Metab Brain Dis https://doi.org/10.1007/s11011-017-0129-7 ORIGINAL ARTICLE Up-regulation of FOS-like antigen 1 contributes to neuronal apoptosis in the cortex of rat following traumatic brain injury Xide Xu 1 & Rui Jiang 2 & Peipei Gong 2 & Qianqian Liu 2 & Yinan Chen 2 & Shiqiang Hou 2 & Debin Yuan 2 & Jiansheng Shi 3 & Qing Lan 1 Received: 1 May 2017 / Accepted: 9 October 2017 # Springer Science+Business Media, LLC 2017 Abstract Neuronal apoptosis is an important process of secondary brain injury which is induced by neurochemical signaling cascades after traumatic brain injury (TBI). Present study was designed to investigate whether FOS-like antigen 1 (Fra-1) is involved in the neuronal apoptosis. Western blot analysis and immunohistochemistry in a rat TBI model revealed a significant increase in the expression of Fra-1 in the ipsilateral brain cortex, which was in parallel with increase in the expression of active caspase-3. With immunofluorescence double-labeling, Fra-1 was colocalized with active caspase-3 and with NeuN, a neuronal marker. In an in vitro cell injury model, H2O2 exposure induced cell apoptosis and reduced cell viability and at the same time, a similar increased expression of active caspase-3, p53 and Fra-1 was found in PC12 cells. Down-regulation of Fra-1 through transfection with Fra-1 siRNA remarkably elevated cell viability, reduced the expression of active caspase-3 and p53, and decreased apoptosis of PC12 cells after H2O2 exposure. Taken together, present findings suggest that Fra-1 may be involved in the induction of neuronal apoptosis through up-regulating p53 signaling Xide Xu and Rui Jiang contributed equally to this work. * Jiansheng Shi [email protected] * Qing Lan [email protected] 1 Department of Neurosurgery, The Second Affiliated Hospital of Suzhou University, 1055 Sanxiang Road, Suzhou, Jiangsu Province 215000, China 2 Department of Neurosurgery, The Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong, Jiangsu Province 226001, China 3 Department of Neurology, The Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong, Jiangsu Province 226001, China pathway and that this action may contribute to the secondary neuropathological process after TBI. Keywords FOS-like antigen 1 . Traumatic brain injury . Neuron . Apoptosis . Caspase-3 . P53 Introduction The secondary injury processes after traumatic brain injury (TBI), including edema, oxidative stress, glial activation, and inflammation responses, will result in considerable delayed neuronal apoptosis (Lewen et al. 2000; Quillinan et al. 2016). Therefore, targeting the neuronal apoptosis has become one of the important strategies to treat TBI or other brain injuries (Kabadi and Faden 2014; Yuan 2009). FOS-like antigen 1 (Fra-1), as one member of the FOS family of proteins, is over-expressed in many cancer cells and participates in growth and migration of cancer cells (Debinski and Gibo 2011; Motrich et al. 2013). Small interfering RNA inhibition of Fra-1 expression was associated with reduced growth and enhanced cisplatin sensitivity in ovarian cancer cells (Casalino et al. 2007; Macleod et al. 2005). However, Shirsat and Shaikh reported that Fra-1 overexpression inhibited proliferation and induced apoptosis of c6 glioma cells (Shirsat and Shaikh 2003). Moreover, Hamdi et al. investigated the role of Fra-1, an AP-1 component, in JNK- and ERK-dependent cell cycle arrest and apoptosis and found that Fra-1 knock-down resulted in decreased cisplatin-induced apoptosis (Debinski and Gibo 2011; Hamdi et al. 2008). Therefore, the role Fra-1 plays in cell apoptosis is still debated. In addition, Fra-1 immunoreactivity in the central nervous system (CNS) has also been found (Faure et al. 2006; Pozas et al. 1999), but its functional role is little understood. Faure Metab Brain Dis observed an increase in the number of Fra-1 expressing neurons after instrumental task training in SNc/VTA and ventral hippocampus, suggesting that a general underlying incentive process regulates Fra-1 (Faure et al. 2006). Pozas et al. found that Fra-1 immunoreactivity in adult rats was restricted to the hippocampus (Pozas et al. 1999). After administration of colchicine, an axonal transport inhibitor, Fra-1 immunoreactivity accumulated in the perikarya of neurons in the dentate gyrus, hippocampus, cerebral cortex, amygdala and thalamus. Moreover, Fra-1 immunoreactivity was also found in the nuclei of reactive astrocytes following either intraperitoneal injection of kainic acid at convulsant doses, intrastriatal injection of quinolinic acid, or intraventricular injection of colchicine. However, the exact role of Fra-1 in neural activity or in neuropathological process and the underlying mechanisms are still unclear. Though Fra-1 is a molecule with some potential functions, its expression and function in the brain after traumatic brain injury (TBI) are still unclear. In current study, we determined the expression and distribution of Fra-1 in the brain cortex in a rat TBI model and especially, investigated the association of Fra-1 with neuronal apoptosis and potential cellular and molecular mechanisms in the brain after TBI. Materials and methods Animals and TBI model Male Sprague-Dawley rats (n = 67) with a body weight of 225-275 g were obtained from the Experimental Animal Center of Nantong University, Nantong, China. All surgical procedures and postoperative animal care were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (National Research Council 1996, USA) and were approved by the Chinese National Committee for the Use of Experimental Animals for Medical Purposes, Jiangsu Branch. TBI model was established as described previously with modifications (Chen et al. 2015). The rats were anesthetized with 10% chloral hydrate (400 mg/kg, i.p.). After placement of the rat in a stereotactic frame, a right parietal craniotomy (3.5 mm posterior and 2.5 mm lateral to the bregma, diameter 5 mm) was made with a drill. A steel rod about 20 g with a flat end and diameter of 4.5 mm was dropped onto a piston resting on the dura from a height of 25 cm. The piston was allowed to compress the brain tissue to a depth of 2.5 mm and was removed immediately after the contusion. The sham-operated rats were surgically treated with right parietal craniotomy alone without brain injury. Rats were housed under a 12 h light/dark cycle at a room temperature of 25 ± 0.5 °C with free access to the food and water. Rats (n = 6 per time-point) were sacrificed for Western blot analysis at 12 h, 1, 3, 5, 7, 14 and 28 days after surgery. The sham-operated rats (n = 6) were sacrificed on the third day. All efforts were made to minimize the number of animals and their suffering. The mortality was totally about 4.5%. Western blot analysis The cortical tissue (extending 3 mm surrounding the incision) were dissected out and stored at −80 °C until Frozen samples were weighed, minced, homogenized in lysis buffer (1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 50 mmol/L Tris, 1% NP-40, pH 7.5, 5 mmol/L EDTA, 1% sodium deoxycholate, 1 lg/mL leupeptin, 10 lg/mL aprotinin, and 1 mmol/L PMSF), and centrifuged at 12,000 g for 20 min at 4 °C. Cell cultures were lysed in sodium lauryl sulfate loading buffer and stored at −80 °C until use. Equal amounts of protein (40 μg per lane) from each sample were loaded on a 10% sodium dodecyl sulphate-polyacrylamide gel, electrophoresed, and transferred onto polyvinyledene difluoride membranes. The membranes were incubated overnight at 4 °C with primary antibodies (diluted in Tris-buffered saline containing 0.1% Tween-20 with 5% non-fat milk) to Fra-1 (rabbit, 1:500, Santa Cruz Biotechnology, Santa Cruz, USA), active caspase3 (mouse, 1:1000, Cell Signaling Technology, Shanghai, China), GAPDH (rabbit, 1:1000, Santa Cruz Biotechnology), polyclonal anti-p53 (rabbit, 1:1000, Santa Cruz Biotechnology). After incubating the blots with goat anti-rabbit IgG (H + L)-HRP and goat anti-mouse IgG (H + L)-HRP (1:10,000, Jackson ImmunoResearch, Pennsylvania, USA) for 2 h at room temperature, the immunoreactive bands were visualized by enhanced chemiluminescence and exposed to X-ray film. The images were captured using an EC3 Imaging System, and the protein levels were quantified using Image-Pro Plus. The samples were analyzed repeatedly for a minimum of two times. Brain sectioning and immunofluorescent staining Rats were anesthetized and perfused through the ascending aorta with 500 mL normal saline followed by 4% paraformaldehyde. The brains were then removed and fixed in the same fixative for 3 h. The fixative was subsequently changed with 20% sucrose for 2–3 days, followed by 30% sucrose for the same time. The brain tissues were coronally sectioned into 6 μm-thick slices. Slices were incubated at 37 °C for 30 min, and rinsed in 0.01 M phosphate buffer solution (PBS) for 5 min twice. After blocking with confining liquid consisting of 10% donkey serum, the slices were incubated with following antibodies: anti-Fra-1 (Rabbit, 1:200, Santa Cruz Biotechnology), anti-active caspase-3 (Mouse, 1:200, Cell Signaling Technology), anti-GFAP (Mouse, 1:200, Sigma-Aldrich Corporation, Saint Louis, USA), anti-NeuN (Mouse, 1:300, Chemicon, Temecula, USA) overnight at Metab Brain Dis 4 °C and followed by incubation in a mixture of FITC- and TRITC-conjugated secondary antibodies (1:1000, Jackson, West Grove, USA) for 2 h at 37 °C. Immunofluorescent analysis of Fra-1 triple-labeling with 4′,6-diamidino-2phenylindole (DAPI, 1:1000, Cell Signaling Technology) and other markers in cultured PC12 cells was similar to above procedures. Immunohistochemistry The brains were sectioned into 30 μm-thick slices and blocked with 10% goat serum supplemented with 0.3% Triton X-100 and 1% bovine serum albumin for 2 h at room temperature. The slices were incubated overnight at 4 °C with anti-Fra-1 antibody (rabbit, 1:100, Santa Cruz Biotechnology) and then with the second antibody for 30 min at 37 °C. These slices were finally incubated with the liquid mixture containing 0.02% diaminobenzidine tetrahydrochloride, 0.1% PBS and 3% H2O2. Strong or moderate brown staining cells were counted as positive, whereas weak or no staining cells were considered negative. Quantification of Fra-1 positive cells Quantification of Fra-1 positive cells was performed by an unbiased approach according to the principle described previously(Konigsmark and Murphy 1970). Every fifth section (50 μm apart) was assayed in order to avoid the possibility of counting the same cell in more than one section. In the Fig. 1 Time-dependent up-regulation of Fra-1 expression in rat cortex. Fra-1 expression in the ipsilateral (a) and contralateral (b) brain cortices surrounding the wound at indicated time after TBI. Semi-quantitative cortex 1 mm away from the lesion site, Fra-1 positive cells were counted under microscope (DM 5000B, Leica). Five sections were examined for each animal. For the quantification of dual-labeled immunofluorescent results, the cortices until 2 mm away from both caudal and rostral to the lesion site were observed. Three non-adjacent sections of each animal were examined. Cell culture and Fra-1 siRNA transfection The rat pheochromocytoma cells (PC12 cells) were obtained from the Institute of Cell Biology, Academy of China, and cultured in a 5% CO2 incubator at 37 °C with Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich Corporation) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, USA). After incubation with DMEM containing 1% FBS for 48 h, PC12 cells were induced for differentiation with 100 ng/ml human recombinant nerve growth factor (R&D Systems, Minneapolis, USA) in DMEM plus 1% FBS for at least 5 days before subsequent experiments. The control and Fra-1 siRNA oligos were obtained from GenePharma (Shanghai, China). The Fra-1 siRNA1 target sequence was 5′-TCAGCTCATCGCAAGAGTA-3′. The Fra-1 siRNA2 target sequence was 5′- GTACCTTGTATCTC CCTTT-3′. The Fra-1 siRNA3 target sequence was 5′CTCTGACCTACCCTCAGTA-3′. The Fra-1 siRNA4 target sequence was 5′-TGGATGGTACAGCCTCATT-3′. All the transfection assays were performed using Lipofectamine analysis of Fra-1 in the ipsilateral (c) and contralateral (d) cortices. *P < 0.05, vs. sham-operated group (n = 6) Metab Brain Dis Fig. 2 Distribution of Fra-1 positive cells in rat cortex. Fra-1 immunohistochemical staining respectively in the cortex of normal rat (a), and contralateral (b) and ipsilateral (c) cortices of TBI rat at 3 d after injury. D, E and F, local magnification view of A-C, respectively. G, negative control with control IgG. Scale bars, 200 μm for A-C, G; 50 μm for D-F. H, Quantitative analysis of Fra-1 positive cells in normal, contralateral, and ipsilateral brain cortices (n = 4). *P < 0.05, vs. two control groups 2000 transfection reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s protocol. were exposed to 200 μM H2O2 for 6 h. The PC12 cells were rinsed with PBS, treated with 1% Triton X-100 in PBS for 2 min on ice and after rinse with PBS, incubated with 50 μL of TUNEL reaction mixture at 37 °C for 60 min. After washing in PBS for three times, the cells were examined with a fluorescence microscope (DM 5000B, Leica). Cell viability measurement Cell viability was evaluated with a Cell Counting Kit-8 (CCK8, Dojindo Laboratories, Kumamoto, Japan). PC12 cells were treated with H2O2 (0–350 μM) in fetal bovine serum-free medium for 24 h, 10 μL of CCK8 agents was then added to the cultures, and the cell viability was measured at 490 nm using an enzyme-linked immune-sorbent assay reader (BioTek, Winooski, USA) according to the manufacturer’s instructions. Terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling Terminal deoxynucleotidyl transferase-mediated biotinylateddUTP nick-end labeling (TUNEL) staining was performed using the In Situ Cell Death Detection Kit (Roche Applied Science, Mannheim, Germany) for cultured PC12 cells that Annexin-V/7AAD apoptosis assay PC12 cells were treated with 200 μM H2O2 for 6 h. A negative control was conducted by incubating cells in the absence of apoptosis inducing agent. The cells were then harvested and washed with cold phosphate-buffered saline (PBS). The supernatant was discarded and the cells were re-suspended in annexin binding buffer at ~1 × 106 cells/mL with 100 μL per assay. Thereafter, 5 μL of Pacific Blue™ annexin V ( C o m p on en t A ) an d 1 μL o f 50 0 μM S Y TO X ® AADvanced™ Dead Cell Stain working solution were added. The cells were then incubated at room temperature for 30 min, and protected from light. After the incubation Metab Brain Dis Fig. 3 Location of Fra-1 in neurons of rat cortex. Fra-1 positive cells in the cortices of sham-operated and injured rats at 3 d after TBI immunostained with anti Fra-1 (a-d), neuron marker NeuN (e and f), and astrocyte marker GFAP (g and h) antibodies. Merged images of colocalization of Fra-1 with cell-specific markers (i-l). M and N, negative controls. O, Fra-1 positive neuron rate in the cortices of shamoperated and TBI rats. *P < 0.05, vs. sham-operated group. Scale bar, 50 μm period, 400 μL of annexin binding buffer were added and mixed gently, and the samples were kept on ice. The stained cells were analyzed with flow cytometry, the fluorescence emission using a 450 nm bandpass or equivalent with 405 nm excitation (Pacific Blue™ dye) and a 670 bandpass or equivalent with 488 nm excitation (SYTOX® AADvanced™) was measured. (Fig. 2h). Data in Fig. 7c were analyzed with two-way ANOVA, and the Fisher’s LSD test was used for post hoc comparisons between the groups. The chi-square test is used for comparison of two rates. Differences were considered statistically significant at P < 0.05. Statistical analysis Results All data were shown as mean ± SEM. Student’s t-test was used for the comparison of two group experiments. One-way ANOVA was used for the comparison of three or more groups, Dunnett’s test was used for multiple comparisons to a single control value. Dunn’s test was used for counted data Time-dependent increase in the expression of Fra-1 in rat cortex after TBI As shown in Fig. 1a and c, Fra-1 protein level in rat cortex after TBI was obviously increased from 1 day, peaked at 3 day Metab Brain Dis Fig. 4 Association of Fra-1 expression with neuronal apoptosis in rat cortex. Active caspase-3 expression in the cortex surrounding the wound at indicated time after TBI (a) and semi-quantitative analysis (b, n = 3). #, *P < 0.05, vs. sham-operated group of each protein. Cortical sections at 3 d after TBI were immunostained with anti active caspase-3 (d and g), NeuN (c), and Fra-1 (f) antibodies. E and H, merged images of NeuN + active caspase-3 and Fra-1 + active caspase-3, respectively. Scale bar, 100 μm and afterwards, recovered to the control level gradually. Weak expression of Fra-1 in contralateral cortex and in the shamoperated group was found (Fig. 1b and d). To examine the distribution of Fra-1 in the cortex of rat, immunohistochemical staining was used. We observed Fra-1 positive cells in the cortex (Fig. 2a, b, d, e) which were significantly increased in the ipsilateral cortex at 3 days following TBI (P < 0.05, Fig. 2c, f, h). Involvement of Fra-1 expression in neuronal apoptosis after TBI Colocalization of Fra-1 with neuronal cells Fra-1 was mainly co-expressed with NeuN in the cortex (Fig. 3i and j) but less with GFAP (Fig. 3k and l). Moreover, the number of Fra-1 positive neurons in the cortex of rat 3 days after TBI was significantly increased (P < 0.05, Fig. 3q). As shown in Fig. 4a and b, a time-dependent elevation in the expression of active caspase-3 were found in the cortex of rat after brain injury, which was closely parallel with the increase in the expression of Fra-1 (Fig. 1a and c). In addition, immunofluorescent double-labeling revealed the co-localization of NeuN with active caspase-3 (Fig. 4c-e) and active caspase-3 with Fra-1 (Fig. 4f-h) in the brain cortex 3 days after TBI, suggesting a potential association of Fra-1 expression with neuronal apoptosis. After treatment of 200 μM H2O2, the expression of Fra-1 was significantly elevated in a time-dependent manner (Fig. 5a and b) and at the same time, the expression of active caspase-3 and p53, another cell apoptosis-related protein, was also enhanced in parallel with the elevation of Fra-1 Metab Brain Dis Fig. 5 Time-dependent up-regulation of Fra-1 expression in H2O2treated PC12 cells. a Fra-1, p53 and active caspase-3 expressions at indicated time in H2O2-treated PC12 cells. b semi-quantitative analysis of Fra-1, active caspase-3, and P53 expressions. *P < 0.05, vs. 0 h group. c immunofluorescent doule-labeling of Fra-1 and active caspase-3 in PC12 cells. Scale bar, 50 μm expression (Fig. 5a and b). Immunofluorescent staining method also revealed an increased expression of Fra-1 and active caspase-3 in PC12 cells after H2O2 treatment (Fig. 5c). TUNEL staining (Fig. 7b). Moreover, when the PC12 cells were exposed to different concentrations of H2O2, cell viability was decreased in a dose-dependent manner. However, Fra1 down-regulation significantly increased the cell viability while the PC12 cells were exposed to 200–350 μM H2O2 (Fig. 7d), suggesting a cell protective effect. Interference with Fra-1 gene expression resulted in H2O2-induced cell apoptosis inhibition and cell protection Four independent siRNA oligos targeting Fra-1 were used and transfection of Fra-1 siRNA#3 (SiFra-1#3, Fig. 6a and c) showed a significant interference effect on Fra-1 expression in PC12 cells. Down-regulation of Fra-1 expression through transfection with Fra-1 siRNA#3 dramatically reduced the expression of both active caspase-3 and p53 in PC12 cells after 200 μM H2O2 treatment (Fig. 6b and d). Moreover, Fra-1 down-regulation further reduced cell apoptosis observed with both annexin-V/7-AAD apoptotic analysis (Fig. 7a) and Discussion For the first time, present results revealed that Fra-1 expression was up-regulated in a time-dependent manner in rat cortex after TBI. Fra-1 was mainly localized in the cortical neurons and its up-regulation was followed by an identical timedependent elevation of active caspase-3 expression in the cortical neurons. Moreover, we also found a time-dependent upregulation of p53 expression in rat cortex after TBI, which was Metab Brain Dis Fig. 6 Influence of Fra-1 knockdown on the expression of p53 and active caspase-3 in H2O2treated PC12 cells. a Fra-1 expression in PC12 cells at 48 h after transfection with nonspecific siRNA or four Fra-1 siRNA oligos and semi-quantitative analysis (C). b influence of Fra-1 down-regulation on the expression of p53 and active caspase-3 in H2O2-treated PC12 cells and semi-quantitative analysis (D). *P < 0.05, vs. control group (n = 3) parallel to Fra-1 and active caspase-3 up-regulations, suggesting a potential association of Fra-1 expression with neuronal apoptosis after TBI. To further explore the function of Fra-1, we used an oxidative stress model in PC12 cells after H2O2 treatment to extrapolate to the in vivo situation because the oxidative stress is one of the most important pathophysiological processes of traumatic brain injury (Cheng et al. 2013; Sheng et al. 2017). Furthermore, we found that the expression of active caspase-3 and p53 in PC12 cells after H2O2 treatment was enhanced in a time-dependent manner in parallel with the elevation of Fra-1 expression. Subsequently, we downregulated Fra-1 expression through transfection with Fra-1 siRNA in PC12 cells and found that Fra-1 down-regulation significantly inhibited the elevated expression of both active caspase-3 and p53 induced by H2O2 exposure. Moreover, Fra1 down-regulation further reduced cell apoptosis detected by annexin-V/7-AAD apoptotic analysis and TUNEL staining. These results suggest that Fra-1 up-regulation after brain injury is potentially involved in neuronal apoptosis possibly through modulating p53 signaling pathway. Furthermore, Fra-1 down-regulation significantly enhanced the viability of PC12 cells after H2O2 exposure. It is suggested that reducing the expression of Fra-1 will provide a cell protective effect in PC12 cells when subjected to a damage insult. Fra-1 is a member of AP-1 transcription factor family just like c-Fos. AP-1 members function as transcription regulators to activate or inhibit target gene transcription and regulate cell proliferation, survival, and death in response to numerous mitotic and stress stimuli(Jochum et al. 2001; Shaulian and Karin 2002). Activation of c-Fos may contribute to neuronal apoptosis in the infarcted thalamus and cortex of rats (Gillardon et al. 1996). Actually, c-Fos and Fra-1 are identified to have maintained functional equivalence during vertebrate evolution (Fleischmann et al. 2000). Transcription factor AP-1 may comprise a collection of related inducible protein complexes that interact with similar sequence motifs (Cohen et al. 1989). Previous studies have found the role Fra-1 plays in tumor cell apoptosis, but the results in different reports are still contradictory (Hamdi et al. 2008; Macleod et al. 2005; Shirsat and Shaikh 2003). In retinal ganglion cell, Fra-1 may be associated with light exposure-induced retinal ganglion cell apoptosis which is regulated by p38 MAPK through cell cycle re-entry mechanism (Liu et al. 2017). Therefore, changes of Fra-1 immunoreactivity in the CNS after physiological activity (Faure et al. 2006) and brain injury (Pozas et al. 1999) suggest potential actions in physiological and pathophysiological processes of the brain, but the exact functions and underlying mechanisms are unclear. Present experiment extends the study on the roles Fra-1 plays in the CNS pathophysiological processes. Activation of p53 can trigger apoptosis in many cell types including neurons (Brynczka and Merrick 2007; Culmsee and Mattson 2005; Hughes et al. 1997). Activation of p53dependent apoptosis will result in mitochondrial apoptotic changes and trigger cell death execution most notably by release of cytochrome C and activation of the caspase cascade (Laposa et al. 2007; Wang et al. 2014). Martin found that retrograde neuronal apoptosis in the thalamus after cortical Metab Brain Dis Fig. 7 Influence of Fra-1 knock-down on cell apoptosis and viability in H2O2-treated PC12 cells. A, apoptotic cell rate in H2O2-treated PC12 cells and statistical analysis (B). C, viability of PC12 cells transfected with non-specific siRNA or SiFra-1#3 after H2O2 exposure at different concentrations. D, TUNEL staining of H2O2-induced apoptotic PC12 cells. *P < 0.05, vs. non-specific siRNA transfection group at the indicated concentration (n = 12). Scale bar, 50 μm injury in adult mouse and rat was modulated by p53 (Martin et al. 2001). Many reports have suggested that p53 may be induced in injured brain regions following experimental traumatic brain injury (Ai et al. 2015; He et al. 2015; Napieralski et al. 1999). Consistent with these reports, the present study also found up-regulation of p53 expression in Metab Brain Dis the cortex of rat after TBI and in PC12 cells after toxic H2O2 exposure accompanied with elevation in Fra-1 expression and active caspase-3 level, and RNA interference of Fra-1 significantly reduced both the levels of active caspase-3 and p53 and inhibited cell apoptosis following H2O2 exposure. These results suggest that Fra-1 up-regulation after brain injury is potentially involved in the induction of neuronal apoptosis possibly through up-regulating p53 expression and its downstream signaling pathway. Whether Fra-1 up-regulation induces p53 expression through other ways such as posttranslational control (Lakin and Jackson 1999) deserves further investigation. Therefore, the present results provide some evidence that Fra-1 is potentially involved in the secondary brain injury of rat after TBI through inducing p53-dependent neuronal apoptosis. If Fra-1 is targeted to induce lower expression or decrease in its activity in the brain, a protective effect on neurons would be obtained, and this action may be translated to clinical use in the treatment of brain injury in the future. Acknowledgments This work was supported by the National Natural Science Foundation of China (81371367, 812713681), the Youth Program of National Natural Science Foundation of China (81401013), 333 High-level Talent Project in Jiangsu Province (BRA2014347), BTalent Innovation Fund^ in Nantong University (CXZR201308), and Nantong Municipal Science and Technology Innovation and Demonstration Project Special Social Programs of 2013 (HS2013068). References Ai Z, Li C, Li L, He G (2015) Resveratrol inhibits beta-amyloid-induced neuronal apoptosis via regulation of p53 acetylation in PC12 cells. Mol Med Rep 11(4):2429–2434. https://doi.org/10.3892/mmr.2014. 3034 Brynczka C, Merrick BA (2007) Nerve growth factor potentiates p53 DNA binding but inhibits nitric oxide-induced apoptosis in neuronal PC12 cells. 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