Ferrous Citrate Up-Regulates the NOS2 through Nuclear Translocation of NFκB Induced by Free Radicals Generation in Mouse Cerebral Endothelial Cells

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.

Estradiol Reduces Ferrous Citrate Complex-Induced NOS2 Up-Regulation in Cerebral Endothelial Cells by Interfering the Nuclear Factor Kappa B Transactivation through an Estrogen Receptor β–Mediated Pathway

“…The mechanisms underlying the ROS-induced neurotoxicity may involve DNA damage and activation of poly adenosine diphosphate (ADP)-ribose synthase with subsequent depletion of glycerol-3-phosphate dehydrogenase and ATP or mitochondrial damage leading to necrosis and apoptosis. Previously, we demonstrated that FC can increase ROS generation and NOS2 gene expression in mouse CEC [31]. Our data also suggest that increases of ROS might contribute to the FC-induced up-regulation of NOS2 through activation and binding of NFκB onto the NOS2 promoter of the mouse CEC.…”

Controversies and evolving new mechanisms in subarachnoid hemorrhage

“…Some of the consequences of oxidative stress after SAH include neuroinflammation, disruption of the BBB, and production of spasmogens (Gaetani et al, 1990; Yun et al, 2013). Intracellular ROS activate NF-κB to upregulate NOS 2 (Chen et al, 2012). In addition, ROS can activate apoptotic signals, including p53, caspase-3 and caspase-9 to promote apoptotic cell death (Lu et al, 2013).…”

Citrate chemistry and biology for biomaterials design

“…All the studies implicate the therapeutic potential of citrate for cancer treatment and further comprehensive studies are needed to determine the toxic concentration range to specific cancer types. In addition, it is worthy to note that although citrate has been found to reduce radical surge [185] or alleviate certain cell dysfunction affected by reactive oxygen species (ROS) [155], the existing results are still preliminary and additional research is needed to confirm the antioxidant effect of citrate, since citrate chelating with ferrous ions was also found to elevate intracellular oxidative stress leading to increased ROS production [186, 187]. Therefore, despite its great importance, this aspect of citrate biology is not discussed in the present review.…”