Effects of FC on NOS2 expression in the CEC.

<p>(A) The levels of NOS2 mRNA in the CEC were significantly increased at 8 h after FC treatment. A peak of NOS2 mRNA level was observed at 8–16 h after FC treatment, and then began to decline. The levels of NOS2 mRNA were determined using quantitative real time-PCR, adjusted with G3PDH mRNA, and expressed as ratio over control. (B) FC (100 µM) treatment increased the levels of NOS2 protein in the CEC. FC (100 µM) time-dependently increased the level of NOS2 protein. Top panel: representative results of NOS2 and G3PDH protein levels determined by Western blot analysis. Bottom panel: quantitative results of NOS2 protein levels, which were adjusted with G3PDH protein level and expressed as fold-induction of its own control. Values represent the means±s.e.mean. (n = 3). ^*<i>P</i> < 0.05 different from corresponding control. (C) The FC-induced increases of the level of NOS2 protein were in a concentration-dependent manner. Top panel: representative results of the levels of NOS2 and G3PDH protein determined by Western blot analysis. Bottom panel: quantitative results of NOS2 protein levels, which were adjusted with G3PDH protein level and expressed as fold of control. Values represent the means±s.e.mean. (n = 3). ^*<i>P</i> < 0.05 different from corresponding control. (D) FC-induced NOS2 expression is mainly located in the CEC. Micrographs show NOS2 (green) and vWF (red) immunoreactivity detected by dual immunofluorescent staining as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046239#s2" target="_blank">Materials and Methods</a>. NOS2, inducible nitric oxide synthase 2; G3PDH, glyceraldehyde3-phosphate dehydrogenase, vWF, von Willebrand Factor.</p

FC induces NFκB activation through increases of the levels of ROS and phosphorylated IκBα.

<p>(A) Pretreatment of the CEC with a ROS scavenger, NAC (5 mM), for 1 h prevented the FC-induced increases of the levels of NOS2 protein. Top panel: representative results of NOS2 and G3PDH protein levels determined by Western blot analysis. Bottom panel: quantitative results of NOS2 protein levels, which were adjusted with G3PDH protein level and expressed as fold of control. Values represent the means±s.e.mean. (n = 3). ^*<i>P</i> < 0.05 different from corresponding control; ^#<i>P</i> < 0.05 different from the FC-treated CEC. (B) The FC (100 µM)-induced an increase of the NFκB (p65) DNA binding onto the NOS2 promoter was completely abolished by pretreatment of the cell with NAC (5 mM). The NOS2 DNA binding activity was assessed by using ChIP assay and detected by semiquantitative PCR (top panel) and quantitative real-time PCR (bottom panel). Values represent the means±s.e.mean. (n = 3). ^*<i>P</i> < 0.05 different from corresponding control; ^#<i>P</i> < 0.05 different from the FC-treated CEC. (C) FC-induced an increase of the NOS2 promoter activity was abolished by pretreatment of the cell with NAC. Values represent the means±s.e.mean. (n = 3). ^*<i>P</i> < 0.05, different from control. (D) Pretreatment of the CEC with NAC (5 mM) for 1 h prevented the FC-induced increases of the levels of phosphorylated IκBα (p-IκBα) protein. Top panel: representative results of the levels of p-IκBα and total IκBα (t-IκBα) protein determined by Western blot analysis. Bottom panel: quantitative results of p-IκBα protein levels, which were adjusted with t-IκBα protein level and expressed as fold of control. Values represent the means±s.e.mean. (n = 3). ^*<i>P</i> < 0.05 different from corresponding control; ^#<i>P</i> <0.05 different from the FC-treated CEC. (E) FC increased phosphorylation of the protein IκBα and NFκB binding on the NOS2 promoter, and these effects were blocked by a ROS scavenger (NAC), an IKK inhibitor (Bay 11-7082), or NFκB translocation inhibitors (PTDC). The levels of NOS2 protein were quantified by Western blot analysis. The NFκB DNA binding activity was assessed by using ChIP assay and quantitative real-time PCR (bottom panel). Values represent the means±s.e.mean. (n = 3). ^*<i>P</i> < 0.05, different from corresponding control; ^#<i>P</i> < 0.05, different from FC-treated without ROS scavenger (NAC).</p

FC increases ROS generation in the CEC.

<p>(A) FC (1–100 µM) concentration-dependently increased ROS generation in the CEC. (B) The ROS generation was observed at 5 min after FC (100 µM) treatment. (C) FC (100 µM)-induced increases of ROS generation in CEC were prevented by pretreatment of the cell with a ROS scavenger, NAC (5 mM). ROS levels were assayed using 5 µM DCF as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046239#s2" target="_blank">Materials and Methods</a>. DCF fluorescence images and DIC images were taken using Leica TCS SP5 fluorescent microscope imaging system (Wetzlar, Germany) and the levels of ROS were quantified by flow cytometric analysis. Values represent the means±s.e.mean. (n = 4). ^*<i>P</i> < 0.05 different from control. ^#<i>P</i> < 0.05 different from FC-treated group. DIC, differential interference contrast; DCF, dichlorodihydrofluorescein diacetate [CM-H2DCF-DA].</p

Involvement of NFκB (p65) in regulating the NOS2 promoter activity in the FC-treated CEC.

<p>(A) FC (100 µM) induced NFκB translocation from the cytosolic fraction into the nuclear fraction in the CEC. After treatment with FC for 4 h, the CEC was fixed and then labeled with an anti-NFκB antibody, followed by an FITC-conjugated secondary antibody. The nuclei were visualized with propidium iodide (50 µg/mL) staining as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046239#s2" target="_blank">Materials and Methods</a>. (B) FC (100 µM) increased nuclear translocation of NFκB in the CEC. Top panel: representative results of NFκB, PARP and G3PDH protein levels determined by Western blot analysis. Bottom panel: quantitative results of NFκB protein levels, which were adjusted with PARP protein level and expressed as fold of control. Values represent the means±s.e.mean. (n = 4). ^*<i>P</i> < 0.05 different from corresponding control. (C) FC (100 µM) induced an increase of NFκB binding onto the NOS2 promoter. The levels of NFκB binding onto the NOS2 promoter were assessed by using ChIP assay (top panel) and quantitative real-time PCR (bottom panel). Values represent the means±s.e.mean. (n = 4). ^*<i>P</i> < 0.05 different from control. P.I., propidium iodide.</p

Proposed signaling pathway associated with FC-induced up-regulation of NOS2 in the CEC.

<p>FC increased ROS generation, which in turn caused IκBα phosphorylation, subsequently increased NFκB (p65) nuclear translocation and binding onto the NOS2 promoter, and finally increased the levels of NOS2 mRNA and protein.</p

NFκB-binding sites are required for the FC-induced NOS2 promoter transactivation.

<p>5′-serial deletions of the mouse NOS2 1.6-kb promoter were generated by PCR as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046239#s2" target="_blank">Materials and Methods</a>” by using the primers indicated in (A). (B) The CEC was transiently transfected with various constructs for 24 h, and then treated with vehicle or FC (100 µM) for 16 h. Subsequently, the cell was processed for the luciferase activity assay. Quantitative results of the NOS2 promoter activity were shown and expressed as fold induction of the CEC transfected with PGL3 (control). Values represent the means±s.e.mean. (n = 4). ^*<i>P</i> < 0.05, different from cells transfected with PGL3; ^#<i>P</i> < 0.05, different from cells transfected with −1211 to −1600. C, Control.</p

Injury and recovery of pyramidal neurons in the rat hippocampus after a single episode of oxidative stress induced by intracerebroventricular injection of ferrous ammonium citrate

10.1051/rnd:2005051Reproduction Nutrition Development455647-66