme catalyzing arachidonic acid to form leukotrienes. We have reported that ischemic-like injury activates 5-LOX in PC12 cells; however, the mechanisms are unknown. To determine whether ischemic-like injury activates 5-LOX mediated by oxidative stress through the p38 MAPK pathway, we transfected GFP-5-LOX into PC12 cells and induced ischemic-like injury by oxygen-glucose deprivation (OGD). We found that the transfected GFP-5-LOX was localized primarily in the nuclei and translocated to the nuclear envelope after OGD/recovery reaching a maximum 2 hr after a 2-hr exposure to OGD. The nonselective 5-LOX inhibitor caffeic acid, 5-LOX-activating protein inhibitor MK886, and selective 5-LOX inhibitor zileuton attenuated the cell injury and reduced the production of 5-LOX products, cysteinyl leukotrienes, after OGD/recovery. However, only caffeic acid inhibited OGD/recovery-induced 5-LOX translocation. OGD/recovery also increased reactive oxygen species (ROS), which was inhibited by caffeic acid only. Hydrogen peroxide, an exogenous ROS, evoked similar cell injury and 5-LOX translocation, and the inhibitors had effects on the changes after H(2)O(2) similar to those after OGD/recovery. Both OGD/recovery and H(2)O(2) increased the phosphorylated p38 MAPK level, which was inhibited by caffeic acid and the ROS scavenger edaravone, but not by MK886 or zileuton. Moreover, SB203580 (a p38 MAPK inhibitor) and edaravone inhibited the cell injury and 5-LOX translocation induced by OGD/recovery and H(2)O(2). Thus, we conclude that OGD/recovery-induced ischemic-like injury induces 5-LOX activation, which is mediated by oxidative stress through activating the p38 MAPK pathway.

Baicalin attenuates oxygen-glucose deprivation-induced injury by inhibiting oxidative stress-mediated 5-lipoxygenase activation in PC12 cells

“…The pEGFP-C2/5-LOX (GFP-5-LOX) and pEGFP-C2 null vectors (gifts from Professor Funk CD, University of Pennsylvania, USA [now in Queen's University, Canada]) were transfected into PC12 cells as we recently reported [14] . PC12 cell lines stably expressing GFP or GFP-5-LOX have been characterized by fluorescence microscopy and Western blot analysis [14] .…”

“…PC12 cell lines stably expressing GFP or GFP-5-LOX have been characterized by fluorescence microscopy and Western blot analysis [14] . To detect GFP-5-LOX translocation, the transfected cells cultured on glass slips were washed with phosphate buffered saline (PBS) and immediately fixed with 4% paraformaldehyde.…”

“…OGD, H 2 O 2 and agent treatments PC12 cells were exposed to OGD as described pre viously [11,12,14] . Briefly, cells were rinsed twice and incubated in glucose-free Earle's solution.…”

“…Specifically, elevation of intracellular calcium that generally occurs after excitotoxicity and mitogen-activated protein kinase (MAPK)-regulated phosphorylation that can be induced by oxidative stress [3] both activate 5-LOX. Previously, we reported that 5-LOX can be activated after ischemic brain injury in rats [8,10] , after oxygen-glucose deprivation (OGD)-induced ischemic injury in cultured neurons and in pheochromocytoma (PC12) cells [11][12][13][14] . In primary neuron cultures, OGD induces release of excitatory amino acids that activate NMDA receptors to elevate intracellular calcium, resulting in 5-LOX translocation to the nuclear envelope and activation of 5-LOX to produce CysLTs [12] .…”

“…In addition, we recently found oxidative stress-induced 5-LOX activation in ischemic PC12 cells. OGD increases the release of reactive oxygen species (ROS), which activate 5-LOX through the p38 MAPK pathway [14] . Therefore, ischemic 5-LOX activation can be regulated by both elevated intracellular calcium after excitotoxicity and the activated MAPK pathway after oxidative stress.…”