Ischemic stroke results in cerebral tissue hypoxia and increased expression of hypoxia-inducible factor (HIF), which is critically implicated in ischemic brain injury. Understanding the mechanisms of HIF-1alpha regulation in the ischemic brain has been an important research focus. The generation of both nitric oxide (NO) and reactive oxygen species (ROS) is increased under hypoxic/ischemic conditions and each of them has been independently shown to regulate HIF-1alpha expression. In this study, we investigated the cross-effects of NO and ROS on the expression of HIF-1alpha in hypoxic astrocytes. Exposure of astrocytes to 2 h-hypoxia remarkably increased HIF-1alpha protein levels, which was accompanied by increased NO and ROS production. Decreasing ROS with NAC, NADPH oxidase inhibitor DPI, or SOD mimetic MnTMPyP decreased hypoxia-induced HIF-1alpha protein accumulation and increased NO level in hypoxic astrocytes. The NO synthase (NOS) inhibitor L-NAME inhibited ROS generation, which led to a reduction in hypoxia-induced HIF-1alpha protein expression. Although NOS inhibitor or ROS scavengers alone reduced HIF-1alpha protein levels, the reduction was reversed when NOS inhibitor and ROS scavenger present together. The NO scavenger PTIO increased hypoxia-induced HIF-1alpha protein expression and ROS production, while HIF-1alpha protein level was decreased in the presence of NO scavenger and ROS scavenger together. These results suggest that ROS, NO, and their interaction critically contribute to the regulation of hypoxia-induced HIF-1alpha protein accumulation under hypoxic condition. Furthermore, our results indicate that hypoxia-induced NO generation may represent an endogenous mechanism for balancing ROS-mediated hypoxic stress, as reflected by inhibiting hypoxia-induced HIF-1alpha protein accumulation.

Cerebral ischemia triggers a cascade of events that contribute to ischemic brain damages. Zinc release and accumulation has been shown to lead to brain cell death following cerebral ischemia. However, the mechanism underlying remains to be elucidated. Our recently published work showed that suppression of mitochondrial-derived reactive oxygen species (ROS) production significantly reduced ischemic stroke related brain damage within 6 h. Herein, we investigated the relationship between zinc accumulation and mitochondrial-derived ROS production in astrocytes after 3-h hypoxia. We found that inhibition of mitochondrial-derived ROS significantly decreased total amount of ROS generation and cell death in primary astrocytes during hypoxia when zinc was overload. In contrast, the inhibition of NADPH oxidase-derived ROS had less of an effect. Our results also showed that zinc and mitochondria were colocalized in hypoxic astrocytes. Moreover, extracellular zinc addition caused zinc accumulation in the mitochondria and decreased mitochondrial membrane potential, leading to mitochondria dysfunction. These findings provide a novel mechanism that zinc accumulation contributes to hypoxia-induced astrocytes death by disrupting mitochondria function, following cerebral ischemia.