We recently found that 5-lipoxygenase (5-LOX) is activated to produce cysteinyl leukotrienes (CysLTs), and CysLTs may cause neuronal injury and astrocytosis through activation of CysLT(1) and CysLT(2) receptors in the brain after focal cerebral ischemia. However, the property of astrocyte responses to in vitro ischemic injury is not clear; whether 5-LOX, CysLTs, and their receptors are also involved in the responses of ischemic astrocytes remains unknown. In the present study, we performed oxygen-glucose deprivation (OGD) followed by recovery to induce ischemic-like injury in the cultured rat astrocytes. We found that 1-h OGD did not injure astrocytes (sub-lethal OGD) but induced astrocyte proliferation 48 and 72 h after recovery; whereas 4-h OGD moderately injured the cells (moderate OGD) and led to death 24-72 h after recovery. Inhibition of phospholipase A(2) and 5-LOX attenuated both the proliferation and death. Sub-lethal and moderate OGD enhanced the production of CysLTs that was inhibited by 5-LOX inhibitors. Sub-lethal OGD increased the expressions of CysLT(1) receptor mRNA and protein, while moderate OGD induced the expression of CysLT(2) receptor mRNA. Exogenously applied leukotriene D(4) (LTD(4)) induced astrocyte proliferation at 1-10 nM and astrocyte death at 100-1,000 nM. The CysLT(1) receptor antagonist montelukast attenuated astrocyte proliferation, the CysLT(2) receptor antagonist BAY cysLT2 reversed astrocyte death, and the dual CysLT receptor antagonist BAY u9773 exhibited both effects. In addition, LTD(4) (100 nM) increased the expression of CysLT(2) receptor mRNA. Thus, in vitro ischemia activates astrocyte 5-LOX to produce CysLTs, and CysLTs result in CysLT(1) receptor-mediated proliferation and CysLT(2) receptor-mediated death.

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.

Gliomas are the most common primary brain tumor in adults, but the efficacy of chemotherapy is limited. Artemisinin and its analogs, such as dihydroartemisinin (DHA), can kill cancer cells via generating free radicals. In the present study, we determined whether DHA at low concentrations potentiates the cytotoxic effect of temozolomide in rat glioma C6 cells. We found that the IC₅₀ values of DHA and temozolomide for cell viability were 23.4 and 560 µmol/l, respectively. The cytotoxic effect of temozolomide was enhanced by 177% at a nontoxic DHA concentration (1 µmol/l), and by 321% at a low-toxic DHA concentration (5 µmol/l). DHA substantially increased temozolomide-induced apoptosis and necrosis. The generation of intracellular reactive oxygen species (ROS) was increased by temozolomide combined with DHA at noneffective concentrations of both agents. Edaravone (20 µmol/l), a ROS scavenger, reversed the effects of temozolomide/DHA on both ROS generation and cell viability reduction. These results indicate that DHA at low concentrations potentiates the cytotoxic effects of temozolomide in C6 cells partly via generating ROS, suggesting a beneficial combination for the chemotherapy of gliomas.

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