Abstract
Previous studies indicate that the inducible nitric oxide synthase 2 (NOS2) of the brain vascular tissue in experimental subarachnoid hemorrhage (SAH) rats is a critical factor for inducing cerebral vasospasm. However, the underlying molecular mechanisms remain to be elucidated. Here, we applied ferrous citrate (FC) complexes to the primary cultured mouse cerebral endothelial cell (CEC) to mimic the SAH conditions and to address the issue how SAH-induced NOS2 up-regulation. Using immunocytochemical staining technique, we demonstrated that NOS2 was expressed in the cultured CEC. Treatment of the CEC with FC induced increases of the intracellular level of ROS, nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) nuclear translocation as well as NFκB binding onto the NOS promoter, and the levels of NOS2 mRNA and protein. These effects were abolished by pre-treatment of the cell with N-Acetyl-Cysteine (NAC), a reactive oxygen species (ROS) scavenger. In the present study, two previously predicted NFκB binding sites were confirmed in the NOS2 promoter within the range of −1529 bp to −1516 bp and −1224 bp to −1210 bp. Interestingly, both NFκB binding sites are involved in the FC-activated NOS2 transcriptional activity; the binding site located at −1529 bp to −1516 bp played a greater role than the other binding site located at −1224 bp to −1210 bp in the mouse CEC. These findings highlight the molecular mechanism underlying FC-induced up-regulation of NOS2 in the mouse CEC.
Highlights
Hemorrhage stroke, which includes intracerebral hemorrhage and subarachnoid hemorrhage (SAH), is associated with high risk of mortality and morbidity
ferrous citrate (FC) Increases reactive oxygen species (ROS) Levels in the cerebral endothelial cell (CEC) It has been indicated that iron can potentiate ROS production and exacerbate oxidative stress [29,30]
To study the effect of iron on the production of ROS, the mouse CEC was treated with various concentrations of FC, small molecular weight iron complex, and the levels of ROS were measured by DCF
Summary
Hemorrhage stroke, which includes intracerebral hemorrhage and SAH, is associated with high risk of mortality and morbidity. Most of the non-heme iron in the brain is bound to ferritin as ferric ion, and can be released only after being reduced to the ferrous state. It has been shown that hypoxia/ischemia conditions cause neuronal cell death and the affected area is accompanied by increased brain levels of iron and ferritin in the cerebral cortex and the hippocampus [11,12,13]. The notion that iron plays an important role in the development of SAH was supported by intracerebroventricular injection with ferrous ammonium citrate causing increases of the level of toxic lipid peroxidation products, such as 4hydroxynonenal (HNE), in the field CA3 of the hippocampus in a rat model [14], and intravenous administration with 2,29dipyridyl, an iron chelator, prevents delayed vasospasm in a primate model of SAH [15]. Desferal chelates iron complex and prevents the iron-catalyzed oxidative stress and brain injury in vivo [16]
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