Background
Traumatic spinal cord injury (SCI) initiates a series of cellular and molecular events, including microglial activation, inflammatory response, and abnormal mitochondrial activities, which induce neuronal death and lead to permanent neurological deficits [
1‐
3]. SCI-induced microglial activation and subsequent release of inflammatory factors, such as interleukin (IL), tumor necrosis factor (TNF), and interferon (INF), cause direct neuronal death while inducing vascular endothelial cells to express a variety of cell adhesion and chemotaxis molecules [
3‐
5]. These pro-inflammatory factors stimulate nitric oxide synthesis, which leads to increased capillary permeability and blood-spinal cord barrier dysfunction while promoting neuronal apoptosis [
2,
3]. The inhibition of SCI-induced, the microglial activation, and the subsequent neuroinflammatory response have been shown to improve the recovery in SCI patients [
6,
7].
Neuroinflammatory responses induced by activated microglia, via the NF-κB pathway, are the critical contributing factors of secondary injury [
8,
9]. The NF-κB signaling pathway is passively released after an injury by necrotic or damaged cells, which activates microglia to secrete a large number of inflammatory cytokines, as well as cascade amplification of inflammatory responses [
8‐
10]. The NF-κB exists in an inactive state sequestered in the cytoplasm by the NF-κB (IκB) inhibitor during non-inflammatory conditions. The activation of the NF-κB is initiated by IκB kinase (IKK), which degrades cytoplasmic IκB protein, which triggers the rapid release of NF-κB from IκB and intranuclear translocation [
11,
12]. The activation process co-exists with signal transducers and activators of transcriptions (STATs) [
13,
14]. The DNA-binding STATs enter from the cytoplasm to the nucleus and activate their target genes at chromosome 19q13. These genes include B cell lymphoma (Bcl)-3, which encodes elements of IκB from the NF-κB pathway [
15‐
17]. Releases of the NF-κB proteins for intranuclear transfer allow them to bind to their target gene promoters that encode chemokines and cytokines, including persistently activated STATs [
13,
16,
17].
Valproic acid (VPA) is commonly used to treat epilepsy. VPA exerts its therapeutic benefits through multiple mechanisms, including enhancement of GABAergic activity, depolarization induced by
N-methyl-
d-aspartic acid (NMDA) receptors, and/or inhibition of calcium channels and voltage-gated sodium [
18‐
21]. Recent studies found that VPA is a class 1/II histone deacetylase inhibitor (HDCAi), where it inhibits histone deacetylase (HDCA) functions [
18,
19]. Acetylated histone proteins exert their neuroprotective effects by reducing inflammation and inhibiting neuronal death [
22,
23], which improve neurological functions in many neurological diseases, including cerebral ischemia [
24], traumatic brain injury [
25], and spinal cord injury [
21,
26]. The role that VPA plays in SCI, as well as if VPA treatment could inhibit the activation of microglia and the subsequent inflammatory response after TBI, has not been studied or established.
The non-histone-binding protein complexes, such as the high-mobility group (HMG) family, the NF-kB, and the signal transducers and activators of transcription (STATs), are modified by post-translational modifications [
14,
27]. Recent studies have confirmed that the transcriptional activities of NF-kB and STATs are closely related to their lysine acetylations, which are regulated by the balance between histone acetyltransferases (HATs) and histone deacetylases (HDACs) [
28‐
30]. HDCAi seems to inhibit NF-kB transcriptional activity by maintaining the NF-kB acetylated (inactive) state and repressing the inflammatory response. Studies from Leus et al. [
31] found that the expression levels of NF-kB are related to HDAC enzymatic activity and the levels of histone acetylation. These findings suggest that HDAC expression may be associated with NF-kB-mediated inflammation. The activation of STATs could alleviate a multitude of NF-kB-driven inflammatory and metabolic disorders [
14,
27,
32]. There is no literature regarding the STAT-mediated nuclear translocation of NF-kB p65 subunit after a traumatic SCI. In the present study, we investigated if VPA, a class 1/II histone deacetylase inhibitor, attenuates the microglia-mediated neuroinflammatory response involving the interactive roles of HDAC, STATs, and NF-kB following a traumatic SCI.
Methods
Animals
Adult male Wistar rats (weighing 230–260 g) were purchased from the Fujian Medical University Experimental Animal Center. The rats were maintained in a clean, temperature-controlled environment (23 ± 2 °C) on a 12:12-h light/dark cycle with free access to food and water. The experimental protocols were in accordance with the guidelines for the care and use of laboratory animals by the Fujian Medical University Experimental Animal Ethics Committee (Fuzhou, China).
Animal model and drug delivery
A total of 192 rats were randomly divided into the sham, the sham + VPA, the SCI, and the SCI + VPA group. Each of these groups had four subgroups (1-, 3-, 7-, and 14-day time points) (
n = 12 per group). Six rats in each group were euthanized for the molecular biological and the biochemical experiments. The remaining rats in each group (
n = 6) were used for evaluating the neurological function and the histological studies. The traumatic SCI rat model was established in accordance with the previous literature [
33], under sodium pentobarbital anesthesia (50 mg/kg via intraperitoneal injection). An incision was made along the middle of the back, which exposed the paravertebral muscles and the vertebral laminas. A laminectomy was performed at the vertebral T9–T10 levels, exposing the dorsal surface of the cord without disrupting the dura. The cord was subjected to weight-drop impact to moderate contusion injury (10 g × 25 mm) using a 10-g metal rod at level T10. The Sham control rat group was subjected to a T9–T10 laminectomy without the weight-drop injury. Following the operation, manual bladder emptying was performed twice daily until the rats were able to urinate by themselves. Approximately 0.5 h after TBI, the rats in the sham + VPA and the SCI + VPA groups were administered VPA by intraperitoneal injection (300 mg/kg/day, diluted in dimethylsulfoxide; Sigma Aldrich, St Louis, MO, USA) once per day for three consecutive days [
21,
34]. Forty microliters per kilogram fludarabine (flu) (5 mmol/L, diluted in dimethylsulfoxide) was administrated into the left lateral ventricle 24 h after intraperitoneal VPA to inhibit STAT1 signaling, which clarified the role of STAT1 in VPA neuroprotection [
35]. The remainder of the groups was injected with the same dose of a dimethylsulfoxide, as a control.
Behavioral assessment
The locomotor recovery was based on the Basso-Beattie-Bresnahan locomotion scale and the inclined plane test, in accordance with the previous reports [
1,
2]. The locomotor recovery was evaluated at 1, 3, 7, and 14 days following the SCI. The Basso-Beattie-Bresnahan (BBB) test was graded on a scale of 0–21. A total score of 0 point indicated severe neurological deficits, and a score of 21 indicated normal performance. The inclined plane test was performed on a testing apparatus. The maximum angle where the rat retained its position for more than 5 s without falling was recorded. Behavioral assessments were performed at different time points by experimenters that were blinded to the group information.
Measurement of blood-spinal cord barrier permeability
The integrity of the blood-spinal cord barrier was established by measuring the extravasation of Evans blue (Sigma Aldrich) [
2]. The Evans blue dye (2% in saline; 4 mL/kg) was injected intravenously 2 h prior to the euthanasia at 1, 3, 7, and 14 days following the SCI. The mice were euthanized and then transcardially perfused with PBS, followed by an additional PBS containing 4% paraformaldehyde. Each tissue sample was immediately weighed and homogenized in a 1-mL 50% trichloroacetic acid solution. The samples were then centrifuged. The absorption of the supernatant was measured by a spectrophotometer (UV-1800 ENG 240V; Shimadzu Corporation, Japan) at a wavelength of 620 nm. The quantity of Evans blue was calculated with a standard curve and expressed as microgram of Evans blue per gram of brain tissue, using a standardized curve.
Nissl staining
The spinal cord specimens near the lesion epicenter were fixed with formaldehyde. The formaldehyde-fixed specimens were embedded in paraffin and cut into 4-μm-thick sections. The sections were deparaffinized with xylene and rehydrated in a graded series of alcohol. Samples were treated with Nissl staining solution (Boster Biotech, Wuhan, China) for 5 min, and then mounted with neutral balsam. The apoptotic neurons were shrunken or contained vacuoles. The normal neurons had a relatively large and full soma, with round, large nuclei. Five areas were randomly selected to be examined with an inverted microscope (Leica, Wetzlar, Germany) by investigators who were blinded to the experimental groups.
Immunohistochemical analysis
Formaldehyde-fixed specimens near the lesion epicenter were embedded in paraffin and cut into 4-μm-thick sections. These sections were deparaffinized with xylene and rehydrated in a graded series of alcohol. Antigen retrieval was performed by microwaving the samples in a citric acid buffer. The sections were incubated with a STAT1 (1:100; Cell Signaling Technology, Danvers, MA, USA) antibody, washed, and incubated with secondary antibody for 1 h at room temperature. These sections were mounted with neutral balsam. The negative control was prepared without the addition of the anti-STAT1 antibody. A total of five sections from each animal were used for quantification, and the signal intensity was evaluated as follows [
36]: 0, no positive cells; 1, very few positive cells; 2, moderate number of positive cells; 3, many positive cells; and 4, the highest number of positive cells.
Immunofluorescence analysis
Formaldehyde-fixed specimens were embedded in paraffin and cut into 4-μm-thick sections. The sections were deparaffinized with xylene and rehydrated in a graded series of alcohol, followed by antigen retrieval. The sections were incubated overnight at 4 °C with CD16 (1:200, Abcam, Cambridge, UK), CD206 (1:200; Abcam), neuronal nuclei (1:100; Boster Biotech, Wuhan, China), ionized calcium-binding adapter molecule (Iba)-1 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and GFAP (1:200; Abcam) antibodies. The sections were washed and incubated with secondary antibodies for 1 h at room temperature. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole. The sections were then mounted with glycerol jelly mounting medium. The immunopositive cells from five randomly selected fields were counted under the inverted microscope (Leica, Wetzlar, Germany) at × 400 magnification by experimenters that were blinded to the experimental group.
Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay
Apoptotic cells were detected with a terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) kit (Roche Diagnostics, Indianapolis, IN, USA) in accordance with the manufacturer’s instructions. Indicators of apoptosis included shrunken cell body, irregular shape, nuclear condensation, and brown diaminobenzidine staining, as observed by the inverted microscope (Leica, Wetzlar, Germany) at × 400 magnification. Average positive cell counts were calculated from the same sections in six rats per group with Image Pro Plus 7.0 by investigators who were blinded to the experimental groups.
Enzyme-linked immunosorbent assay
Inflammatory factors in the brain tissue were detected with ELISA kits for TNF-α, IL-1β, IL-6, and IFN-γ (all from Boster Biotech). The Multiplex Microplate Reader (Molecular Devices, SpectraMax M5) was used to measure the absorbance value of ELISAs. The measured OD values were converted into a concentration value.
Nuclear and cytoplasmic proteins extraction
The tissue samples were subjected to subcellular fractionation using the cytoplasmic and nuclear protein extraction kit (KeyGEN Biotech, KGP150), using hypotonic lysis buffer (20 mM HEPES (pH 7.4), 2 mM EGTA, 2 mM MgCl2) to extract the cytosolic protein, and using hypertonic lysis buffer (20 mM Tris/HCl, pH 7.6, 100 mM NaCl, 20 mM KCl, 1.5 mM MgCl2, 0.5% Nonidet P-40, and protease inhibitors) to extract the nuclear protein. The protein of the lysates was determined separately via Western blot by stripping the PVDF membranes and re-probed with LaminB1 (Cell Signaling Technology) as the nuclear control protein and β-actin (Boster Biotech) antibodies as the cytosolic control.
Western blotting
Proteins were extracted with a radioimmunoprecipitation assay lysis buffer (sc-24948; Santa Cruz Biotechnology). The BCA method (KeyGEN Biotech, KGPBCA) was used for the protein quantitation. A total of 25 μg protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane by wet transfer. The membrane was then blocked in 10% skim milk at room temperature for 2 h and then incubated at 4 °C overnight with primary antibodies against the following proteins: B cell lymphoma (Bcl)-2 (1:400), Bcl-2-associated X factor (Bax) (1:200), CD16 (1:200), and CD206 (1:200) (all from Abcam), and cleaved caspase-3 (1:200), Iba-1 (1:100), and NF-κB p65 (1:200) primary antibodies (all from Cell Signaling Technology), followed by incubation with the appropriate secondary antibodies (goat anti-rabbit IgG- HRP or goat anti-mouse IgG-HRP, 1:3500) at room temperature for 1 h. Immunoreactivity was visualized with the ECL Western Blotting Detection System (Millipore, Billerica, MA, USA). A gray value analysis was conducted with the UN-Scan-It 61 software (Silk Scientific Inc., Orem, UT, USA). Expression levels were normalized against β-actin (1:5000, Boster Biotech) or Lamin B1 (1:3000, Cell Signaling Technology).
Co-immunoprecipitation
The spinal cord specimens near the lesion epicenter were incubated for 2 h at 4 °C with either 1 μg of STAT1 (Cell Signaling Technology) or 1 μg NF-κB p65 anti-acetylated lysine antibody (Cell Signaling Technology). A 10-μl volume of protein A/G agarose beads (Roche, Mannheim, Germany) was added to the sample, followed by overnight incubation. The agarose beads were washed three times with a lysis buffer after immunoprecipitation and centrifugation. The degree of acetylation of the STAT1 or the NF-κB p65 was analyzed with Western blotting, using an anti-acetylated lysine antibody (Cell Signaling Technology).
HDAC activity assay and NF-κB DNA-binding activity assay
Nucleoproteins were extracted, where their concentrations were determined by bicinchoninic acid assay. A colorimetric HDAC fluorometric assay kit (BioVision, Mountain View, CA, USA) was used to detect the HDAC activity by measuring the absorbance at 405 nm on a microplate reader. A transcription factor binding assay colorimetric ELISA kit (Cayman Chemical, Ann Arbor, MI) was used to detect NF-κB p65 DNA-binding activity by measuring the absorbance at 450 nm on a microplate reader.
Statistical analysis
The data was analyzed with SPSS v 19.0 software (SPSS Inc., Chicago, IL, USA). The results are expressed as the mean ± standard deviation. Comparisons between groups were made with the unpaired Student’s t test. Multiple group comparisons were assessed with one-way ANOVA. Post hoc multiple comparisons were performed using Student-Newman-Keuls tests. P < 0.05 was considered statistically significant.
Discussion
Accumulating evidence have demonstrated the benefits of VPA against SCI-induced neural damage and secondary pathological processes [
18,
21]. The present study also supports the view that VPA is a suitable therapeutic candidate against SCI-induced neurological deficits. VPA treatment reduced brain edema and BBB permeability and improved neurological function after SCI. Furthermore, VPA treatment protected neurons against SCI-induced neuronal apoptosis by inhibiting the expression of the pro-apoptotic factors, cleaved caspase-3 and Bax, and increasing expression of the anti-apoptotic factor, Bcl-2. The VPA works as a histone deacetylase inhibitor to directly inhibit HDACs, which have been implicated in many biological activities [
21,
38,
39]. The present study confirmed that the HDAC3 expression was upregulated in the lesioned spinal cord, suggesting that the histone deacetylation played an important role in post-traumatic secondary spinal cord injury. The VPA treatment reduced the nuclear HDAC3 expression and inhibited its activity following the SCI, thereby suppressing neuronal apoptosis. These findings indicated that the VPA exerted neuroprotective effects by inhibiting HDAC3 expression and activity.
The microglia-mediated inflammatory response plays an important role in secondary spinal cord injury after a SCI [
4,
40,
41]. A phenotypic transition of microglia from the anti-inflammatory (M2)-like to the pro-inflammatory (M1)-like play a crucial role in the microglial activation and its mediation of neuroinflammatory response [
4,
40,
41]. Our study showed that the levels of activated microglia and the inflammatory cytokines (TNF-α, IL-1β, IL-6, and IFN-γ) in the spinal cord tissue increased after the SCI, which were linked to the BSCB permeability and the neurological function scores. The inhibition of HDAC activity following the VPA treatment promoted the phenotypic shift of microglia from the M1 to the M2 phenotype, as well as inhibiting microglial activation and reducing the expressions of inflammatory cytokines in vivo. The NF-κB is considered to be the central transcription factor of inflammatory mediators, where it plays a crucial role in inflammation [
8,
9]. The VPA treatment significantly weakened the NF-κB p65 nuclear translocation and its transcriptional activity following the SCI, which showed that the VPA exerted neuroprotective effects.
The non-histone-binding protein complexes, including the HMG family, the NF-kB, and the STATs, are modified by post-translational modifications [
27]. The NF-κB signaling pathway is activated by post-translational modification. The phosphorylation and the methylation on serine residues within two nuclear localization signals (NLS) of the NF-κB in monocytes/macrophages accelerate its nuclear translocation, as well as its transcriptional activity [
3,
14,
42]. The post-translational acetylation of NF-κB is also reported to promote its transcriptional activity, which is regulated by the balance between the HDACs and the histone acetyltransferases (HATs) [
28‐
30]. The HDCAi seems to inhibit NF-kB transcriptional activity by maintaining the NF-kB acetylated (inactive) state, which represses the inflammatory response [
18,
38]. The Co-IP analysis showed that the VPA increased the NF-kB p65 acetylation, which inhibited the NF-kB p65 nuclear translocation and its transcriptional activity. The HDCAi has been shown to alter the equilibrium between the HDACs and the HATs while inducing the STAT1 acetylation on the Lys 410 and the Lys 413 sites [
14,
38]. The Co-IP analysis also showed that the presence of the STAT1 protein in acetyl-lysine immunoprecipitate fractions confirmed increased STAT1 acetylation following the VPA treatment. The VPA-mediated upregulation of the acetylation and the expression of STAT1 was likely to be due to the reduced HDAC3 translocation to the nucleus and the activity.
The STAT1 pathway plays a critical role in mediating the NF-kB p65 nuclear translocation [
13,
14]. The lysine acetylation of the STAT1 seemed to be dispensable for the NF-kB releasing from its inhibitor IκB inhibition in response to damage stimuli [
14]. The evidence regarding the cross-talk association of the STAT1 and the NF-kB pathways should be further investigated. The current study provided a novel counter-regulatory relationship between the attenuation of the SCI-mediated NF-kB p65 activity, which seemed to be regulated by the interactions of STAT1 and HDACs. The Co-IP assays indicated that the VPA induced significant interactions between the STAT1 and the NF-κB p65 after the SCI. The acetylated STAT1 formed a complex with the nuclear NF-κB p65, which inhibited its DNA-binding activity and weakened the central inflammatory response following the SCI. The inhibitory effects of the VPA treatment on the inflammatory response and on the STAT1/NF-κB axis activation were reversed by the pharmacological inhibition of STAT1, suggesting that the anti-inflammation effect of the VPA was dependent on STAT1 expression. Future studies involving STAT1 knockout mice should be performed to investigate the mechanisms involved in the VPA-mediated activation of STAT1 and the subsequent inhibition of the NF-κB pathway. Additional in vitro experiments are required to establish the direct effect of the VPA treatment on neuron and microglia activation.