Male C57BL/6 mice (25 ± 2 g, 8 weeks) were subjected to SCI. All animal studies were conducted according to the Animal Welfare Guidelines of the United States National Institutes of Health with supervision and ratification from the Animal Care and Use Committee of the General Hospital of the Chinese People’s Liberation Army under standard conditions, including adequate temperature and humidity (60%) and a 12 h light/12 h dark cycle. The mice had free access to water and food.

The SCI model was established as previously described (Jiao et al., 2020). The mice were anesthetized using 4% isoflurane with a rate of 1 L/min, exposed to the T6–T7 spinous process, and underwent laminectomy. Then, epidural compression of the spinal cord with a 24-g closure force was applied for 1 min at the T6–T7 level, resulting in SCI, while the mice that underwent laminectomy were regarded as the sham group. We assessed SCI levels at baseline and postoperative days 1, 2, 3, 7, 14, 21, and 28.

As previously reported (Bombardo et al., 2018; Mota et al., 2020), we dissolved entinostat (Selleck, Shanghai, China) in 0.1 M phosphate-buffered saline (PBS, as the vehicle) (pH 7.4), and it was administered to the animals (1, 5, or 10 mg/kg, gavage) at 4 h after SCI and again 24 and 48 h later. The sham group was treated with equal PBS volumes.

The forelimb grip strength was assessed by using a Chatillon digital force meter (LTCM-100, Wintop Co., Shanghai, China). The mice tails were lifted and placed near the bars of a grip-measuring machine so they could grasp the bar. The mice were then gently pulled back horizontally away from the crossbar with a device that measured the grip strength in both front paws. While evaluating the grip of the other front paw, one front paw was taped. If the mouse cannot catch the crossbar, a score of 0 was obtained.

The BMS assessed the hind limb movements during locomotion in an open field, with a 0-to 9-point scale (a high score means better hind limb function). This measurement was conducted independently by two blinded experimenters over 5 min at pre-SCI conditions and 1, 2, 3, 7, 14, 21, and 28 days after SCI (n = 8 mice/group).

The tissues were immersed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. The tissue sections were stained with hematoxylin and eosin (H&E). Microscopic observation of the histologic slides was performed using a light microscope.

The tissues were obtained, fixed with 4% paraformaldehyde solution, dehydrated, and embedded in paraffin. 4-µm paraffin coronal sections (having proximity to and located in the injury site) were stained by using a DeadEnd™ Fluorometric terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) System (Solarbio, Beijing, China, and then incubated with secondary antibodies. The cell nuclei were stained with DAPI (Solarbio). Images were obtained under the fluorescence microscope (Olympus, Osaka, Japan).

The samples were obtained and fixed with 4% paraformaldehyde, blocked with 5% BSA, and then incubated with primary antibodies, namely, NLRP3 (Thermo Fisher Scientific, Waltham, MA, United States; 1:100) and caspase-1 (Proteintech, 1:100) and then incubated with secondary antibodies. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich), 6 random high-power fields (200 ×) were chosen, and images were obtained using a fluorescence microscope (Leica, Oskar-Barnack, Germany).

Spinal neurons were obtained from E16 mouse embryos with a modified protocol from Jiao et al. (2020). Briefly, the neonatal pups of C57BL/6 mice were anesthetized by isoflurane, decapitated, and vertebral columns were removed. The spinal cord was isolated and cut into 1-mm³ pieces after gently peeling off the meninges. The tissue pieces were digested by 0.25% trypsin for 10 min at 37°C, and then the cells were resuspended in a serum-free medium, including Neurobasal, B27, and penicillin/streptomycin (50 U/mL). 2.5 × 10⁵ cells were seeded in a 24-well plate at 37°C with 5% CO₂, and supplemented with 10 μM AraC (Sigma-Aldrich, St Louis, MO, United States) after 48 h. On the next day, the medium was replaced with Neurobasal containing supplements, excluding AraC, and changed every three days. The cells were cultured for 10 days and then OGD treatment was performed on them. Briefly, the spinal cord neurons were incubated in DMEM and cultured in an incubator containing 95% N₂/5% CO₂ for 4 h. Then, the DMEM was replaced with a standard neuronal culture medium for 24 h. The cells cultured in a standard neuronal culture medium in the presence of ambient 5% CO₂ served as the control. After OGD treatment, the neurons were either immediately treated with entinostat or not. The vehicle group received an equal volume of PBS.

Cell damage was measured using the CellTiter 96^® Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, United States). The cells were plated in 96-well plates in triplicate at approximately 3 × 10⁴ cells per well and cultured in the growth medium. After treatment, MTS was added to the culture medium and incubated for 2 h at 37°C in 95% humidified air and 5% CO₂. The absorbance was measured at 490 nm using a microplate reader (Thermo Fisher Scientific).

After being fixed in 4% paraformaldehyde for 5 min, the treated cells were washed thrice with PBS and then stained with 500 nM of propidium iodide (PI; Sigma-Aldrich) for 5 min. DAPI (Sigma-Aldrich) was used to stain nuclei, and images were obtained with a fluorescence microscope.

The spinal tissues and the cell culture medium supernatant were collected. A bicinchoninic acid (BCA) assay kit (Invitrogen, Carlsbad, CA, United States) was used to measure the protein concentration. ELISA analysis was performed using the TNF-α, IL-1β, and IL-18 ELISA kit (Beyotime Biotechnology, Shanghai, China) following the manufacturer’s instructions.

Caspase-1 activity was detected using the Caspase-1 Activity Assay Kit (Solarbio). The specific steps were performed according to the manufacturer’s instructions (Solarbio). The protein concentration was ensured from 1 μg/μl to 3 μg/μl. A standard curve was prepared using the pNA standard. Then, the optical density of the specimen was read on a microplate reader (Thermo Fisher Scientific) at 405 nm. The percentage of changes in caspase-1 activity was calculated by the ratio of OD₄₀₅ of the experimental well to that of the normal well.

HDAC enzyme activity was conducted by the EpiQuik HDAC Activity/Inhibition Assay Kit (Colorimetric) (Epigentek, Farmingdale, NY, United States). Briefly, nuclear cell lysates were extracted using the Nuclear Protein Extraction Kit (Solarbio), 50 µg of the lysates was incubated with the HDAC colorimetric substrate, and the color was immediately quantified through an ELISA reaction. HDAC activity was inversely proportional to the amount of the remaining acetylated histone substrates. HDAC activity or inhibition was calculated using the following formula: HDAC activity (OD/min/mg) = ((Sample OD–Blank OD)/ (Protein Amount (50 µg) ×60 min)) × 1,000.

Spinal cord tissue and primary neurons were lysed by lysis buffer (Beyotime, Shanghai, China). After protein quantification, electrophoresis (12% SDS-PAGE gels), and Western transfer, the membranes were blocked with 5% BSA and incubated with the appropriate primary antibodies, such as HDAC1 (CST, 1:1,000), HDAC2 (Abcam, 1:800), HDAC3 (Abcam, 1:1,000), NLRP3 (Thermo Fisher Scientific, 1:1,000), caspase-1 (Proteintech, 1:800), and caspase-1 p20 (CST, 1:1,000) at 4°C overnight and then incubated with secondary antibodies (Abgent, 1: 20,000) followed by the hypersensitivity chemiluminescent substrate (Bio-Rad, Hercules, CA, United States), and protein bands were photographed by using a ChemiDoc™ MP imaging system (Bio-Rad).

Total RNA was lysed by using the TRIzol reagent (Invitrogen) and reversely transcripted to cDNA using a HiFi-MMLV cDNA First-Strand Synthesis Kit (CW-Bio, Beijing, China). QRT-PCR analysis was executed using GoTaq qPCR Master Mix (Tiangen, Beijing, China) by the CFX96TM Real-Time System (Bio-Rad, Hercules, CA, United States). QPCR was performed on samples and standards in triplicate, and fold changes were calculated by applying the relative quantification (2^−ΔΔCt) method. GAPDH was used as an internal control, and primer sequences are listed as follows.

Data were expressed as the mean ± SEM. SPSS 22.0 was used for Student’s t-test or one-way ANOVA followed by Tukey’s multiple comparisons test (two-way ANOVA analysis for grip strength assessment). P < 0.05 was considered statistically significant.

After the onset of SCI within 4 weeks, the motor function of mice was assessed by the forelimb grip strength test and BMS score. Before the experiment, all the mice showed consistent behavioral evaluations. SCI directly impaired motor function with decreased grip strength both of left, right, and paired forepaws (Figures 1A–C), and the grip strength had a slow recovery process during the 1 week following SCI. Entinostat improved the motor function in a concentration-dependent manner, at 7–28 days after SCI (but not during the 7 days following SCI) (Figures 1A–C), where 5 mg/kg and 10 mg/kg of entinostat showed significant protection of motor dysfunction (not the 1 mg/kg group) (p<0.05, Figures 1A–C), but there was still a gap compared with the characteristics in the sham group (Figures 1A–C). At 7–28 days after SCI, the BMS score was significantly improved in the entinostat 5 mg/kg and 10 mg/kg groups than the sham group (p<0.05, Figure 1D). But, the entinostat 1 mg/kg group at 28 days post-SCI showed a statistically significant BMS score alteration compared with that of the sham group (p<0.05, Figure 1D). The data showed that high concentrations of entinostat (5 mg/kg and 10 mg/kg) treatment presented the protection of motor function following SCI.

To explore the protection of entinostat on histopathologic damage, H&E and TUNEL staining were performed to assess the pathologic alteration after treating with 5 mg/kg entinostat. The H&E staining results showed that the structure of the spinal cord was clear, the nucleus was complete and inerratic, and there was hypochromatic nucleus, neutrophil infiltration (as the black arrow indicating), neuronal disruption, and diffuse hemorrhage (Figure 2A). However, entinostat relieved histopathologic damage after SCI (Figure 2A). By TUNEL staining, the results found more TUNEL-positive cells (about 64.3%) in the SCI group compared with the sham group, and entinostat treatment significantly suppressed cell death (about 27.5% TUNEL positive cell) (Figure 2B). These results indicated that entinostat attenuates histopathologic damage and showed protection post-SCI.

After SCI at 7 days, HDACs were measured. As shown in Figure 3A, SCI increased class I HDAC levels, including HDAC1, HDAC2, HDAC3, HDAC6, HDAC8, and HDAC11 mRNA, while entinostat significantly suppressed SCI-induced class I HDAC levels (p < 0.05, Figure 3A). Total HDAC activation was also upregulated after SCI and decreased after entinostat treatment (p < 0.05, Figure 3B). WB results showed that the expressions of HDAC1, HDAC2, and HDAC3 were induced by SCI in the spinal cord and entinostat inhibited the expressions of HDAC1 and HDAC3, but the variation in HDAC2 expression was weak following treatment with entinostat (Figure 3C). The data showed that class I HDAC involved in the pathologic processes of SCI and entinostat suppressed HDAC1 and HDAC3 expression in the spinal cord. Furthermore, we analyzed NLRP3 inflammasome activation after SCI. As shown in Figure 4A, NLRP3 mRNA was increased with time dependence, which peaked at 24 h following SCI, then decreased gradually from 2 to 7 days, but was significantly upregulated at 7 days (p < 0.05, Figure 4A). Then, NLRP3 expression was measured at 7 days post-TBI in the spinal cord of entinostat-treated mice. Undoubtedly, SCI not only induced NLRP3 expression and caspase-1 p 10 fragment but also was significantly suppressed by entinostat compared with the SCI group (p < 0.05, Figure 4B). IF results demonstrated that entinostat inhibited NLRP3/caspase-1 staining (Figure 4C). Furthermore, NLPR3 inflammasome–mediated inflammation was also measured in the spinal cord. ELISA results showed that SCI increased the level of inflammatory factors, such as TNF-α, IL-1β, and IL-18 at 7 days post-SCI, whereas entinostat significantly decreased the levels of TNF-α (p<0.05), IL-1β (p<0.01), and IL-18 (p<0.01) (Figure 4D). Caspase-1 activity assay also demonstrated that entinostat reversed the increase of caspase-1 activity after SCI (p<0.01, Figure 4E). Thus, the results demonstrated that entinostat alleviated NLPR3 inflammasome activation in the spinal cord following SCI.

MTS was used to detect the cell viability of primal neurons. At the beginning of OGD, spinal cord neurons were immediately treated with entinostat (1, 2, and 5 μM) for 24 h or not. As shown in Figure 5A, OGD downregulated cell viability, and high concentration of entinostat (2 and 5 μM) could inhibit the decrease of cell activity, but the effect of 1 μM entinostat was weak (Figure 5A). Then, 2 μM entinostat was treated for 12, 24, 36, and 48 h. The longer the treatment time of entinostat, the better the cell activity of neurons (Figure 5B). So, the treated condition of entinostat was selected as 2 μM for 24 h. As shown in Figures 5C,D, PI staining showed that OGD induced the increase of PI-positive cells, and 2 μM entinostat significantly decreased the PI-positive cells. After entinostat treatment in OGD-induced primal neurons, total HDAC activation and caspase-1 activation were analyzed at 24 h. OGD significantly enhanced the total HDAC (p<0.05, Figure 6A) and caspase-1 (p<0.05, Figure 6B) activation, and entinostat reduced the total HDAC and caspase-1 activation (p<0.05, Figures 6A,B). ELISA results showed that OGD increased the level of inflammatory factors, IL-1β and IL-18, whereas entinostat significantly decreased the levels of IL-1β and IL-18 (p<0.05, Figure 6C). WB results further found that entinostat significantly decreased HDAC3 expression and NLRP3 inflammasome activation (including NLRP3 expression and caspase-1 p 10 level), related to the OGD group (p<0.05, Figure 6D). Furthermore, IF results showed that OGD induced NLRP3 expression and entinostat suppressed NLRP3 expression in the OGD model (Figure 6E). The results demonstrated that entinostat alleviated NLPR3 inflammasome activation and neuronal damage in the OGD model.