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reperfusion (IR) injury by reducing inflammation and apoptosis via the activation of Nrf-2
Biochemical and Biophysical Research Communications 509 (2019) 713e721
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DUSP14 rescues cerebral ischemia/reperfusion (IR) injury by reducing inflammation and apoptosis via the activation of Nrf-2 Song Jianrong a, Zhao Yanjun b , Yu Chen b , Xu Jianwen c
*
a
Department of Neurosurgery, Baoji Municipal Central Hospital, Baoji City, 721008, China Department of Anesthesiology, Beijing Tsinghua Changgung Hospital, No. 168, Li Tang Road, Changping District, Beijing 102218, China c Department of Anesthesiology, Zaozhuang Municipal Hospital, Zaozhuang, Shandong 277100, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 December 2018 Accepted 27 December 2018 Available online 9 January 2019
Ischemic stroke is the second most common cause of death, a major cause of acquired disability in adults. However, the pathogenesis that contributes to ischemic stroke has not been fully understood. Dualspecificity phosphatase 14 (DUSP14, also known as MKP6) is a MAP kinase phosphatase, playing important role in regulating various cellular processes, including oxidative stress and inflammation. However, its effects on cerebral ischemia/reperfusion (IR) are unclear. In the study, we found that DUSP14 expression was decreased responding to IR surgery. Over-expressing DUSP14 reduced the infarction volume of cerebral IR mice. Cognitive dysfunction was also improved in mice with DUSP14 overexpression. Promoting DUSP14 expression markedly reduced the activation of glial cells, as evidenced by the decreases in GFAP and Iba-1 expressions in mice with cerebral IR injury. In addition, inflammatory response induced by cerebral IR injury was inhibited in DUSP14 over-expressed mice, as proved by the reduced expression of tumor necrosis factor (TNF)-a and interleukin 1b (IL-1b). Furthermore, oxidative stress was markedly reduced by DUSP14 over-expression through elevating nuclear factor-erythroid 2 related factor 2 (Nrf-2) signaling pathway. Importantly, we found that DUSP14 could interact with Nrf-1, which thereby protected mice against cerebral IR injury. In vitro, we also found that repressing Nrf-2 expression abrogated DUSP14 over-expression-reduced inflammation and ROS generation. Consistent with the anti-inflammatory effect of DUSP14, reducing the production of reactive oxygen species (ROS) also down-regulated TNF-a and IL-1b expressions. Collectively, elevated DUSP14 alleviated brain damage from cerebral IR injury through Nrf-2-regulated anti-oxidant signaling pathway, and the restraining of inflammatory response. These results suggested that DUSP14 might be a potential therapeutic target to prevent ischemic stroke. © 2018 Published by Elsevier Inc.
Keywords: DUSP14 Cerebral IR injury Inflammation Oxidative stress Nrf-2
1. Introduction Ischemic stroke is the second most common cause of death, which is a leading cause of acquired disability in adults. Approximately 15 million people suffer from strokes each year, and about 5 million die as a result in the world [1,2]. Various basic and clinical experiments have suggested that neuroinflammation plays an essential role in the primary and secondary injury phases of stroke [3,4]. In addition, ROS-associated oxidative damage has been considered as the important pathogenesis of neuronal loss and the subsequent memory impairment following brain IR injury [5,6].
* Corresponding author. Department of Anesthesiology, Zaozhuang Municipal Hospital, Zaozhuang, Shandong 277100, China. E-mail address: [email protected] (X. Jianwen). https://doi.org/10.1016/j.bbrc.2018.12.170 0006-291X/© 2018 Published by Elsevier Inc.
Redox disturbance has been linked to the deteriorating clinical outcomes in acute ischemic stroke patients [7]. However, presently, the underlying molecular mechanisms remain unclear. Thus, further study is still required to better understand the pathogenesis of cerebral IR injury for developing effective therapeutic strategy against ischemic stroke progression. DUSP14, also known as MKP6, is an atypical DUSP [8]. DUSP14 contains the consensus C-terminal catalytic domain but lacks the N-terminal CH2 domain. DUSP14 could dephosphorylate mitogen activated protein kinases (MAPKs), including ERK1/2, p38 and c-Jun N-terminal kinase (JNK), which regulate various cellular responses, such as differentiation, stress response, proliferation and immune defense [9e12]. Oxidative stress leads to activation of JNK and p38 and directly affects DUSP-activity, either by oxidizing their catalytic cysteine or by elevating their degradation [13]. Phosphorylation of
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DUSP8 by JNK has been indicated under arsenite-triggered oxidative stress [14]. Herein, DUSP14 might also have anti-oxidant activity. In addition, DUSP14-knockout mice showed enhanced immune responses and were more susceptible to experimental autoimmune encephalomyelitis induction [15]. Over-expression of DUSP14 suppresses TNF- and IL-1-triggered nuclear factor-kB (NFkB) activation, subsequently limiting inflammatory response [16]. Oxidative stress, TNF-a and IL-1b have crucial roles in the progression of cerebral I/R injury [17]. Thus, DUSP14 might possess a key role in the progression of cerebral IR injury. In the study, we explored the effects of DUSP14 on the development of cerebral IR injury in mice with middle cerebral artery occlusion (MCAO). Our findings provided evidence that DUSP14 could be a protective modulator in cerebral IR injury by preventing inflammation and oxidative stress through interacting with Nrf-2 pathway.
2.3. Cognitive analysis The neurologic severity score (mNSS) was used to calculate neurological deficits 24 h after reperfusion as previously described [19]. The scoring system involved three tests, including motor, sensory and beam balance test. Here, 0 represented no deficit and 14 represented maximal deficits. Spatial learning and memory were assessed through Morris Water Maze (MWM) tests at day 22e28 after MCAO surgery according to previous study [20]. The escape latency to find the platform, the swim path length, the time spent in the target quadrant and platform crossovers were analyzed. Open field test was carried out to determine spontaneous activity and adaptability for at day 21 after MCAO by a computerized tracking system (Noldus EthoVision XT, shanghai, China) [21]. Total distance traveled, rearing activities, time spent and frequency of entry into the central area were recorded. 2.4. Co-immunoprecipitation analysis
2. Materials and methods 2.1. Animals Male C57BL/6J mice (weighting 20e25 g), purchased from Beijing Vital River Laboratories (Beijing, China) and housed under controlled conditions (temperature 23 ± 2 C; humidity 55e60%; 12-h-light-dark cycle) with access to food and water. All experiments were approved by Experimental Animal Ethic Committee of the South China Hospital Affiliated to University of South China (Hunan, China) and were implemented following National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication, revised in 1996). Vector construction was carried out by Cyagen Bioscience Inc (Guangzhou, China) to obtain DUSP14-encoding vector. The vector pLentiviruses LV (pLV) [Exp]EGFP was used as control. Virus was packaged through Lenti-XTM HTX packaging system (Clontech, USA). Purified lentiviruses were injected into the right lateral ventricle (dosage: 5 ml) and hippocampus (dosage: 3 ml) of mice with stereotaxic apparatus. Transfection efficiency was measured with RT-qPCR and western blot. Then, MCAO operation was conducted 2 weeks after transfection. All mice were divided into 4 groups: 1) Sham group; 2) MACO group; 3) MCAOþLV-GFP group and 4) MCAOþLV-DUSP14 group. Transient focal cerebral ischemia was performed through intraluminal MCAO surgery according to previous study [18]. After occlusion for 90 min, monofilament was withdrawn for reperfusion. A heating pad was used to keep the body temperature during the surgery. The sham group of mice was subjected to the same procedures except that MCA was not occluded after neck incision.
2.2. Microglia cells and culture Mouse microglial BV2 cells were obtained from the Cell Culture Center at the Institute of Basic Medical Sciences of Chinese Academy of Medical Sciences (Beijing, China) and were incubated in DMEM (GIBCO, USA) with 10% heat-inactivated fetal bovine serum (FBS, GIBCO), 2 mM glutamine (Gbico), 100 mg/ml streptomycin and 100 U/mL penicillin at 37 C with 5% CO2. The DUSP14 plasmid (pcDUSP14) or negative control plasmid (NC-DNA) were constructed by Cobioer (Nanjing, China) and transfected into BV2 cells for 24 h with lipofectamine 2000 (Invitrogen, USA). Nrf-2 siRNA and corresponding negative siRNA (siNC) were purchased from Santa Cruz (USA). Lipopolysaccharides (LPS), ROS scavenger of N-acetylcysteine (NAC) and Nrf-2 activator of tert-butylhydroquinone (tBHQ) were purchased from Sigma Aldrich (USA). Nrf-2 inhibitor, ML385, was purchased from MedChemExpress (USA).
Immunoprecipitation was performed with a Pierce Classic IP kit (Pierce Biotechnology, Inc., USA) according to the manufacturer's instructions and analyzed through western blot method. 2.5. Cerebral infarct volume, brain edema and neurological deficit measurements 2, 3, 5-triphenyltetrazoliumchloride (TTC, Sigma Aldrich, USA) staining was performed 24 h after reperfusion to calculate infarct volume. The 1-mm-thick brain slices were stained with 2% TTC for 15 min in dark at 37 C, followed by fixing with 4% paraformaldehyde overnight at 4 C. Then, slices were scanned, and the infarct volume and edema ratio were analyzed as reported [18]. 2.6. Immunofluorescent analysis Frozen brain sections and LPS-incubated BV2 cells were subjected to immunofluorescence staining according to previous study [22]. Primary antibodies used in immunofluorescence analysis were GFAP (sc-33673, Santa Cruz, USA), Iba-1 (sc-32725, Santa Cruz), DUSP14 (ab168969, Abcam, USA), 8-OHdG (ab48508, Abcam), Nrf-2 (ab31163, Abcam) and DUSP14 (ab168969, Abcam) at 1:100 dilutions. Alexa Fluor 488- or 594-labeled anti-rabbit or -goat secondary antibodies (Beyotime) and DAPI (Sigma Aldrich) to identify cell nuclei. Images were captured with a fluorescent microscope. 2.7. Statistical analysis Data analysis was conducted using Graphpad Prism software (USA). Data were presented as mean ± standard error of mean (SEM). The one-way analysis of variance (ANOVA) and t-test were conducted among multi-groups or between two groups. P < 0.05 was served as statistical significance. 3. Results 3.1. DUSP14 is down-regulated in the infarcted area of mice following ischemic stroke As shown in Fig. 1AeD, DUSP14 expression was markedly downregulated from 2 h to 168 h after reperfusion in ischemic core and ischemic penumbra. To further investigate the effects of DUSP14 on ischemic stroke, LV-GFP and LV-DUSP14 were subjected to the hippocampus and right lateral ventricle of mice. Cells were doublelabeled with DUSP14 combing with Iba1 (microglial marker). The
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Fig. 1. DUSP14 is down-regulated in the infarcted area of mice following ischemic stroke. The mRNA and protein expression pattern and time course of DUSP14 in (A,B) ischemic core and (C,D) ischemic penumbra. (E) Double-immunofluorescence staining for microglial (Iba1), and DUSP14 expression in the ischemic penumbra area after MCAO operation. (F,G) LV-GFP and LV-DUSP14 were injected into the right hippocampus and right lateral ventricle of mice 2 weeks before MCAO operation. RT-qPCR and western blot analysis of DUSP14 in brain extracts of the described groups. (H) RT-qPCR analysis of pro-inflammatory cytokines (IL-b, IL-6, TNF-a and COX2) in brain extracts before MCAO treatment. (I) Representative image of TTC staining 1 day after reperfusion. Quantification of (J) infarct volume and (K) edema area. (L) Calculation of neurological score. Data are presented as the means ± SEM (n ¼ 6, for each group). þP < 0.05, þþP < 0.01 and þþþP < 0.001 versus Sham group; **P < 0.01 versus MCAO group.
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DUSP14 was down-regulated in microglia (Iba1/DUSP14, Fig. 1E) 24 h after MCAO operation, as evidenced by the weaker fluorescent intensity (green). Thus, DUSP14 expression was detected in microglia of the brain. Microglia cells are active contributors to the inflammation in ischemic stroke [23,24]. DUSP14 expression was significantly up-regulated after LV-DUSP14 transfection (Fig. 1F and G). No difference was observed in the change of pro-inflammatory cytokines (IL-b, IL-6, TNF-a and COX2) before MCAO operation, indicating that lentivirus injection did not influence inflammatory response (Fig. 1H). TTC analysis suggested that DUSP14 overexpression markedly reduced the infarction volume, and brain edema volume when compared with MCAO group of mice (Fig. 1IeK). Furthermore, neurological deficits in MCAO mice were significantly alleviated by LV-DUSP14 transfection (Fig. 1L). 3.2. Over-expression of DUSP14 improves cerebral IR-induced cognitive deficits In this regard, the cognitive function was measured using MWM. Longer distance and time spent to find the platform were observed in MCAO mice, which were attenuated by DUSP14 overexpression (Fig. 2AeC). MCAO-reduced platform crossovers and time spent in target quadrant were significantly rescued due to DUSP14 over-expression (Fig. 2A, D and E). OFT analysis suggested that the spontaneous locomotion in center, periphery and the total locomotion reduced by MCAO were markedly improved by DUSP14 over-expression (Fig. F and G). In addition, the entries into the center, the time spent in center and the rearing activities were markedly decreased by MCAO, while being alleviated in DUSP14over-expressed mice (Fig. 2F, H-J). 3.3. DUSP14 up-regulation inhibits inflammation and oxidative stress in brain of cerebral IR mice GFAP and Iba-1 are essential gliosis markers, playing crucial role in promoting cerebral IR injury [25,26]. In the peri-infarct region, MCAO led to a significant increase of GFAP and Iba-1 expression, which was, however, down-regulated by DUSP14 over-expression (Fig. 3A and B). In addition, IL-b and TNF-a expression in brain extracts of the peri-infarct region was markedly up-regulated by MCAO. The process was reversed due to LV-DUSP transfection (Fig. 3C). SOD activities in the peri-infarct region were decreased in MCAO mice, whereas MDA levels were increased, which were markedly alleviated by DUSP14 over-expression (Fig. 3D). Consistently, NADPH oxidase activity and ROS production in the periinfarct region were obviously up-regulated by MCAO, whereas being reduced in DUSP14-over-expressed mice (Fig. 3E and F). Immunofluorescent analysis suggested that MCAO-induced 8OHdG expression was diminished in DUSP14 over-expressed mice (Fig. 3G). Moreover, western blot analysis suggested that SOD2, HO1, NQO-1 and Nrf-2 expressions reduced by MCAO were rescued by LV-DUSP14 transfection. In contrast, expression levels of Keap-1 and XO, as Nrf-2 suppressor and major source of ROS production [27,28], were increased by MCAO, which were markedly decreased by DUSP14 over-expression (Fig. 3H). Immunofluorescent analysis showed weaker Nrf-2 fluorescent intensity in MCAO group of mice, which was contrast to Iba-1; however, DUSP14 over-expression promoted Nrf-2 fluorescence in the peri-infarct region of mice along with reduced Iba-1, suggesting that Nrf-2 accumulation was detected in microglia (Fig. 3I). 3.4. DUSP14 evokes Nrf-2-meditated anti-oxidative stress and inflammatory response Co-immunoprecipitation
assay
indicated
an
interaction
between DUSP14 and Nrf-2 (Fig. 4A). In vitro, LPS stimulation decreased DUSP14 and Nrf-2 expressions in microglia cells (Fig. 4B). Next, DUSP14 was over-expressed in vitro by transfecting DUSP14 pcDNA (Fig. 4C). LPS-reduced Nrf-2 and DUSP14 expressions was restored by DUSP14 over-expression (Fig. 4D). Promoting DUSP14 expression rescued Nrf-2, NQO-1 and HO-1 expressions in LPSstimulated microglia, which were further down-regulated by ML385, Nrf-2 inhibitor. An opposite expression change was observed in Keap-1 (Fig. 4E). In addition, ROS, NADPH oxidase activity and MDA levels induced by LPS were alleviated by DUSP14 over-expression, while being recovered in BV2 cells pre-treated with Nrf-2 inhibitor of ML385 or with Nrf-2 knockdown (Fig. 4F and G, Supplementary Figs. 1AeC). Suppressing Nrf-2 expression abolished DUSP14 over-expression-reduced IL-b and TNF-a expression in LPS-treated microglial cells (Fig. 4H and Supplementary Fig. 1D). Then, both over-expressing DUSP14 and NAC pre-treatment decreased LPS-induced ROS generation, IL-b and TNF-a expression, further indicating that DUSP14 exhibited suppressive role in oxidative stress and subsequently suppressing inflammatory response (Fig. 4I and J). 4. Discussion Ischemic stroke is an essential cause of death and acquired disability in adults, which is linked to various risk factors, such as hypertension, atherosclerosis, thrombosis, diabetes, and even smoking [1,3,29]. Increasing studies regarded to ischemic stroke have been conducted in recent decades [5e7,30]. Unfortunately, the pathogenesis of ischemic stroke has not been fully understood. In our study, DUSP14 expression was found to be down-regulated in ischemic core and ischemic penumbra of mice with MCAO surgery. These observations indicated that DUSP14 might have an essential role in cerebral IR injury. Furthermore, DUSP14 over-expressed mice exhibited reduced infarction area, and showed better cognitive performance after MCAO operation. Attenuated inflammatory response and oxidative stress were observed in mice following MCAO. These findings indicated that DUSP14 could attenuate brain injury via the regulation of inflammatory response and oxidative stress after cerebral IR injury. Consistent with the in vivo data, in vitro cell culture experiments suggested a protective function of DUSP14 in LPS-treated microglia, which was tightly associated with Nrf-2 pathway activation. These findings elucidated that DUSP14 could be a target to develop effective therapeutic strategy against ischemic stroke. The inflammatory response has an essential role in the pathogenesis of cerebral I/R injury, and suppression of inflammation could be an effective strategy for the prevention of, or intervention in, this disease [31,32]. Pro-inflammatory cytokines could promote various signaling pathways to regulate inflammation. In addition, oxidative stress has been identified as one of the key contributing factors in the pathogenesis of brain damage [5e7,33]. Excessive production of ROS enhances brain injury post I/R injury via various molecular mechanisms, such as inflammation, apoptosis, and brain-blood barrier disruption [34e36]. Therefore, suppressing inflammation and reducing the production of ROS, or repairing oxidative damage are the significant strategies to combat against cerebral IR injury. Studies have suggested that DUSP14 plays a critical role in modulating inflammation and oxidative stress [16,37]. DUSP14 targets MAPKs to regulate the functions of immune cells [38]. DUSP14 negatively regulates TNF-a- or IL-1b-induced NFkB activation, restraining inflammatory response in hepatic IR injury [16]. DUSP14 knockout accelerated oxidative stress in myocardial IR injury [37]. In our study, we also found that overexpressing DUSP14 down-regulated TNF-a and IL-1b expression. Therefore, repressing inflammatory response was a major
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Fig. 2. Over-expression of DUSP14 improves cerebral IR-induced cognitive deficits. (A) The swim path traces of mice in hidden platform test, including learning (up) and memory (bottom) traces. Calculation of (B) the swim path and (C) escape latency from day 23 to day 27 after MCAO surgery. Recording of (D) platform crossovers and (E) the time spent in the target quadrant. (F) Tracks of movements of mice from the indicated groups for 30 min in an open field box. (G) Cumulative distance traveled in each zone. (H) The frequency entry into the center area, (I) the time spent and (J) rearing activities during the 30 min session in the open field test. Data are presented as the means ± SEM (n ¼ 6, for each group). þþP < 0.01 versus Sham group; *P < 0.05 versus MCAO group.
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Fig. 3. DUSP14 up-regulation inhibits inflammation and oxidative stress in brain of cerebral IR mice. (A,B) Immunofluorescent analysis of GFAP and Iba-1 in the peri-infarct region of mice, and the quantified analysis of GFAP and Iba-1 expressions. (C) RT-qPCR analysis of IL-b and TNF-a in brain extracts. Measurements of (D) SOD activity, MDA levels, (E) NADPH oxidase activity and (F) relative ROS production in the peri-infarct region of mice. (G) Immunofluorescent analysis of 8-OHdG in the peri-infarct region of mice, and the quantified analysis of 8-OHdG expressions. (H) Western blot analysis of SOD2, HO-1, NQO-1, Nrf-2, Keap-1 and XO protein expressions in the peri-infarct region of mice. (I) Immunofluorescent analysis of Iba-1 and Nrf-2 in the peri-infarct region of mice. Data are presented as the means ± SEM (n ¼ 6, for each group). þþP < 0.01 and þþþP < 0.001 versus Sham group; *P < 0.05 and **P < 0.01 versus MCAO group.
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Fig. 4. DUSP14 evokes Nrf-2-meditated anti-oxidative stress and inflammatory response. (A) DUSP14 binds to Nrf-2. Brain lysates were immunoprecipitated with anti-DUSP14 (up) or anti-Nrf-2 (bottom) antibodies. Next, total lysates and immunoprecipitates were calculated using immunoblotting analysis with anti-DUSP14 and anti-Nrf-2. (B) Micorglia cells were treated with 100 ng/ml LPS for 24 h, followed by western blot analysis of DUSP14 and Nrf-2. (C) Microglia cells were transfected with pcDNA DUSP14 or the negative control (NC) pcDNA to over-express (OE) DUSP14 expressions. Western blot analysis was performed to determine the efficacy of transfection. (D) Microglia cells were transfected with pcDNA DUSP14 for 24 h, followed by LPS (100 ng/ml) treatment for another 24 h, and then cells were harvested for immunofluorescent analysis of Nrf-2 and DUSP14. Microglia cells were pre-treated with Nrf-2 inhibitor ML385 (10 mM) or activator tBHQ (10 mM) for 2 h. Then, cells were subjected to pcDNA DUSP14 transfection for another 24 h. Finally, LPS was challenged to cells for an additional 24 h. (E) Western blot analysis of Nrf-2, NQO-1, HO-1 and Keap-1. (F) DCF analysis for ROS production and calculation of NADPH oxidase activity. (G) MDA levels in cells treated as indicated. (H) RT-qPCR analysis of IL-b and TNF-a in cells. Microglia cells were pre-treated with or without NAC (5 mM) for 2 h, followed by pcDNA DUSP14 or negative control pcDNA transfection for another 24 h. Finally, LPS was subjected to cells for an additional 24 h. (I) DCF analysis for ROS production. (J) RT-qPCR analysis of IL-b and TNF-a in cells. (K) Proposed schematic diagram of Nrf-2-meditated anti-oxidative and anti-inflammatory response regulated by DUSP14. Data are presented as the means ± SEM (n ¼ 6, for each group).
pathology by which DUSP14 protected against cerebral IR injury development. Nrf-2 is a master regulatory element meditating a diverse set of
antioxidant defense machineries. Nrf-2 regulates more than 200 genes that encode cytoprotective phase II detoxification and antioxidant enzymes [39]. NADPH oxidase consisting of membrane-
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bound subunits is an essential source of ROS [40]. 8-OHdG is a significant marker of DNA oxidative stress [39]. In the study, we found that MCAO-induced oxidative stress was attenuated by DUSP14 over-expression, as evidenced by the reduced MDA levels, ROS generation, NADPH oxidase and 8-OHdG expressions. Moreover, the increased anti-oxidants, including SOD2, HO-1 and NQO1, regulated by the activation of Nrf-2 were observed in mice transfected with LV-DUSP14. In contrast, the expression of Keap-1 and XO-1, significant molecules in promoting the generation of ROS, was markedly decreased in DUSP14 over-expressed mice. Intriguingly, we found that Nrf-2 was a critical target of DUSP14. Consistently, suppressing Nrf-2 abrogated DUSP14 overexpression-reduced inflammatory response and ROS production. Increased production of ROS could stimulate the NF-kB pathway, leading to the release of pro-inflammatory cytokines and subsequent inflammation [41,43]. Also, we found that reducing ROS with its scavenger of NAC markedly decreased TNF-a and IL-1b expressions. Thus, in our study, Nrf-2 anti-inflammatory function was ROS-dependent. Consistently, Nrf-2 has been indicated to inhibit inflammation through ROS-dependent and/or -independent manner [44,45]. Therefore, DUSP14 could interact with Nrf-2 to restrain oxidative stress and inflammation, subsequently attenuating cerebral IR injury (Fig. 4K). However, further study is still required to determine how DUSP14 interacts with Nrf-2, and if anti-inflammatory function of Nrf-2 regulated by DUSP14 could be ROS-independent. In summary, the study indicated that DUSP14 protected cerebral IR injury. The neuroprotective effects of DUSP14 were regulated by its interaction with Nrf-2 to inhibit oxidative stress and inflammatory response, as well as attenuate cognitive dysfunction. The results encourages further study on DUSP14 to prevent ischemic stoke or even other neurodegenerative disease. Acknowledgments
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This work was supported by Fundamental Research Funds for the Central Universities (grant number: 06123143497).
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Appendix A. Supplementary data
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Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.12.170.
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Transparency document [30]
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Xing, et al., Ischemic postconditioning inhibits apoptosis after focal cerebral ischemia/reperfusion injury in the rat, Stroke 39 (2008) 2362e2369. X. Chen, et al., Neutralizing anti-interleukin-1b antibodies modulate fetal bloodebrain barrier function after ischemia, Neurobiol. Dis. 73 (2015) 118e129. B. Lin, et al., DUSP14 knockout accelerates cardiac ischemia reperfusion (IR) injury through activating NF-kB and MAPKs signaling pathways modulated by ROS generation, Biochem. Biophys. Res. Commun. 501 (2018) 24e32. X. Wang, et al., Dusp14 protects against hepatic ischaemiaereperfusion injury via Tak1 suppression, J. Hepatol. 68 (2018) 118e129. M.A. Riedl, et al., Oral sulforaphane increases Phase II antioxidant enzymes in the human upper airway, Clin. Immunol. 130 (2009) 244e251. S.A. Manea, et al., Epigenetic regulation of vascular NADPH oxidase expression
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
DUSP14 rescues cerebral ischemia/reperfusion (IR) injury by reducing inflammation and apoptosis via the activation of Nrf-2 Song Jianrong a, Zhao Yanjun b , Yu Chen b , Xu Jianwen c
*
a
Department of Neurosurgery, Baoji Municipal Central Hospital, Baoji City, 721008, China Department of Anesthesiology, Beijing Tsinghua Changgung Hospital, No. 168, Li Tang Road, Changping District, Beijing 102218, China c Department of Anesthesiology, Zaozhuang Municipal Hospital, Zaozhuang, Shandong 277100, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 December 2018 Accepted 27 December 2018 Available online 9 January 2019
Ischemic stroke is the second most common cause of death, a major cause of acquired disability in adults. However, the pathogenesis that contributes to ischemic stroke has not been fully understood. Dualspecificity phosphatase 14 (DUSP14, also known as MKP6) is a MAP kinase phosphatase, playing important role in regulating various cellular processes, including oxidative stress and inflammation. However, its effects on cerebral ischemia/reperfusion (IR) are unclear. In the study, we found that DUSP14 expression was decreased responding to IR surgery. Over-expressing DUSP14 reduced the infarction volume of cerebral IR mice. Cognitive dysfunction was also improved in mice with DUSP14 overexpression. Promoting DUSP14 expression markedly reduced the activation of glial cells, as evidenced by the decreases in GFAP and Iba-1 expressions in mice with cerebral IR injury. In addition, inflammatory response induced by cerebral IR injury was inhibited in DUSP14 over-expressed mice, as proved by the reduced expression of tumor necrosis factor (TNF)-a and interleukin 1b (IL-1b). Furthermore, oxidative stress was markedly reduced by DUSP14 over-expression through elevating nuclear factor-erythroid 2 related factor 2 (Nrf-2) signaling pathway. Importantly, we found that DUSP14 could interact with Nrf-1, which thereby protected mice against cerebral IR injury. In vitro, we also found that repressing Nrf-2 expression abrogated DUSP14 over-expression-reduced inflammation and ROS generation. Consistent with the anti-inflammatory effect of DUSP14, reducing the production of reactive oxygen species (ROS) also down-regulated TNF-a and IL-1b expressions. Collectively, elevated DUSP14 alleviated brain damage from cerebral IR injury through Nrf-2-regulated anti-oxidant signaling pathway, and the restraining of inflammatory response. These results suggested that DUSP14 might be a potential therapeutic target to prevent ischemic stroke. © 2018 Published by Elsevier Inc.
Keywords: DUSP14 Cerebral IR injury Inflammation Oxidative stress Nrf-2
1. Introduction Ischemic stroke is the second most common cause of death, which is a leading cause of acquired disability in adults. Approximately 15 million people suffer from strokes each year, and about 5 million die as a result in the world [1,2]. Various basic and clinical experiments have suggested that neuroinflammation plays an essential role in the primary and secondary injury phases of stroke [3,4]. In addition, ROS-associated oxidative damage has been considered as the important pathogenesis of neuronal loss and the subsequent memory impairment following brain IR injury [5,6].
* Corresponding author. Department of Anesthesiology, Zaozhuang Municipal Hospital, Zaozhuang, Shandong 277100, China. E-mail address: [email protected] (X. Jianwen). https://doi.org/10.1016/j.bbrc.2018.12.170 0006-291X/© 2018 Published by Elsevier Inc.
Redox disturbance has been linked to the deteriorating clinical outcomes in acute ischemic stroke patients [7]. However, presently, the underlying molecular mechanisms remain unclear. Thus, further study is still required to better understand the pathogenesis of cerebral IR injury for developing effective therapeutic strategy against ischemic stroke progression. DUSP14, also known as MKP6, is an atypical DUSP [8]. DUSP14 contains the consensus C-terminal catalytic domain but lacks the N-terminal CH2 domain. DUSP14 could dephosphorylate mitogen activated protein kinases (MAPKs), including ERK1/2, p38 and c-Jun N-terminal kinase (JNK), which regulate various cellular responses, such as differentiation, stress response, proliferation and immune defense [9e12]. Oxidative stress leads to activation of JNK and p38 and directly affects DUSP-activity, either by oxidizing their catalytic cysteine or by elevating their degradation [13]. Phosphorylation of
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DUSP8 by JNK has been indicated under arsenite-triggered oxidative stress [14]. Herein, DUSP14 might also have anti-oxidant activity. In addition, DUSP14-knockout mice showed enhanced immune responses and were more susceptible to experimental autoimmune encephalomyelitis induction [15]. Over-expression of DUSP14 suppresses TNF- and IL-1-triggered nuclear factor-kB (NFkB) activation, subsequently limiting inflammatory response [16]. Oxidative stress, TNF-a and IL-1b have crucial roles in the progression of cerebral I/R injury [17]. Thus, DUSP14 might possess a key role in the progression of cerebral IR injury. In the study, we explored the effects of DUSP14 on the development of cerebral IR injury in mice with middle cerebral artery occlusion (MCAO). Our findings provided evidence that DUSP14 could be a protective modulator in cerebral IR injury by preventing inflammation and oxidative stress through interacting with Nrf-2 pathway.
2.3. Cognitive analysis The neurologic severity score (mNSS) was used to calculate neurological deficits 24 h after reperfusion as previously described [19]. The scoring system involved three tests, including motor, sensory and beam balance test. Here, 0 represented no deficit and 14 represented maximal deficits. Spatial learning and memory were assessed through Morris Water Maze (MWM) tests at day 22e28 after MCAO surgery according to previous study [20]. The escape latency to find the platform, the swim path length, the time spent in the target quadrant and platform crossovers were analyzed. Open field test was carried out to determine spontaneous activity and adaptability for at day 21 after MCAO by a computerized tracking system (Noldus EthoVision XT, shanghai, China) [21]. Total distance traveled, rearing activities, time spent and frequency of entry into the central area were recorded. 2.4. Co-immunoprecipitation analysis
2. Materials and methods 2.1. Animals Male C57BL/6J mice (weighting 20e25 g), purchased from Beijing Vital River Laboratories (Beijing, China) and housed under controlled conditions (temperature 23 ± 2 C; humidity 55e60%; 12-h-light-dark cycle) with access to food and water. All experiments were approved by Experimental Animal Ethic Committee of the South China Hospital Affiliated to University of South China (Hunan, China) and were implemented following National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication, revised in 1996). Vector construction was carried out by Cyagen Bioscience Inc (Guangzhou, China) to obtain DUSP14-encoding vector. The vector pLentiviruses LV (pLV) [Exp]EGFP was used as control. Virus was packaged through Lenti-XTM HTX packaging system (Clontech, USA). Purified lentiviruses were injected into the right lateral ventricle (dosage: 5 ml) and hippocampus (dosage: 3 ml) of mice with stereotaxic apparatus. Transfection efficiency was measured with RT-qPCR and western blot. Then, MCAO operation was conducted 2 weeks after transfection. All mice were divided into 4 groups: 1) Sham group; 2) MACO group; 3) MCAOþLV-GFP group and 4) MCAOþLV-DUSP14 group. Transient focal cerebral ischemia was performed through intraluminal MCAO surgery according to previous study [18]. After occlusion for 90 min, monofilament was withdrawn for reperfusion. A heating pad was used to keep the body temperature during the surgery. The sham group of mice was subjected to the same procedures except that MCA was not occluded after neck incision.
2.2. Microglia cells and culture Mouse microglial BV2 cells were obtained from the Cell Culture Center at the Institute of Basic Medical Sciences of Chinese Academy of Medical Sciences (Beijing, China) and were incubated in DMEM (GIBCO, USA) with 10% heat-inactivated fetal bovine serum (FBS, GIBCO), 2 mM glutamine (Gbico), 100 mg/ml streptomycin and 100 U/mL penicillin at 37 C with 5% CO2. The DUSP14 plasmid (pcDUSP14) or negative control plasmid (NC-DNA) were constructed by Cobioer (Nanjing, China) and transfected into BV2 cells for 24 h with lipofectamine 2000 (Invitrogen, USA). Nrf-2 siRNA and corresponding negative siRNA (siNC) were purchased from Santa Cruz (USA). Lipopolysaccharides (LPS), ROS scavenger of N-acetylcysteine (NAC) and Nrf-2 activator of tert-butylhydroquinone (tBHQ) were purchased from Sigma Aldrich (USA). Nrf-2 inhibitor, ML385, was purchased from MedChemExpress (USA).
Immunoprecipitation was performed with a Pierce Classic IP kit (Pierce Biotechnology, Inc., USA) according to the manufacturer's instructions and analyzed through western blot method. 2.5. Cerebral infarct volume, brain edema and neurological deficit measurements 2, 3, 5-triphenyltetrazoliumchloride (TTC, Sigma Aldrich, USA) staining was performed 24 h after reperfusion to calculate infarct volume. The 1-mm-thick brain slices were stained with 2% TTC for 15 min in dark at 37 C, followed by fixing with 4% paraformaldehyde overnight at 4 C. Then, slices were scanned, and the infarct volume and edema ratio were analyzed as reported [18]. 2.6. Immunofluorescent analysis Frozen brain sections and LPS-incubated BV2 cells were subjected to immunofluorescence staining according to previous study [22]. Primary antibodies used in immunofluorescence analysis were GFAP (sc-33673, Santa Cruz, USA), Iba-1 (sc-32725, Santa Cruz), DUSP14 (ab168969, Abcam, USA), 8-OHdG (ab48508, Abcam), Nrf-2 (ab31163, Abcam) and DUSP14 (ab168969, Abcam) at 1:100 dilutions. Alexa Fluor 488- or 594-labeled anti-rabbit or -goat secondary antibodies (Beyotime) and DAPI (Sigma Aldrich) to identify cell nuclei. Images were captured with a fluorescent microscope. 2.7. Statistical analysis Data analysis was conducted using Graphpad Prism software (USA). Data were presented as mean ± standard error of mean (SEM). The one-way analysis of variance (ANOVA) and t-test were conducted among multi-groups or between two groups. P < 0.05 was served as statistical significance. 3. Results 3.1. DUSP14 is down-regulated in the infarcted area of mice following ischemic stroke As shown in Fig. 1AeD, DUSP14 expression was markedly downregulated from 2 h to 168 h after reperfusion in ischemic core and ischemic penumbra. To further investigate the effects of DUSP14 on ischemic stroke, LV-GFP and LV-DUSP14 were subjected to the hippocampus and right lateral ventricle of mice. Cells were doublelabeled with DUSP14 combing with Iba1 (microglial marker). The
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Fig. 1. DUSP14 is down-regulated in the infarcted area of mice following ischemic stroke. The mRNA and protein expression pattern and time course of DUSP14 in (A,B) ischemic core and (C,D) ischemic penumbra. (E) Double-immunofluorescence staining for microglial (Iba1), and DUSP14 expression in the ischemic penumbra area after MCAO operation. (F,G) LV-GFP and LV-DUSP14 were injected into the right hippocampus and right lateral ventricle of mice 2 weeks before MCAO operation. RT-qPCR and western blot analysis of DUSP14 in brain extracts of the described groups. (H) RT-qPCR analysis of pro-inflammatory cytokines (IL-b, IL-6, TNF-a and COX2) in brain extracts before MCAO treatment. (I) Representative image of TTC staining 1 day after reperfusion. Quantification of (J) infarct volume and (K) edema area. (L) Calculation of neurological score. Data are presented as the means ± SEM (n ¼ 6, for each group). þP < 0.05, þþP < 0.01 and þþþP < 0.001 versus Sham group; **P < 0.01 versus MCAO group.
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DUSP14 was down-regulated in microglia (Iba1/DUSP14, Fig. 1E) 24 h after MCAO operation, as evidenced by the weaker fluorescent intensity (green). Thus, DUSP14 expression was detected in microglia of the brain. Microglia cells are active contributors to the inflammation in ischemic stroke [23,24]. DUSP14 expression was significantly up-regulated after LV-DUSP14 transfection (Fig. 1F and G). No difference was observed in the change of pro-inflammatory cytokines (IL-b, IL-6, TNF-a and COX2) before MCAO operation, indicating that lentivirus injection did not influence inflammatory response (Fig. 1H). TTC analysis suggested that DUSP14 overexpression markedly reduced the infarction volume, and brain edema volume when compared with MCAO group of mice (Fig. 1IeK). Furthermore, neurological deficits in MCAO mice were significantly alleviated by LV-DUSP14 transfection (Fig. 1L). 3.2. Over-expression of DUSP14 improves cerebral IR-induced cognitive deficits In this regard, the cognitive function was measured using MWM. Longer distance and time spent to find the platform were observed in MCAO mice, which were attenuated by DUSP14 overexpression (Fig. 2AeC). MCAO-reduced platform crossovers and time spent in target quadrant were significantly rescued due to DUSP14 over-expression (Fig. 2A, D and E). OFT analysis suggested that the spontaneous locomotion in center, periphery and the total locomotion reduced by MCAO were markedly improved by DUSP14 over-expression (Fig. F and G). In addition, the entries into the center, the time spent in center and the rearing activities were markedly decreased by MCAO, while being alleviated in DUSP14over-expressed mice (Fig. 2F, H-J). 3.3. DUSP14 up-regulation inhibits inflammation and oxidative stress in brain of cerebral IR mice GFAP and Iba-1 are essential gliosis markers, playing crucial role in promoting cerebral IR injury [25,26]. In the peri-infarct region, MCAO led to a significant increase of GFAP and Iba-1 expression, which was, however, down-regulated by DUSP14 over-expression (Fig. 3A and B). In addition, IL-b and TNF-a expression in brain extracts of the peri-infarct region was markedly up-regulated by MCAO. The process was reversed due to LV-DUSP transfection (Fig. 3C). SOD activities in the peri-infarct region were decreased in MCAO mice, whereas MDA levels were increased, which were markedly alleviated by DUSP14 over-expression (Fig. 3D). Consistently, NADPH oxidase activity and ROS production in the periinfarct region were obviously up-regulated by MCAO, whereas being reduced in DUSP14-over-expressed mice (Fig. 3E and F). Immunofluorescent analysis suggested that MCAO-induced 8OHdG expression was diminished in DUSP14 over-expressed mice (Fig. 3G). Moreover, western blot analysis suggested that SOD2, HO1, NQO-1 and Nrf-2 expressions reduced by MCAO were rescued by LV-DUSP14 transfection. In contrast, expression levels of Keap-1 and XO, as Nrf-2 suppressor and major source of ROS production [27,28], were increased by MCAO, which were markedly decreased by DUSP14 over-expression (Fig. 3H). Immunofluorescent analysis showed weaker Nrf-2 fluorescent intensity in MCAO group of mice, which was contrast to Iba-1; however, DUSP14 over-expression promoted Nrf-2 fluorescence in the peri-infarct region of mice along with reduced Iba-1, suggesting that Nrf-2 accumulation was detected in microglia (Fig. 3I). 3.4. DUSP14 evokes Nrf-2-meditated anti-oxidative stress and inflammatory response Co-immunoprecipitation
assay
indicated
an
interaction
between DUSP14 and Nrf-2 (Fig. 4A). In vitro, LPS stimulation decreased DUSP14 and Nrf-2 expressions in microglia cells (Fig. 4B). Next, DUSP14 was over-expressed in vitro by transfecting DUSP14 pcDNA (Fig. 4C). LPS-reduced Nrf-2 and DUSP14 expressions was restored by DUSP14 over-expression (Fig. 4D). Promoting DUSP14 expression rescued Nrf-2, NQO-1 and HO-1 expressions in LPSstimulated microglia, which were further down-regulated by ML385, Nrf-2 inhibitor. An opposite expression change was observed in Keap-1 (Fig. 4E). In addition, ROS, NADPH oxidase activity and MDA levels induced by LPS were alleviated by DUSP14 over-expression, while being recovered in BV2 cells pre-treated with Nrf-2 inhibitor of ML385 or with Nrf-2 knockdown (Fig. 4F and G, Supplementary Figs. 1AeC). Suppressing Nrf-2 expression abolished DUSP14 over-expression-reduced IL-b and TNF-a expression in LPS-treated microglial cells (Fig. 4H and Supplementary Fig. 1D). Then, both over-expressing DUSP14 and NAC pre-treatment decreased LPS-induced ROS generation, IL-b and TNF-a expression, further indicating that DUSP14 exhibited suppressive role in oxidative stress and subsequently suppressing inflammatory response (Fig. 4I and J). 4. Discussion Ischemic stroke is an essential cause of death and acquired disability in adults, which is linked to various risk factors, such as hypertension, atherosclerosis, thrombosis, diabetes, and even smoking [1,3,29]. Increasing studies regarded to ischemic stroke have been conducted in recent decades [5e7,30]. Unfortunately, the pathogenesis of ischemic stroke has not been fully understood. In our study, DUSP14 expression was found to be down-regulated in ischemic core and ischemic penumbra of mice with MCAO surgery. These observations indicated that DUSP14 might have an essential role in cerebral IR injury. Furthermore, DUSP14 over-expressed mice exhibited reduced infarction area, and showed better cognitive performance after MCAO operation. Attenuated inflammatory response and oxidative stress were observed in mice following MCAO. These findings indicated that DUSP14 could attenuate brain injury via the regulation of inflammatory response and oxidative stress after cerebral IR injury. Consistent with the in vivo data, in vitro cell culture experiments suggested a protective function of DUSP14 in LPS-treated microglia, which was tightly associated with Nrf-2 pathway activation. These findings elucidated that DUSP14 could be a target to develop effective therapeutic strategy against ischemic stroke. The inflammatory response has an essential role in the pathogenesis of cerebral I/R injury, and suppression of inflammation could be an effective strategy for the prevention of, or intervention in, this disease [31,32]. Pro-inflammatory cytokines could promote various signaling pathways to regulate inflammation. In addition, oxidative stress has been identified as one of the key contributing factors in the pathogenesis of brain damage [5e7,33]. Excessive production of ROS enhances brain injury post I/R injury via various molecular mechanisms, such as inflammation, apoptosis, and brain-blood barrier disruption [34e36]. Therefore, suppressing inflammation and reducing the production of ROS, or repairing oxidative damage are the significant strategies to combat against cerebral IR injury. Studies have suggested that DUSP14 plays a critical role in modulating inflammation and oxidative stress [16,37]. DUSP14 targets MAPKs to regulate the functions of immune cells [38]. DUSP14 negatively regulates TNF-a- or IL-1b-induced NFkB activation, restraining inflammatory response in hepatic IR injury [16]. DUSP14 knockout accelerated oxidative stress in myocardial IR injury [37]. In our study, we also found that overexpressing DUSP14 down-regulated TNF-a and IL-1b expression. Therefore, repressing inflammatory response was a major
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Fig. 2. Over-expression of DUSP14 improves cerebral IR-induced cognitive deficits. (A) The swim path traces of mice in hidden platform test, including learning (up) and memory (bottom) traces. Calculation of (B) the swim path and (C) escape latency from day 23 to day 27 after MCAO surgery. Recording of (D) platform crossovers and (E) the time spent in the target quadrant. (F) Tracks of movements of mice from the indicated groups for 30 min in an open field box. (G) Cumulative distance traveled in each zone. (H) The frequency entry into the center area, (I) the time spent and (J) rearing activities during the 30 min session in the open field test. Data are presented as the means ± SEM (n ¼ 6, for each group). þþP < 0.01 versus Sham group; *P < 0.05 versus MCAO group.
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Fig. 3. DUSP14 up-regulation inhibits inflammation and oxidative stress in brain of cerebral IR mice. (A,B) Immunofluorescent analysis of GFAP and Iba-1 in the peri-infarct region of mice, and the quantified analysis of GFAP and Iba-1 expressions. (C) RT-qPCR analysis of IL-b and TNF-a in brain extracts. Measurements of (D) SOD activity, MDA levels, (E) NADPH oxidase activity and (F) relative ROS production in the peri-infarct region of mice. (G) Immunofluorescent analysis of 8-OHdG in the peri-infarct region of mice, and the quantified analysis of 8-OHdG expressions. (H) Western blot analysis of SOD2, HO-1, NQO-1, Nrf-2, Keap-1 and XO protein expressions in the peri-infarct region of mice. (I) Immunofluorescent analysis of Iba-1 and Nrf-2 in the peri-infarct region of mice. Data are presented as the means ± SEM (n ¼ 6, for each group). þþP < 0.01 and þþþP < 0.001 versus Sham group; *P < 0.05 and **P < 0.01 versus MCAO group.
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Fig. 4. DUSP14 evokes Nrf-2-meditated anti-oxidative stress and inflammatory response. (A) DUSP14 binds to Nrf-2. Brain lysates were immunoprecipitated with anti-DUSP14 (up) or anti-Nrf-2 (bottom) antibodies. Next, total lysates and immunoprecipitates were calculated using immunoblotting analysis with anti-DUSP14 and anti-Nrf-2. (B) Micorglia cells were treated with 100 ng/ml LPS for 24 h, followed by western blot analysis of DUSP14 and Nrf-2. (C) Microglia cells were transfected with pcDNA DUSP14 or the negative control (NC) pcDNA to over-express (OE) DUSP14 expressions. Western blot analysis was performed to determine the efficacy of transfection. (D) Microglia cells were transfected with pcDNA DUSP14 for 24 h, followed by LPS (100 ng/ml) treatment for another 24 h, and then cells were harvested for immunofluorescent analysis of Nrf-2 and DUSP14. Microglia cells were pre-treated with Nrf-2 inhibitor ML385 (10 mM) or activator tBHQ (10 mM) for 2 h. Then, cells were subjected to pcDNA DUSP14 transfection for another 24 h. Finally, LPS was challenged to cells for an additional 24 h. (E) Western blot analysis of Nrf-2, NQO-1, HO-1 and Keap-1. (F) DCF analysis for ROS production and calculation of NADPH oxidase activity. (G) MDA levels in cells treated as indicated. (H) RT-qPCR analysis of IL-b and TNF-a in cells. Microglia cells were pre-treated with or without NAC (5 mM) for 2 h, followed by pcDNA DUSP14 or negative control pcDNA transfection for another 24 h. Finally, LPS was subjected to cells for an additional 24 h. (I) DCF analysis for ROS production. (J) RT-qPCR analysis of IL-b and TNF-a in cells. (K) Proposed schematic diagram of Nrf-2-meditated anti-oxidative and anti-inflammatory response regulated by DUSP14. Data are presented as the means ± SEM (n ¼ 6, for each group).
pathology by which DUSP14 protected against cerebral IR injury development. Nrf-2 is a master regulatory element meditating a diverse set of
antioxidant defense machineries. Nrf-2 regulates more than 200 genes that encode cytoprotective phase II detoxification and antioxidant enzymes [39]. NADPH oxidase consisting of membrane-
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bound subunits is an essential source of ROS [40]. 8-OHdG is a significant marker of DNA oxidative stress [39]. In the study, we found that MCAO-induced oxidative stress was attenuated by DUSP14 over-expression, as evidenced by the reduced MDA levels, ROS generation, NADPH oxidase and 8-OHdG expressions. Moreover, the increased anti-oxidants, including SOD2, HO-1 and NQO1, regulated by the activation of Nrf-2 were observed in mice transfected with LV-DUSP14. In contrast, the expression of Keap-1 and XO-1, significant molecules in promoting the generation of ROS, was markedly decreased in DUSP14 over-expressed mice. Intriguingly, we found that Nrf-2 was a critical target of DUSP14. Consistently, suppressing Nrf-2 abrogated DUSP14 overexpression-reduced inflammatory response and ROS production. Increased production of ROS could stimulate the NF-kB pathway, leading to the release of pro-inflammatory cytokines and subsequent inflammation [41,43]. Also, we found that reducing ROS with its scavenger of NAC markedly decreased TNF-a and IL-1b expressions. Thus, in our study, Nrf-2 anti-inflammatory function was ROS-dependent. Consistently, Nrf-2 has been indicated to inhibit inflammation through ROS-dependent and/or -independent manner [44,45]. Therefore, DUSP14 could interact with Nrf-2 to restrain oxidative stress and inflammation, subsequently attenuating cerebral IR injury (Fig. 4K). However, further study is still required to determine how DUSP14 interacts with Nrf-2, and if anti-inflammatory function of Nrf-2 regulated by DUSP14 could be ROS-independent. In summary, the study indicated that DUSP14 protected cerebral IR injury. The neuroprotective effects of DUSP14 were regulated by its interaction with Nrf-2 to inhibit oxidative stress and inflammatory response, as well as attenuate cognitive dysfunction. The results encourages further study on DUSP14 to prevent ischemic stoke or even other neurodegenerative disease. Acknowledgments
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This work was supported by Fundamental Research Funds for the Central Universities (grant number: 06123143497).
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Appendix A. Supplementary data
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Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.12.170.
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