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NF-κB and MAPKs signaling pathways
Biochemical and Biophysical Research Communications xxx (2018) 1e8
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HDAC9 promotes brain ischemic injury by provoking IkBa/NF-kB and MAPKs signaling pathways Shan Lu a, Hang Li b, Kai Li a, Xiao-Di Fan a, * a b
Department of Anesthesiology, China Japan Union Hospital of Jilin University, Jilin, 130033, China Department of Hepatobiliary and Pancreas Surgery, China Japan Union Hospital of Jilin University, Jilin, 130033, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 June 2018 Accepted 9 July 2018 Available online xxx
Ischemic stroke is an acute cerebrovascular disease due to poor blood flow to the brain. Nevertheless, there is still no effective therapy for it and the pathology contributing to ischemic stroke is not fully understood. Histone Deacetylase 9 (HDAC9) is a class IIa chromatin-modifying enzyme. HDAC9 gene region is a leading risk locus for large artery atherosclerotic stroke. However, the mechanisms linking HDAC9 to ischemic remain elusive. In the study, we attempted to explore HDAC9-associated inflammatory response using the wild type (WT) and HDAC9-knockout (KO) mice with brain ischemic injury. The results indicated that WT mice with ischemia brain exhibited higher expression levels of HDAC9. HDAC9 depletion resulted in a decreased infarct volume and an improved neurological function in mice after ischemic reperfusion (I/R) injury. I/R injury markedly enhanced GFAP and Iba-1 expressions in cortex and HDAC9 knockout significantly reversed this up-regulation. Loss of HDAC9 inhibited the release of inducible NO-synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin 1b (IL-1b), IL-6, tumor necrosis factor-a (TNF-a), and IL-18 in cortex, hippocampus and hypothalamus of mice with I/R injury, which occurred at the transcription levels. Furthermore, the inhibitory actions of HDAC9 deficiency were associated with the down-regulation of phosphorylated-IkBa, phosphorylated-nuclear factor-kappa B (NF-kB), and p-mitogen-activated protein kinases (MAPKs), including phosphorylated-p38, phosphorylated-extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphorylated-c-Jun N-terminal kinase (JNK). Importantly, the in vitro study indicated that HDAC9 inhibition-reduced inflammation and activation of IkBa/NF-kB were restored by promoting MAPKs activity in LPS-stimulated cells. Our findings suggest that HDAC9 inhibition showed neuroprotective effects on ischemic stroke by restraining inflammation, which might help develop new and effective strategies for the therapeutic interventions in ischemic stroke. © 2018 Published by Elsevier Inc.
Keywords: Ischemic stroke HDAC9 Inflammation IkBa/NF-kB MAPKs
1. Introduction Stroke is an acute cerebrovascular disease due to poor blood flow to the brain [1]. Increasing studies suggested that clinical trials have failed to indicate a benefit in treating stroke, illustrating that the physiopathological mechanisms of ischemic stroke are far more complex realized previously [2,3]. In addition to the insufficient oxygen and glucose delivery, other detrimental factors, including acidotoxicity, excitotoxicity, nitrative stress and particularly, postischemic neuroinflammation, result in the last outcome of
* Corresponding author. Department of Anesthesiology, China Japan Union Hospital of Jilin University, Jilin, 130033, China. E-mail address: [email protected] (X.-D. Fan).
ischemic stroke [4,5]. Furthermore, the circulating factors secreted by brain may also influence the pathogenesis of stroke [6]. Histone deacetylases (HDACs) are suggested as promising therapeutic targets for preventing neurodegenerative diseases and stroke through epigenetic and non-epigenetic molecular mechanisms [7]. Various HDAC inhibitors exhibit beneficial effects in a variety of brain disorders, involving ischemic stroke [8]. HDAC9 is a class IIa chromatin-modifying enzyme and regulates diverse normal and abnormal physiological functions [9,10]. Class IIa HDACs exert tissue specific expression and lead to differentiation and development [11]. HDAC9 is over-expressed in striated muscle, where it functions as a negative modulator of differentiation and growth [12]. In brain, HDAC9 is associated with neuronal morphogenesis. Accumulating evidence demonstrate that HDAC9 is an essential target for neuroprotection. For example, HDAC9 is
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Please cite this article in press as: S. Lu, et al., HDAC9 promotes brain ischemic injury by provoking IkBa/NF-kB and MAPKs signaling pathways, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.07.043
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expressed in the brain to meditate neocortical neuronal development [13]. Recently, though studies have identified a variant in HDAC9 related to large-vessel ischaemic stroke and further indicated that HDAC9 variant linked to ischaemic stroke enhances risk through enhancing carotid atherosclerosis, the molecular mechanisms of HDAC9 in regulating ischaemic stroke are not fully understood. In the present study, the animal model with brain I/R injury in WT and HDAC9-KO mice was established in vivo. The results indicated that HDAC9-KO mice exerted a decreased infarct volume and an improved neurological function, along with a significant reduced expression of pro-inflammatory cytokines. The process was associated with the inactivation of IkBa/NF-kB and MAPKs signaling pathways in HDAC9-KO mice with experimental ischaemic stroke. We also used the in vitro cell cultures through LPS treatment. Of note, we found that promoting MAPKs activity abrogated HDAC9 inhibition-induced inflammatory response in LPS-stimulated cells. Thus, targeting HDAC9 could be an effective strategy for combating ischaemic stroke. 2. Materials and methods 2.1. Animals and treatments Male 8e12 weeks old wild type (WT) C57BL/6 or HDAC9knockout (KO) mice were purchased from Laboratory Animal Center, Jilin University and Cyagen BIOSCIENCES (Guangzhou, China), respectively. Animals were housed in a facility with controlled temperature (23 ± 2 C) and lighting (08: 00 to 20: 00 h), with free access to tap water. All animal experiments were approved by the Animal Ethics Committee of China Japan Union Hospital of Jilin University and were performed in compliance with the principles stated in the Guide for the Care and Use of Laboratory Animals, NIH Publication, 1996 edition. All mice were divided into 4 groups, including 1) WT/Sham; 2) KO/Sham; 3) WT/I/R and 4) KO/I/ R. Focal cerebral ischemia and reperfusion (I/R) was induced by intraluminal middle cerebral artery occlusion (MCAO) as described previously [14]. Before operation, mice were anesthetized using pentobarbital sodium (30 mg/kg). Next, mice were placed on a heating panel, maintaining at 37 C during operation. Then, a midline incision was made to expose the right common carotid artery, external carotid artery, and internal carotid artery. MCAO was triggered through advancing a 6/0 surgical nylon monofilament using a rounded tip into the lumen of internal carotid artery carefully from the right external carotid artery until the rounded tip blocked the origin of the middle cerebral artery (MCA). After 1 h of MCAO, the surgical nylon monofilament was retracted to allow reperfusion for differ rent durations (3, 6, 12, 24 and 48 h). The sham-operated group of mice was treated with similar operations without the surgical nylon monofilament insertion. 2.2. Cells and culture PC12 and BV2 cells were obtained from the Chinese Academy of Medical Sciences (Beijing, China). Astrocytes (AST) were isolated from mice using the methods as described previously [15]. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) medium (Gbico, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), 2 mM glutamine (Gbico), 100 mg/ml streptomycin (Gbico) and 100 U/ml penicillin (Gbico) at 37 C with 5% CO2. HDAC9 siRNA and negative control siRNA (NC) sequences were purchased from Santa Cruz (USA), which were transfected to BV2 cells using lipofectamine 2000 reagent (Invitrogen, USA). P79350 (p38 activator) was purchased from Calbiochem (USA). Lysophosphatidic Acid (LPA) was obtained from Santa Cruz (USA).
Anisomycin (ANI, JNK activator) was purchased from Beyotime (Nanjing, China).
2.3. Cognitive function analysis Motion function and locomotor activity were measured by open-field test as previously described [14]. The average velocity and time spent in the center square or side was recorded. To calculate motor coordination and learning before and after I/R operation, an accelerating rotarod test was performed (SD Instruments, USA) as previously described [16]. Tests were carried out at 1, 7, 14, 21, 28 days after I/R. The morris water maze (MWM) task included a circle water tank filled with opaque water and a round platform, which was submerged 1 cm beneath the surface of water at the center of the second quadrant. Hidden platform training was performed within 90 s at 6 consecutive days with four trials each session. Escape latency to search for the hidden platform, swimming paths, swimming velocity were recorded. Data was analyzed using a TM-Vision video-tracking system (Chengdu Taimeng Software Co. LTD, China).
2.4. 5-Triphenyltetrazolium chloride (TTC) staining and infarct volume measurement Mice were euthanized. Brain tissues were removed to froze at 25 C for 20 min after 24 h of reperfusion or sham operations. Then, the brains were sliced into serial coronal sections (2 mm apart) and stained with 2% TTC (Sigma-Aldrich, USA) for 30 min at 37 C. Infarction volume was the sum of all lesion areas multiplied by slice thickness using Image J software (National Institutes of Health, USA). Neurological status was calculated based on a neurologic deficit score following ischemia at 24 h reperfusion [17].
2.5. RNA extraction and qRT-PCR analysis Real-time quantitative PCR was performed as described previously [18]. Total RNA for real-time quantitative RT-PCR was isolated from tissues or cells using RNAiso Reagent (TaKaRa, Japan) and reverse transcripted into the single strand cDNA. The primers for targeting genes were listed in Supplementary Table 1. Gene expression of GAPDH was used for control. Quantification of mRNA was carried out using the ABI Prism 7500 (Applied Biosystems, USA) by PrimeScript™ RT-PCR Kit (TaKaRa).
2.6. Western blot assay Cortex, hippocampus and hypothalamus, and cells were lysed in modified RIPA buffer (10 mM Na2HPO4 pH 7.2, 150 mM NaCl, 1% Nonidet P-40 [NP-40], 0.5% Na-deoxycholate) containing protease inhibitors (2 mM PMSF, Beyotime). Total proteins (20e50 mg) were separated by SDS-PAGE, and transferred to PVDF (Millipore, USA) membranes. Membranes were then incubated with primary antibodies, followed by incubation with horseradish peroxidase (HRP)conjugated secondary antibodies. Protein bands were then visualized with an enhanced chemiluminescence system (ECL, Beyotime). Th eprimary anti-bodies used in the present study were shown as followings: HDAC9 (1:500, sc-398003, Santa Cruz); p-IkBa (1:1000, #2859, CST, USA); IkBa (1:1000, #4814, CST); p-NF-kB/p65 (1:1000, #3033, CST); NF-kB/p65 (1:1000, #8242, CST); p-p38 (1:1000, #4511, CST); p-ERK1/2 (1:1000, #9101, CST); p-JNK (1:1000, #9255, CST); p38 (1:1000, #8690, CST); ERK1/2 (1:1000, #4695, CST); JNK (1:1000, #9252, CST) and GAPDH (1:1000, #5174, CST).
Please cite this article in press as: S. Lu, et al., HDAC9 promotes brain ischemic injury by provoking IkBa/NF-kB and MAPKs signaling pathways, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.07.043
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2.7. Immunofluorescence (IF) staining Immunofluorescence were performed as described previously [19]. Cultured cells or brain sections were fixed in 4% paraformaldehyde, blocked by 10% normal goat serum (Solarbio, China), and incubated in specific primary antibodies as follows: HDAC9 (1:100, Santa Cruz), NeuN (1:200, #24307, CST), GFAP (1:200, ab7260, Abcam, USA), and Iba-1 (1:200, ab5076, Abcam). After being washed three times with PBS, the sections and cells were incubated with Alexa 488-conjugated secondary antibodies (Abcam). Images were obtained by fluorescence microscope with a digital camera.
2.8. Statistical analysis Data are expressed as mean ± SEM. Differences were evaluated by one-tailed Student's t-test or ANOVA followed by Tukey's posthoc test with GraphPad Prism 5.0 version. Statistical significance was set at P < 0.05.
3. Results 3.1. HDAC9 expression levels were increased in the ischemic brain As shown in Fig. 1A and B, HDAC9 mRNA and protein levels in brain penumbra area of I/R mice was significantly higher than that in non-ischemia brain area, which reached peak at 24 h post I/R operation. Expression of HDAC9 in cortex of brain penumbra area was also enhanced at 24 h post I/R operation via IF analysis (Fig. 1C). Moreover, HDAC9 mRNA and protein levels were also markedly increased by LPS treatment in PC12, BV2 and AST cells (Fig. 1D and E). IF analysis showed that LPS exposure enhanced the number of HDAC9 positive cells in vitro (Fig. 1F). These results indicated that brain HDAC9 was up-regulated by experimental cerebral ischemia.
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3.2. Loss of HDAC9 protects against I/R-induced ischemic brain injury in vivo There was no significant difference observed in the motion function and locomotor activity in each group of mice before I/R operation (Fig. 2A). After I/R surgery, WT mice showed a lower velocity and spent less time in the center area of the apparatus than WT/Sham mice, which was restored by HDAC9-knockout, indicating an alleviated locomotor activity and anxiety-like behavior (Fig. 2B). It was also noteworthy that loss of HDAC9 led to better recovery of motor coordination in rotarod test throughout the 4week observation period after I/R operation (Fig. 2C). MWM analysis suggested that long-term learning and memory deficits were apparently improved in HDAC9-knockout mice after I/R, as manifested by a reduced latency to find the hidden platform (Fig. 2D). TTC staining indicated that HDAC9 deletion significantly downregulated the cerebral infarct volume compared with the volumes observed in the WT mice after I/R operation (Fig. 2E and F). In line with the smaller infarct sizes, HDAC9-knockout resulted in prominent improvements in neurological function 24 h after I/R injury (Fig. 2G). As calculated by NeuN staining, I/R operation for 24 h induced severe neuronal cell death in the cortex, and HDAC9knockout significantly improved neuronal survival (Fig. 2H). In contrast, a significant increase of GFAP and Iba-1 expressions was observed in the cortex of I/R mice, which was markedly reversed by the loss of HDAC9 (Fig. 2I). 3.3. HDAC9 ablation down-regulates inflammation in brain of mice subjected to I/R 24 h I/R operation significantly elevated mRNA expression levels of iNOS, COX-2, IL-1b, IL-6, TNF-a, and IL-18 in cortex, hippocampus and hypothalamus of mice, which was partly blocked by HDAC9deficiency (Fig. 3AeC). Also, HDAC9-knockout partly inhibited the p-IkBa and p-NF-kB protein up-regulation in infarcted brain tissue samples (cortex, hippocampus and hypothalamus) of mice (Fig. 3DeF). Similar phenotypes were observed in MAPKs. The
Fig. 1. HDAC9 expression levels were increased in the ischemic brain. (A) RT-qPCR and (B) western blotting analysis for HDAC9 in the ischemic brain tissues 3, 6, 12, 24 and 48 h after focal cerebral ischemia. (C) IF staining of HDAC9 in cortex of mice subjected to I/R 24 h (D,E,F) RT-qPCR, western blotting and IF staining of HDAC9 in PC12, BV2 and AST cells stimulated by 80 ng/ml of LPS for 24 h. Values were mean ± SEM (n ¼ 6e9). **P < 0.01 and ***P < 0.001 versus Sham or Ctrl group.
Please cite this article in press as: S. Lu, et al., HDAC9 promotes brain ischemic injury by provoking IkBa/NF-kB and MAPKs signaling pathways, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.07.043
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Fig. 2. Loss of HDAC9 protects against I/R-induced ischemic brain damage in vivo. The motion function and locomotor activity of the WT mice and KO mice were measured by open-field test (A) before and (B) after 24 h of I/R operation. Quantifications of average velocity, time spent in the side areas and in the center square were exhibited for 10 min during each session. (C) The performance on rotarod of mice was analyzed before I/R operation and throughout the 4-week period after 24 h of I/R operation. (D) The performance on MWM mice was analyzed 3 days after 24 h of I/R operation. Hidden platform training was performed within 90 s at 6 consecutive days. Curve lines exhibits the escape latency to find the hidden platform. (E) Brain infarction was determined by TTC staining on Day 3 after 24 h of I/R operation. (F) Quantification of infarct volume. (G) Neurologic deficit score of mice subjected to I/R 24 h or sham-treated mice. (H) NeuN and (I) GFAP and Iba-1 expression levels in cortex of mice subjected to I/R 24 h were measured using IF staining. Values were mean ± SEM (n ¼ 6e9). *P < 0.05, **P < 0.01 and ***P < 0.001 versus WT/Sham group; þP < 0.05 and þþP < 0.01 versus WT/I/R group.
protein levels of p-p38, p-ERK1/2 and p-JNK in cortex, hippocampus and hypothalamus of I/R mice were increased, while HDAC9 deletion partly depressed these changes (Fig. 3GeI).
3.4. HDAC9 knockdown alleviates LPS-induced inflammation in BV2 cells partially by MAPKs pathway To further explore the effects of HDAC9 on brain ischemic injury, the in vitro study was carried out using BV2 cells with HDAC9 knockdown by transfecting HDAC9 siRNA sequence. The transfection efficacy was confirmed using western blot analysis (Fig. 4A). As shown in Fig. 4B, LPS treatment markedly up-regulated the mRNA expression levels of iNOS, COX-2, IL-1b, IL-6, TNF-a, and IL18 in BV2 cells, which were significantly repressed by HDAC9silence. The expression levels of p-IkBa and p-NF-kB stimulated by LPS were evidently reduced by HDAC9 knockdown in BV2 cells (Fig. 4C). Treatment of HDAC9-knockdown significantly attenuated the protein levels of p-p38, p-ERK1/2 and p-JNK in LPS-treated cells (Fig. 4D). Further, the activation of p38, ERK1/2 and JNK was considerably increased by P79350, LPA and ANI treatment, respectively (Fig. 4E). Moreover, we found that the loss of HDAC9induced reduction of iNOS, COX-2, IL-1b, IL-6, TNF-a, and IL-18 in LPS-treated BV2 cells was promoted by the pre-treatment of P79350, LPA or ANI (Fig. 4F). Consistently, HDAC9 knockdowntriggered inactivation of IkBa and NF-kB was recovered by the pre-treatment with P79350, LPA or ANI in LPS-stimulated cells (Fig. 4G). Therefore, the findings here demonstrated that HDAC9 suppression-attenuated inflammation could be impeded by promoting MAPKs activation in LPS-incubated cells.
4. Discussion HDAC9 is a member of class II HDACs, modulating a wide variety of normal and abnormal physiological functions [9,10]. Recently, through studies have implied the importance of HDAC-regulated epigenetic processes in the progression of ischemic stroke, the molecular events to trigger cerebral injury are not fully explained. Accumulating evidence has indicated that HDAC9 is linked to neuronal physiology and pathology [11e14]. In the present study, we provided the evidence that loss of HDAC9 ameliorated cerebral ischemia injury in vivo and in vitro. Firstly, we found that brain HDAC9 was up-regulated by ischemic insult. HDAC9 knockout showed neuroprotection effects, as evidenced by the reduced brain infarcted volume and neurological deficit score. Further, neuronal death was suppressed by HDAC9 deletion. Glial cells activity was also blocked in HDAC9-KO mice. HDAC9 ablation inhibited NF-kB and MAPKs signaling activation during ischemic stroke and therefore neuroinflammation (iNOS, COX-2, IL-1b, IL-18, TNF-a and IL-6 production). At last, promoting AMPKs activity compromised the neuroprotection of HDAC9-suppression, and consequently enhancing inflammatory response. The brain in response to ischemic damage shows an acute and prolonged inflammatory activation, characterized by both rapid activation of resident microglia and subsequent infiltration of different types of inflammatory cells into the ischemic area [20,21]. The pro-inflammatory factors, including IL-18, IL-1b, TNF-a and IL6 are secreted by the activated microglia or other inflammatory cells [22,23]. In patients with ischemic stroke, IL-6 and TNF-a levels in plasma and cerebrospinal fluid are related to early clinical deterioration, suggesting pro-inflammatory cytokines and early
Please cite this article in press as: S. Lu, et al., HDAC9 promotes brain ischemic injury by provoking IkBa/NF-kB and MAPKs signaling pathways, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.07.043
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Fig. 3. HDAC9 ablation down-regulates inflammation in brain of mice subjected to I/R 24 h. RT-qPCR analysis of iNOS, COX-2, IL-1b, IL-6, TNF-a, and IL-18 in (A) cortex, (B) hippocampus and (C) hypothalamus of mice. (D,E,F) Western blot analysis of p-IkBa and p-NF-kB expression levels in cortex, hippocampus and hypothalamus of mice. (G,H,I) Western blot analysis of p-p38, p-ERK1/2 and p-JNK in cortex, hippocampus and hypothalamus of mice. Values were mean ± SEM (n ¼ 6e9). ***P < 0.001 versus WT/Sham group; þ P < 0.05, þþP < 0.01 and þþþP < 0.001 versus WT/I/R group.
Please cite this article in press as: S. Lu, et al., HDAC9 promotes brain ischemic injury by provoking IkBa/NF-kB and MAPKs signaling pathways, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.07.043
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Fig. 4. HDAC9 knockdown alleviates LPS-induced inflammation in BV2 cells partially by MAPKs pathway. (A) BV2 cells were transfected HDAC9 si-RNA (si-HDAC9) for 24 h. Transfection efficacy was confirmed using western blot analysis. BV2 cells were transfected with si-HDAC9 for 24 h first, and then were subjected to LPS (80 ng/ml) treatment for an additional 24 h (B) RT-qPCR analysis was used to determine iNOS, COX-2, IL-1b, IL-6, TNF-a, and IL-18 mRNA expression levels in cells. (C,D) Western blot analysis of protein expression levels of p-IkBa, p-NF-kB, p-p38, p-ERK1/2 and p-JNK in cells. (E) BV2 cells were treated with P79350 (50 mM), LPA (20 mM) and ANI (10 mM) for 2 h. Then, western blot analysis was performed to calculate p-p38, p-ERK1/2 and p-JNK in cells. BV2 cells were pre-treated with P79350, LPA and ANI for 2 h, followed by si-HDAC9 transfection for another 24 h. Subsequently, all cells were stimulated by LPS for an additional 24 h (F) RT-qPCR analysis was used to determine iNOS, COX-2, IL-1b, IL-6, TNF-a, and IL-18 mRNA expression levels in cells. (G) Western blot analysis of protein expression levels of p-IkBa and p-NF-kB in clls treated as indicated. (H) Schematic model of pro-neuroinflammatory effects of HDAC9 on ischemic brain injury. Values were mean ± SEM (n ¼ 6). **P < 0.01 and ***P < 0.001 versus LPS/Ctrl group; þP < 0.05, þþP < 0.01 and þþþP < 0.001 versus LPSþ/Ctrl group; $ P < 0.05 and $$P < 0.01 versus LPSþ/si-HDAC9 group.
neurological worsening in ischemic stroke [24]. Several studies indicate a direct relationship between HDAC9 and inflammatory diseases [25,26]. HDAC9 deletion in a CD4þ T cell-regulated autoimmunity mice had down-regulated inflammation and generated less cytokines and chemokine [27]. HDAC9-deficient Treg cells proliferate faster and results in stronger immune repression [28]. Depletion of HDAC9 significantly suppressed the expression of ox-
LDL-induced inflammatory factors, such as TNF-a [29]. Vascular HDAC9 could modulate atherogenicprocesses and enhances NF-kB activation [30]. NF-kB signaling pathway regulates inflammation through promoting the expression of IL-1b, IL-6, TNF-a, and other pro-inflammatory cytokines [31]. NF-kB binds to the inhibitor IkBa; dissociation of the complexes could be activated by cytokines, free radicals, stress and bacterial and viral antigens [32]. Once
Please cite this article in press as: S. Lu, et al., HDAC9 promotes brain ischemic injury by provoking IkBa/NF-kB and MAPKs signaling pathways, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.07.043
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stimulated, IkBa kinase engages in phosphorylation of NF-kB. Activated NF-kB enters the nucleus and triggers the expression of numerous genes involved in inflammatory response [33]. In our study, HDAC9 knockout attenuated neuroinflammation in cortex, hippocampus and hypothalamus of mice after I/R operation, supported by the reduced expression levels of iNOS, COX-2, IL-1b, IL-6, TNF-a and IL-18, which was accompanied with the down-regulated p-IkBa and p-NF-kB expression levels. Therein, HDAC9 deficiency inhibited the neuroinflammation to attenuate brain ischemic injury. MAPKs comprise three major members, p38, ERK1/2, and JNK, which convey extracellular signals to their intracellular targets to modulate cellular activities via various signaling pathways [34]. p38 MAPK has been suggested to be implicated in mitochondrial dynamics in cerebral ischemic injury, indicating a potential neuroprotective benefit of p38 suppression [35]. ERK1/2 and JNK cascades have also been involved in neuronal apoptosis after brain ischemia injury [36,37]. HDAC9c could transcriptionally modulate p38-mediated mesenchymal stem cell differentiation into osteoblasts [38]. Consistently, in our study, mice with I/R injury showed higher activation of p38, ERK1/2 and JNK in brain tissue samples, which were, however, reduced by HDAC9 knockout. Blocking MAPKs pathway has been implicated in attenuating various diseases significantly associated with inflammation suppression [39]. Previous studies have suggested that MAPKs, as the up-streaming regulators of NF-kB, play an important role in inflammatory factor release [40]. In the present study, we found that promoting MAPKs activation abrogated HDAC9 inhibition-reduced inflammation, which was evidenced by the increased expressions of proinflammatory cytokines, and the activation of p-IkBa and p-NF-kB in LPS-treated BV2 cells. The findings illustrated that HDAC9 could partly modulate MAPKs to influence inflammatory response. Therefore, HDAC9 promoted inflammatory response through activating IkBa/NF-kB and MAPKs pathways, contributing to brain ischemic injury. However, further study is still necessary in future to investigate if other pathways are potentially involved in HDAC9regulated brain ischemic injury. In conclusion, our results demonstrated that HDAC9 was overexpressed in brain tissue samples, leading to inflammatory response by activating IkBa/NF-kB and MAPKs signaling pathways, which consequently resulted in brain ischemic injury (Fig. 4H). Herein, our study provided that HDAC9 may be used as a potential neurotherapeutic target to treat the inflammatory components of acute brain injury. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2018.07.043. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.07.043. References [1] R.N. Kalaria, Cerebrovascular disease and mechanisms of cognitive impairment: evidence from clinicopathological studies in humans, Stroke 43 (9) (2012) 2526e2534. [2] J. Borst, et al., Value of computed tomographic perfusionebased patient selection for intra-arterial acute ischemic stroke treatment, Stroke 46 (12) (2015) 3375e3382. [3] P.M. Meyers, et al., Current status of endovascular stroke treatment, Circulation 123 (22) (2011) 2591e2601. [4] K.P. Doyle, et al., Mechanisms of ischemic brain damage, Neuropharmacology 55 (3) (2008) 310e318.
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HDAC9 promotes brain ischemic injury by provoking IkBa/NF-kB and MAPKs signaling pathways Shan Lu a, Hang Li b, Kai Li a, Xiao-Di Fan a, * a b
Department of Anesthesiology, China Japan Union Hospital of Jilin University, Jilin, 130033, China Department of Hepatobiliary and Pancreas Surgery, China Japan Union Hospital of Jilin University, Jilin, 130033, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 June 2018 Accepted 9 July 2018 Available online xxx
Ischemic stroke is an acute cerebrovascular disease due to poor blood flow to the brain. Nevertheless, there is still no effective therapy for it and the pathology contributing to ischemic stroke is not fully understood. Histone Deacetylase 9 (HDAC9) is a class IIa chromatin-modifying enzyme. HDAC9 gene region is a leading risk locus for large artery atherosclerotic stroke. However, the mechanisms linking HDAC9 to ischemic remain elusive. In the study, we attempted to explore HDAC9-associated inflammatory response using the wild type (WT) and HDAC9-knockout (KO) mice with brain ischemic injury. The results indicated that WT mice with ischemia brain exhibited higher expression levels of HDAC9. HDAC9 depletion resulted in a decreased infarct volume and an improved neurological function in mice after ischemic reperfusion (I/R) injury. I/R injury markedly enhanced GFAP and Iba-1 expressions in cortex and HDAC9 knockout significantly reversed this up-regulation. Loss of HDAC9 inhibited the release of inducible NO-synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin 1b (IL-1b), IL-6, tumor necrosis factor-a (TNF-a), and IL-18 in cortex, hippocampus and hypothalamus of mice with I/R injury, which occurred at the transcription levels. Furthermore, the inhibitory actions of HDAC9 deficiency were associated with the down-regulation of phosphorylated-IkBa, phosphorylated-nuclear factor-kappa B (NF-kB), and p-mitogen-activated protein kinases (MAPKs), including phosphorylated-p38, phosphorylated-extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphorylated-c-Jun N-terminal kinase (JNK). Importantly, the in vitro study indicated that HDAC9 inhibition-reduced inflammation and activation of IkBa/NF-kB were restored by promoting MAPKs activity in LPS-stimulated cells. Our findings suggest that HDAC9 inhibition showed neuroprotective effects on ischemic stroke by restraining inflammation, which might help develop new and effective strategies for the therapeutic interventions in ischemic stroke. © 2018 Published by Elsevier Inc.
Keywords: Ischemic stroke HDAC9 Inflammation IkBa/NF-kB MAPKs
1. Introduction Stroke is an acute cerebrovascular disease due to poor blood flow to the brain [1]. Increasing studies suggested that clinical trials have failed to indicate a benefit in treating stroke, illustrating that the physiopathological mechanisms of ischemic stroke are far more complex realized previously [2,3]. In addition to the insufficient oxygen and glucose delivery, other detrimental factors, including acidotoxicity, excitotoxicity, nitrative stress and particularly, postischemic neuroinflammation, result in the last outcome of
* Corresponding author. Department of Anesthesiology, China Japan Union Hospital of Jilin University, Jilin, 130033, China. E-mail address: [email protected] (X.-D. Fan).
ischemic stroke [4,5]. Furthermore, the circulating factors secreted by brain may also influence the pathogenesis of stroke [6]. Histone deacetylases (HDACs) are suggested as promising therapeutic targets for preventing neurodegenerative diseases and stroke through epigenetic and non-epigenetic molecular mechanisms [7]. Various HDAC inhibitors exhibit beneficial effects in a variety of brain disorders, involving ischemic stroke [8]. HDAC9 is a class IIa chromatin-modifying enzyme and regulates diverse normal and abnormal physiological functions [9,10]. Class IIa HDACs exert tissue specific expression and lead to differentiation and development [11]. HDAC9 is over-expressed in striated muscle, where it functions as a negative modulator of differentiation and growth [12]. In brain, HDAC9 is associated with neuronal morphogenesis. Accumulating evidence demonstrate that HDAC9 is an essential target for neuroprotection. For example, HDAC9 is
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expressed in the brain to meditate neocortical neuronal development [13]. Recently, though studies have identified a variant in HDAC9 related to large-vessel ischaemic stroke and further indicated that HDAC9 variant linked to ischaemic stroke enhances risk through enhancing carotid atherosclerosis, the molecular mechanisms of HDAC9 in regulating ischaemic stroke are not fully understood. In the present study, the animal model with brain I/R injury in WT and HDAC9-KO mice was established in vivo. The results indicated that HDAC9-KO mice exerted a decreased infarct volume and an improved neurological function, along with a significant reduced expression of pro-inflammatory cytokines. The process was associated with the inactivation of IkBa/NF-kB and MAPKs signaling pathways in HDAC9-KO mice with experimental ischaemic stroke. We also used the in vitro cell cultures through LPS treatment. Of note, we found that promoting MAPKs activity abrogated HDAC9 inhibition-induced inflammatory response in LPS-stimulated cells. Thus, targeting HDAC9 could be an effective strategy for combating ischaemic stroke. 2. Materials and methods 2.1. Animals and treatments Male 8e12 weeks old wild type (WT) C57BL/6 or HDAC9knockout (KO) mice were purchased from Laboratory Animal Center, Jilin University and Cyagen BIOSCIENCES (Guangzhou, China), respectively. Animals were housed in a facility with controlled temperature (23 ± 2 C) and lighting (08: 00 to 20: 00 h), with free access to tap water. All animal experiments were approved by the Animal Ethics Committee of China Japan Union Hospital of Jilin University and were performed in compliance with the principles stated in the Guide for the Care and Use of Laboratory Animals, NIH Publication, 1996 edition. All mice were divided into 4 groups, including 1) WT/Sham; 2) KO/Sham; 3) WT/I/R and 4) KO/I/ R. Focal cerebral ischemia and reperfusion (I/R) was induced by intraluminal middle cerebral artery occlusion (MCAO) as described previously [14]. Before operation, mice were anesthetized using pentobarbital sodium (30 mg/kg). Next, mice were placed on a heating panel, maintaining at 37 C during operation. Then, a midline incision was made to expose the right common carotid artery, external carotid artery, and internal carotid artery. MCAO was triggered through advancing a 6/0 surgical nylon monofilament using a rounded tip into the lumen of internal carotid artery carefully from the right external carotid artery until the rounded tip blocked the origin of the middle cerebral artery (MCA). After 1 h of MCAO, the surgical nylon monofilament was retracted to allow reperfusion for differ rent durations (3, 6, 12, 24 and 48 h). The sham-operated group of mice was treated with similar operations without the surgical nylon monofilament insertion. 2.2. Cells and culture PC12 and BV2 cells were obtained from the Chinese Academy of Medical Sciences (Beijing, China). Astrocytes (AST) were isolated from mice using the methods as described previously [15]. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) medium (Gbico, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), 2 mM glutamine (Gbico), 100 mg/ml streptomycin (Gbico) and 100 U/ml penicillin (Gbico) at 37 C with 5% CO2. HDAC9 siRNA and negative control siRNA (NC) sequences were purchased from Santa Cruz (USA), which were transfected to BV2 cells using lipofectamine 2000 reagent (Invitrogen, USA). P79350 (p38 activator) was purchased from Calbiochem (USA). Lysophosphatidic Acid (LPA) was obtained from Santa Cruz (USA).
Anisomycin (ANI, JNK activator) was purchased from Beyotime (Nanjing, China).
2.3. Cognitive function analysis Motion function and locomotor activity were measured by open-field test as previously described [14]. The average velocity and time spent in the center square or side was recorded. To calculate motor coordination and learning before and after I/R operation, an accelerating rotarod test was performed (SD Instruments, USA) as previously described [16]. Tests were carried out at 1, 7, 14, 21, 28 days after I/R. The morris water maze (MWM) task included a circle water tank filled with opaque water and a round platform, which was submerged 1 cm beneath the surface of water at the center of the second quadrant. Hidden platform training was performed within 90 s at 6 consecutive days with four trials each session. Escape latency to search for the hidden platform, swimming paths, swimming velocity were recorded. Data was analyzed using a TM-Vision video-tracking system (Chengdu Taimeng Software Co. LTD, China).
2.4. 5-Triphenyltetrazolium chloride (TTC) staining and infarct volume measurement Mice were euthanized. Brain tissues were removed to froze at 25 C for 20 min after 24 h of reperfusion or sham operations. Then, the brains were sliced into serial coronal sections (2 mm apart) and stained with 2% TTC (Sigma-Aldrich, USA) for 30 min at 37 C. Infarction volume was the sum of all lesion areas multiplied by slice thickness using Image J software (National Institutes of Health, USA). Neurological status was calculated based on a neurologic deficit score following ischemia at 24 h reperfusion [17].
2.5. RNA extraction and qRT-PCR analysis Real-time quantitative PCR was performed as described previously [18]. Total RNA for real-time quantitative RT-PCR was isolated from tissues or cells using RNAiso Reagent (TaKaRa, Japan) and reverse transcripted into the single strand cDNA. The primers for targeting genes were listed in Supplementary Table 1. Gene expression of GAPDH was used for control. Quantification of mRNA was carried out using the ABI Prism 7500 (Applied Biosystems, USA) by PrimeScript™ RT-PCR Kit (TaKaRa).
2.6. Western blot assay Cortex, hippocampus and hypothalamus, and cells were lysed in modified RIPA buffer (10 mM Na2HPO4 pH 7.2, 150 mM NaCl, 1% Nonidet P-40 [NP-40], 0.5% Na-deoxycholate) containing protease inhibitors (2 mM PMSF, Beyotime). Total proteins (20e50 mg) were separated by SDS-PAGE, and transferred to PVDF (Millipore, USA) membranes. Membranes were then incubated with primary antibodies, followed by incubation with horseradish peroxidase (HRP)conjugated secondary antibodies. Protein bands were then visualized with an enhanced chemiluminescence system (ECL, Beyotime). Th eprimary anti-bodies used in the present study were shown as followings: HDAC9 (1:500, sc-398003, Santa Cruz); p-IkBa (1:1000, #2859, CST, USA); IkBa (1:1000, #4814, CST); p-NF-kB/p65 (1:1000, #3033, CST); NF-kB/p65 (1:1000, #8242, CST); p-p38 (1:1000, #4511, CST); p-ERK1/2 (1:1000, #9101, CST); p-JNK (1:1000, #9255, CST); p38 (1:1000, #8690, CST); ERK1/2 (1:1000, #4695, CST); JNK (1:1000, #9252, CST) and GAPDH (1:1000, #5174, CST).
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2.7. Immunofluorescence (IF) staining Immunofluorescence were performed as described previously [19]. Cultured cells or brain sections were fixed in 4% paraformaldehyde, blocked by 10% normal goat serum (Solarbio, China), and incubated in specific primary antibodies as follows: HDAC9 (1:100, Santa Cruz), NeuN (1:200, #24307, CST), GFAP (1:200, ab7260, Abcam, USA), and Iba-1 (1:200, ab5076, Abcam). After being washed three times with PBS, the sections and cells were incubated with Alexa 488-conjugated secondary antibodies (Abcam). Images were obtained by fluorescence microscope with a digital camera.
2.8. Statistical analysis Data are expressed as mean ± SEM. Differences were evaluated by one-tailed Student's t-test or ANOVA followed by Tukey's posthoc test with GraphPad Prism 5.0 version. Statistical significance was set at P < 0.05.
3. Results 3.1. HDAC9 expression levels were increased in the ischemic brain As shown in Fig. 1A and B, HDAC9 mRNA and protein levels in brain penumbra area of I/R mice was significantly higher than that in non-ischemia brain area, which reached peak at 24 h post I/R operation. Expression of HDAC9 in cortex of brain penumbra area was also enhanced at 24 h post I/R operation via IF analysis (Fig. 1C). Moreover, HDAC9 mRNA and protein levels were also markedly increased by LPS treatment in PC12, BV2 and AST cells (Fig. 1D and E). IF analysis showed that LPS exposure enhanced the number of HDAC9 positive cells in vitro (Fig. 1F). These results indicated that brain HDAC9 was up-regulated by experimental cerebral ischemia.
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3.2. Loss of HDAC9 protects against I/R-induced ischemic brain injury in vivo There was no significant difference observed in the motion function and locomotor activity in each group of mice before I/R operation (Fig. 2A). After I/R surgery, WT mice showed a lower velocity and spent less time in the center area of the apparatus than WT/Sham mice, which was restored by HDAC9-knockout, indicating an alleviated locomotor activity and anxiety-like behavior (Fig. 2B). It was also noteworthy that loss of HDAC9 led to better recovery of motor coordination in rotarod test throughout the 4week observation period after I/R operation (Fig. 2C). MWM analysis suggested that long-term learning and memory deficits were apparently improved in HDAC9-knockout mice after I/R, as manifested by a reduced latency to find the hidden platform (Fig. 2D). TTC staining indicated that HDAC9 deletion significantly downregulated the cerebral infarct volume compared with the volumes observed in the WT mice after I/R operation (Fig. 2E and F). In line with the smaller infarct sizes, HDAC9-knockout resulted in prominent improvements in neurological function 24 h after I/R injury (Fig. 2G). As calculated by NeuN staining, I/R operation for 24 h induced severe neuronal cell death in the cortex, and HDAC9knockout significantly improved neuronal survival (Fig. 2H). In contrast, a significant increase of GFAP and Iba-1 expressions was observed in the cortex of I/R mice, which was markedly reversed by the loss of HDAC9 (Fig. 2I). 3.3. HDAC9 ablation down-regulates inflammation in brain of mice subjected to I/R 24 h I/R operation significantly elevated mRNA expression levels of iNOS, COX-2, IL-1b, IL-6, TNF-a, and IL-18 in cortex, hippocampus and hypothalamus of mice, which was partly blocked by HDAC9deficiency (Fig. 3AeC). Also, HDAC9-knockout partly inhibited the p-IkBa and p-NF-kB protein up-regulation in infarcted brain tissue samples (cortex, hippocampus and hypothalamus) of mice (Fig. 3DeF). Similar phenotypes were observed in MAPKs. The
Fig. 1. HDAC9 expression levels were increased in the ischemic brain. (A) RT-qPCR and (B) western blotting analysis for HDAC9 in the ischemic brain tissues 3, 6, 12, 24 and 48 h after focal cerebral ischemia. (C) IF staining of HDAC9 in cortex of mice subjected to I/R 24 h (D,E,F) RT-qPCR, western blotting and IF staining of HDAC9 in PC12, BV2 and AST cells stimulated by 80 ng/ml of LPS for 24 h. Values were mean ± SEM (n ¼ 6e9). **P < 0.01 and ***P < 0.001 versus Sham or Ctrl group.
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Fig. 2. Loss of HDAC9 protects against I/R-induced ischemic brain damage in vivo. The motion function and locomotor activity of the WT mice and KO mice were measured by open-field test (A) before and (B) after 24 h of I/R operation. Quantifications of average velocity, time spent in the side areas and in the center square were exhibited for 10 min during each session. (C) The performance on rotarod of mice was analyzed before I/R operation and throughout the 4-week period after 24 h of I/R operation. (D) The performance on MWM mice was analyzed 3 days after 24 h of I/R operation. Hidden platform training was performed within 90 s at 6 consecutive days. Curve lines exhibits the escape latency to find the hidden platform. (E) Brain infarction was determined by TTC staining on Day 3 after 24 h of I/R operation. (F) Quantification of infarct volume. (G) Neurologic deficit score of mice subjected to I/R 24 h or sham-treated mice. (H) NeuN and (I) GFAP and Iba-1 expression levels in cortex of mice subjected to I/R 24 h were measured using IF staining. Values were mean ± SEM (n ¼ 6e9). *P < 0.05, **P < 0.01 and ***P < 0.001 versus WT/Sham group; þP < 0.05 and þþP < 0.01 versus WT/I/R group.
protein levels of p-p38, p-ERK1/2 and p-JNK in cortex, hippocampus and hypothalamus of I/R mice were increased, while HDAC9 deletion partly depressed these changes (Fig. 3GeI).
3.4. HDAC9 knockdown alleviates LPS-induced inflammation in BV2 cells partially by MAPKs pathway To further explore the effects of HDAC9 on brain ischemic injury, the in vitro study was carried out using BV2 cells with HDAC9 knockdown by transfecting HDAC9 siRNA sequence. The transfection efficacy was confirmed using western blot analysis (Fig. 4A). As shown in Fig. 4B, LPS treatment markedly up-regulated the mRNA expression levels of iNOS, COX-2, IL-1b, IL-6, TNF-a, and IL18 in BV2 cells, which were significantly repressed by HDAC9silence. The expression levels of p-IkBa and p-NF-kB stimulated by LPS were evidently reduced by HDAC9 knockdown in BV2 cells (Fig. 4C). Treatment of HDAC9-knockdown significantly attenuated the protein levels of p-p38, p-ERK1/2 and p-JNK in LPS-treated cells (Fig. 4D). Further, the activation of p38, ERK1/2 and JNK was considerably increased by P79350, LPA and ANI treatment, respectively (Fig. 4E). Moreover, we found that the loss of HDAC9induced reduction of iNOS, COX-2, IL-1b, IL-6, TNF-a, and IL-18 in LPS-treated BV2 cells was promoted by the pre-treatment of P79350, LPA or ANI (Fig. 4F). Consistently, HDAC9 knockdowntriggered inactivation of IkBa and NF-kB was recovered by the pre-treatment with P79350, LPA or ANI in LPS-stimulated cells (Fig. 4G). Therefore, the findings here demonstrated that HDAC9 suppression-attenuated inflammation could be impeded by promoting MAPKs activation in LPS-incubated cells.
4. Discussion HDAC9 is a member of class II HDACs, modulating a wide variety of normal and abnormal physiological functions [9,10]. Recently, through studies have implied the importance of HDAC-regulated epigenetic processes in the progression of ischemic stroke, the molecular events to trigger cerebral injury are not fully explained. Accumulating evidence has indicated that HDAC9 is linked to neuronal physiology and pathology [11e14]. In the present study, we provided the evidence that loss of HDAC9 ameliorated cerebral ischemia injury in vivo and in vitro. Firstly, we found that brain HDAC9 was up-regulated by ischemic insult. HDAC9 knockout showed neuroprotection effects, as evidenced by the reduced brain infarcted volume and neurological deficit score. Further, neuronal death was suppressed by HDAC9 deletion. Glial cells activity was also blocked in HDAC9-KO mice. HDAC9 ablation inhibited NF-kB and MAPKs signaling activation during ischemic stroke and therefore neuroinflammation (iNOS, COX-2, IL-1b, IL-18, TNF-a and IL-6 production). At last, promoting AMPKs activity compromised the neuroprotection of HDAC9-suppression, and consequently enhancing inflammatory response. The brain in response to ischemic damage shows an acute and prolonged inflammatory activation, characterized by both rapid activation of resident microglia and subsequent infiltration of different types of inflammatory cells into the ischemic area [20,21]. The pro-inflammatory factors, including IL-18, IL-1b, TNF-a and IL6 are secreted by the activated microglia or other inflammatory cells [22,23]. In patients with ischemic stroke, IL-6 and TNF-a levels in plasma and cerebrospinal fluid are related to early clinical deterioration, suggesting pro-inflammatory cytokines and early
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Fig. 3. HDAC9 ablation down-regulates inflammation in brain of mice subjected to I/R 24 h. RT-qPCR analysis of iNOS, COX-2, IL-1b, IL-6, TNF-a, and IL-18 in (A) cortex, (B) hippocampus and (C) hypothalamus of mice. (D,E,F) Western blot analysis of p-IkBa and p-NF-kB expression levels in cortex, hippocampus and hypothalamus of mice. (G,H,I) Western blot analysis of p-p38, p-ERK1/2 and p-JNK in cortex, hippocampus and hypothalamus of mice. Values were mean ± SEM (n ¼ 6e9). ***P < 0.001 versus WT/Sham group; þ P < 0.05, þþP < 0.01 and þþþP < 0.001 versus WT/I/R group.
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Fig. 4. HDAC9 knockdown alleviates LPS-induced inflammation in BV2 cells partially by MAPKs pathway. (A) BV2 cells were transfected HDAC9 si-RNA (si-HDAC9) for 24 h. Transfection efficacy was confirmed using western blot analysis. BV2 cells were transfected with si-HDAC9 for 24 h first, and then were subjected to LPS (80 ng/ml) treatment for an additional 24 h (B) RT-qPCR analysis was used to determine iNOS, COX-2, IL-1b, IL-6, TNF-a, and IL-18 mRNA expression levels in cells. (C,D) Western blot analysis of protein expression levels of p-IkBa, p-NF-kB, p-p38, p-ERK1/2 and p-JNK in cells. (E) BV2 cells were treated with P79350 (50 mM), LPA (20 mM) and ANI (10 mM) for 2 h. Then, western blot analysis was performed to calculate p-p38, p-ERK1/2 and p-JNK in cells. BV2 cells were pre-treated with P79350, LPA and ANI for 2 h, followed by si-HDAC9 transfection for another 24 h. Subsequently, all cells were stimulated by LPS for an additional 24 h (F) RT-qPCR analysis was used to determine iNOS, COX-2, IL-1b, IL-6, TNF-a, and IL-18 mRNA expression levels in cells. (G) Western blot analysis of protein expression levels of p-IkBa and p-NF-kB in clls treated as indicated. (H) Schematic model of pro-neuroinflammatory effects of HDAC9 on ischemic brain injury. Values were mean ± SEM (n ¼ 6). **P < 0.01 and ***P < 0.001 versus LPS/Ctrl group; þP < 0.05, þþP < 0.01 and þþþP < 0.001 versus LPSþ/Ctrl group; $ P < 0.05 and $$P < 0.01 versus LPSþ/si-HDAC9 group.
neurological worsening in ischemic stroke [24]. Several studies indicate a direct relationship between HDAC9 and inflammatory diseases [25,26]. HDAC9 deletion in a CD4þ T cell-regulated autoimmunity mice had down-regulated inflammation and generated less cytokines and chemokine [27]. HDAC9-deficient Treg cells proliferate faster and results in stronger immune repression [28]. Depletion of HDAC9 significantly suppressed the expression of ox-
LDL-induced inflammatory factors, such as TNF-a [29]. Vascular HDAC9 could modulate atherogenicprocesses and enhances NF-kB activation [30]. NF-kB signaling pathway regulates inflammation through promoting the expression of IL-1b, IL-6, TNF-a, and other pro-inflammatory cytokines [31]. NF-kB binds to the inhibitor IkBa; dissociation of the complexes could be activated by cytokines, free radicals, stress and bacterial and viral antigens [32]. Once
Please cite this article in press as: S. Lu, et al., HDAC9 promotes brain ischemic injury by provoking IkBa/NF-kB and MAPKs signaling pathways, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.07.043
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stimulated, IkBa kinase engages in phosphorylation of NF-kB. Activated NF-kB enters the nucleus and triggers the expression of numerous genes involved in inflammatory response [33]. In our study, HDAC9 knockout attenuated neuroinflammation in cortex, hippocampus and hypothalamus of mice after I/R operation, supported by the reduced expression levels of iNOS, COX-2, IL-1b, IL-6, TNF-a and IL-18, which was accompanied with the down-regulated p-IkBa and p-NF-kB expression levels. Therein, HDAC9 deficiency inhibited the neuroinflammation to attenuate brain ischemic injury. MAPKs comprise three major members, p38, ERK1/2, and JNK, which convey extracellular signals to their intracellular targets to modulate cellular activities via various signaling pathways [34]. p38 MAPK has been suggested to be implicated in mitochondrial dynamics in cerebral ischemic injury, indicating a potential neuroprotective benefit of p38 suppression [35]. ERK1/2 and JNK cascades have also been involved in neuronal apoptosis after brain ischemia injury [36,37]. HDAC9c could transcriptionally modulate p38-mediated mesenchymal stem cell differentiation into osteoblasts [38]. Consistently, in our study, mice with I/R injury showed higher activation of p38, ERK1/2 and JNK in brain tissue samples, which were, however, reduced by HDAC9 knockout. Blocking MAPKs pathway has been implicated in attenuating various diseases significantly associated with inflammation suppression [39]. Previous studies have suggested that MAPKs, as the up-streaming regulators of NF-kB, play an important role in inflammatory factor release [40]. In the present study, we found that promoting MAPKs activation abrogated HDAC9 inhibition-reduced inflammation, which was evidenced by the increased expressions of proinflammatory cytokines, and the activation of p-IkBa and p-NF-kB in LPS-treated BV2 cells. The findings illustrated that HDAC9 could partly modulate MAPKs to influence inflammatory response. Therefore, HDAC9 promoted inflammatory response through activating IkBa/NF-kB and MAPKs pathways, contributing to brain ischemic injury. However, further study is still necessary in future to investigate if other pathways are potentially involved in HDAC9regulated brain ischemic injury. In conclusion, our results demonstrated that HDAC9 was overexpressed in brain tissue samples, leading to inflammatory response by activating IkBa/NF-kB and MAPKs signaling pathways, which consequently resulted in brain ischemic injury (Fig. 4H). Herein, our study provided that HDAC9 may be used as a potential neurotherapeutic target to treat the inflammatory components of acute brain injury. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2018.07.043. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.07.043. References [1] R.N. Kalaria, Cerebrovascular disease and mechanisms of cognitive impairment: evidence from clinicopathological studies in humans, Stroke 43 (9) (2012) 2526e2534. [2] J. Borst, et al., Value of computed tomographic perfusionebased patient selection for intra-arterial acute ischemic stroke treatment, Stroke 46 (12) (2015) 3375e3382. [3] P.M. Meyers, et al., Current status of endovascular stroke treatment, Circulation 123 (22) (2011) 2591e2601. [4] K.P. Doyle, et al., Mechanisms of ischemic brain damage, Neuropharmacology 55 (3) (2008) 310e318.
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Please cite this article in press as: S. Lu, et al., HDAC9 promotes brain ischemic injury by provoking IkBa/NF-kB and MAPKs signaling pathways, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.07.043