2.1 Animals

The experiments were performed using adult C57BL/6J mice (half males and half females, 20-25 g, 8 weeks old) in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996) and the Guidelines and Policies for Rodent Survival Surgery established by the Animal Care and Use Committee of Jinzhou Medical College and were approved by the Animal Care and Use Committees of Jinzhou Medical College. These animals were maintained at 22°C-24°C with a 12-h/12-h light-dark cycle with free access to food and water for 1 week to allow acclimatization to the environment prior to experimentation.

2.2 SCI model and administration of zinc

All animals were randomly divided into three groups: the sham group, SCI + vehicle group, and SCI + zinc group. We chose to use the method described by Allen to establish a spinal contusion injury model.²⁹ We anesthetized all animals with 10% chloral hydrate (0.3 mL/100 g) combined with 0.03 mg/kg fentanyl,³⁰ and a laminectomy was performed at the T9-T10 level without damaging the dura mater. Moderate contusive injury was made by dropping an impactor (RWD, Cat#Model Ⅲ, USA, 2 mm diameter, 12.5 g, 20 mm in height) onto the surface of the T9-T10 spinal cord.²⁶ The muscles, fascia, and skin were sutured layer by layer and disinfected with iodine, and the mice were placed at a constant suitable temperature in a moderately illuminated chamber. Sham group mice underwent the same surgical procedure except contusion. Postoperative monitoring included manual bladder emptying three times daily until reflexive bladder control was reestablished. Two hours after the injury, zinc gluconate was first injected into the zinc group (30 mg/kg i.p; Biotopped, Beijing, China), and 30 mg/kg zinc (0.1 mL) (Figure 2A) was then given daily until day three; the vehicle group mice were injected with an equivalent volume of isotonic glucose (0.1 mL) as a control.²⁶

2.3 Western blot analysis

Tissue (a 1.0 cm piece from the injury epicenter of the spinal cord) or cells were physically sheared on ice using RIPA lysis containing PMSF buffer for 30 minutes to obtain protein. The debris was removed by centrifugation at 12 000 rpm (25 minutes, 4°C), and the supernatant was immediately stored at −80°C. Then, the proteins obtained from the tissues or cells were quantified by BCA reagents, and equivalent amounts of protein (40 µg) were separated on 8%-15% SDS-PAGE gels, transferred to polyvinylidene fluoride (PVDF) membranes and incubated with the appropriate primary antibodies overnight. The PVDF membranes were washed with TBST and incubated with secondary antibodies (1:5000; Proteintech, USA) for 90 minutes at room temperature. Finally, the bands were detected by BeyoECL Plus, and the signals were visualized via a Imaging System. Primary antibodies were as follows: anti-LC3B (1:1000; Abcam, UK); anti-p62 (1:1,000; Abcam, UK); anti-Beclin1 (1:1000; CST, USA); anti-NLRP3 (1:1000; Affinity, USA); anti-ASC (1:100; Affitiny, USA); anti-Caspase-1 (1:100; CST, USA), anti-IL1β (1:1,000; Abcam, UK); anti-cl-IL1β (1:1,000; Abcam, UK); anti-IL6 (1:1,000; Abcam, UK); anti-Ubiquitin (1:1000; Abcam, UK); anti-β-Actin (1:1000; Proteintech, USA); anti-GAPDH (1:1000; Proteintech, USA).

2.4 Enzyme-linked immunosorbent assay

The levels of IL1, IL-6, and NLRP3 were detected by following the protocol of a commercial ELISA kit. Briefly, the sample, the standard, and a HRP-labeled antibody were added to microwells coated with the antibody, incubated at 37°C and washed thoroughly. The color was developed with TMB substrate, which was converted to blue by peroxidase catalysis and ultimately converted to yellow by the action of acid. The intensity of the color was positively correlated with the expression of the target protein in the sample. The absorbance (OD value) was measured at 450 nm with a microplate reader, and the target protein content in the final sample was calculated from the standard curve.

2.5 Tissue preparation

Animals were anesthetized as described above at a specific time after operation and then intracardially perfused with 0.9% normal saline followed by 4% sodium methyl benzoate. Then, 0.5 cm-long pieces of tissue from above and below the epicenter of the damaged spinal cord were collected. The collected spinal cord tissues were fixed for 3 days and then moved to 30% sucrose in 4% paraformaldehyde until they sunk. Frozen sections (10 µm thick) of the region of the spinal cord that was 3 mm away from the injury epicenter were prepared with a cryostat and stored at −80°C.

Paraffin section's preparation is similar to frozen section. After fixed in 4% paraformaldehyde, the tissues were collected from the same position and length and put it into the dehydration box. Next, sequentially gradient alcohol in the dehydrator for dehydration: 75% alcohol 4 hours, 85% alcohol 2 hours, 95% alcohol 1 hour, anhydrous ethanol twice 30 minutes each time, alcohol benzene 5-10 minutes, xylene twice 5-10 minutes each time, finally dipped in wax for 3 hours. After the dehydration process is completed, embed the wax-soaked tissue in the embedding machine. Finally, place the wax block on a paraffin microtome and got the slices (4 µm thick) and stored at −4°C.

2.6 Immunofluorescence analysis

Mice were sacrificed at the third day after zinc administration. The tissue was prepared as described above. The frozen sections were dried at room temperature for 2 hours. The sections were then incubated at room temperature with 5% normal goat serum containing 0.3% Triton X-100 for 2 hours. Then, 1x PBS was used to wash the section three times for 5 minutes each. Primary antibody was added, and the sections were incubated with gentle shaking for 12 hours at 4°C. On the following day, 1x PBS was used to wash the section three times for 5 minutes each. Next, the sections were incubated with secondary antibody for 8 hours on a shaker at 4°C. 4'6-Diamidino-2-phenylindole (DAPI) solution was used to stain the nuclei. The slices were selected from 0.5 cm-long pieces of tissue from above and below the epicenter of the damaged spinal cord. For each mouse, select every other continuous section and count three sections in total. In the ventral horn area on both sides of the spinal cord, a 200 × 200 µm area was selected to count the number of neuron-positive cell, IBA-1-positive cell, and NLRP3-positive cell. The antibodies used are listed as followed: anti-NeuN (1:1000; abcam, UK), anti-Iba-1 (1:500; Wako, JP), and anti-NLRP3 (1:250, Affinity, USA).

2.7 Nissl staining

The tissue was prepared as described above. We follow the procedure of the Nissl staining kit (Solarbio, Cat#G1436, CN) for staining and the brief description is as follows: First, the paraffin sections were dried at room temperature for 2 hours. Next, the sections were dewaxed twice for 10 minutes each in xylene. Then, the sections were hydrated in 100% ethanol, 95% ethanol, and 70% ethanol for 5 minutes. The slices were rinsed in tap water and later in distilled water. Finally, the sections were stained with 0.5% cresol violet solution for 3-10 minutes and immediately rinsed in distilled water. Next, the sections were differentiated in 95% ethanol for 20 minutes. Then, 100% ethanol was used to dehydrate the sections twice for 5 minutes each. Finally, the sections were washed in xylene for 10 minutes and permanently sealed, and the numbers of Nissl-positive cells were observed and counted under a microscope. The slices were selected from 0.5-cm-long pieces of tissue from above and below the epicenter of the damaged spinal cord. For each mouse, select every other continuous section and count three sections in total. In the ventral horn area on both sides of the spinal cord, a 200 × 200 µm area was selected to count the number of classical Nissl bodies and the average was calculated. The classic Nissl-positive motor neuron is plaque-like as described in the previous article.³¹

2.8 Transmission electron microscope analysis

The mice were sacrificed on the third day after zinc administration and then subjected to special tissue processing required for TEM analysis. The brief description is as follows: All animals were anesthetized as described above at a specific time after operation. After fixing the mouse, infuse the animal with 50 mL of pre-cooled 3% glutaraldehyde fixative solution through the left ventricle. A sharp blade was used to quickly and completely remove a piece of the damaged spinal cord and the tissue was placed in glutaraldehyde fixative for long-term storage. Next, the tissue sample was removed from the fixative solution, washed three times with PBS for five minutes each, and then fixed in 1% acetic acid fixative for 1 hour. After washing three times with PBS, the tissues were placed in 50%, 70%, 80%, 90%, and 100% acetone and double distilled water for dehydration. After dehydration was completed, a permeabilization buffer with a 3:1 ratio of embedding agent to acetone was prepared, and the tissue was permeabilized for 2 hours. Next, embedding was performed under a temperature gradient in an oven at 45°C for 24 hours and at 60°C for 48 hours. After successful embedding, 60-nm tissue slices were cut with an ultrathin microtome, stained with 1% uranium acetate-lead citrate, and finally observed the autophagosome under a transmission electron microscope. A typical autophagosome is a vesicle structure with a double-layer membrane around 500 nm. The slices were selected from 0.5 cm-long pieces of tissue from above and below the epicenter of the damaged spinal cord. Observe the number of typical autophagosomes around a single cell nucleus under the EM4200X field of view. Three consecutive discontinuous slices were taken for each tissue sample, three fields of view were selected for statistics and the average was taken.

2.9 Behavioral assessment

On days 1, 3, 7, 14, 21, and 28, the Basso Mouse Scale (BMS) locomotor rating scale was used to evaluate the functional recovery of the injured animals. Scoring ranging from 0 to 9 points (9 meaning completely normal and 0 meaning completely paralyzed) were assigned. The mice were placed in an open field arena and allowed to move freely for 5 minutes. The range of motion, coordination, paw posture, trunk stability, and posture of the hind limbs and tail of the mice were observed and scored. Observations and scoring were performed independently by three examiners who were unaware of the grouping of the mice, and the experiment was repeated three times.

2.10 Cell culture and treatment

BV2 cells (a microglia cell line) were cultured in DMEM supplemented with 10% FBS and 50 µg/mL penicillin/streptomycin at 37°C and 5% CO₂. BV2 cells were stimulated with LPS (1 µg/mL) and ATP (5 mmol/L) for 6 hours to induce a proinflammatory phenotype involving NLRP3 protein expression. After successfull induction of an increase in NLRP3 inflammasome expression, the cells were treated with zinc gluconate (100 µmol/L) for 6 hours. LPS-primed BV2 cells were cultured with 3-MA (5 mmol/L) or bafilomycin A1 (100 nmol/L) for 2 hours before being treated with zinc. LPS-primed BV2 cells were treated with MG-132 (10 µmol/L) 2 hours before collection. 3-MA and Bafilomycin A1 are two autophagy inhibitors widely used in research. The former function is to prevent the formation of vesicle double-layer membrane, while the latter is to prevent the binding of autophagosomes and lysosomes to inhibit autophagy.¹⁶ In the study of ubiquitination modification, MG-132 is a class of highly effective inhibitors that can inhibit cell ubiquitination.³²

2.11 Confocal fluorescence microscopy of cells

Cells were cultured in confocal culture dishes in the manner described above, removed, placed on a slide at 4°C, washed three times with 1x PBS for 5 minutes each, fixed with 4% PFA for 30 minutes, washed again with PBS three times, permeabilized with 1‰ Triton X-100 for 30 minutes, blocked with sheep serum for 2 hours, washed with PBS three times, and incubated with an appropriate dilution of primary antibody in a 4°C refrigerator overnight. On the second day, after three washes in PBS, the cells were incubated with a suitable dilution of secondary antibody at room temperature for 4 hours, washed with PBS and incubated with DAPI for 30 minutes. Finally, the cells were incubated with ghost dye at room temperature in the dark, incubated for 30 minutes, and observed and imaged with a high-resolution confocal microscope.

2.12 Immunoprecipitation assay

A total of 100 µL 1x IP lysis buffer (containing protease inhibitor) was added to the collected cell, and the cells were lysed on ice for 30 minutes. The supernatant was centrifuged at 12 000 g for 30 minutes. After that, the precleaned lysates were mixed with 1 µg primary antibody and 50 µL protein A/G beads. The mixture was shaken gently at 4°C and incubated overnight. After the immunoprecipitation reaction, the samples were centrifuged at a speed of 3000 g for 5 minutes at 4°C to collect the protein A/G beads at the bottom of each tube. The supernatant was carefully removed. The protein A/G beads were washed with 1 mL 0.1x IP buffer three to four times. A total of 50 µL of 2x SDS sample buffer was added, and the samples were boiled for 10 minutes. Finally, 20 µL of each sample was separated by SDS-PAGE for Western blot analysis.

2.13 Statistical analysis

SPSS 21.0 was used for analysis, and all the data are expressed as the means ± SD Shapiro-Wilk test was used to assess data distribution. Two experimental groups were analyzed by using Mann-Whitney U test, when more than two groups were compared, one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test was used, except for BMS scoring analyzed by two-way analysis of variance (ANOVA) following Tukey's post hoc test. The Kruskal-Wallis test was used instead when the variance was not equal. In all analyses, P < .05 indicated a statistically significant difference.

3.1 NLRP3 inflammasome cytokine activation after SCI

Inflammatory cytokines were detected in injured spinal cord segment samples by WB (Figure 1A) and ELISA on days 1, 3, 5, and 7 after trauma, and sham group samples were used as negative controls. The protein expression of IL-1 and IL-6 reached a peak on the third day after SCI (Figure 1B). ELISA analysis showed that the levels of IL-1 and IL-6 reached the highest point on the third day levels (Figure 1C,D). On this basis, we analyzed the expression of the four main inflammasomes, the NLRP1, AIM2, NLRP3, and NLRC4 inflammasome, in the spinal cord (Figure 1E) by WB, and the results showed that the NLRP3 inflammasome was the most prominently expressed inflammasome (Figure 1F). ELISA analysis showed that the level of NLRP3 in the SCI group was higher than that in the sham group on the third day (Figure 1G).

3.2 Zinc promotes the recovery of motor neurons following SCI

Zinc gluconate is a kind of organic compound (Figure 2A). According to the design of this study (Figure 2B), we evaluated the neuroprotective effects of zinc by using BMS scores. The scores of the vehicle group were not statistically different from those of the zinc treatment group until three weeks postcontusion (Figure 2C). To confirm these results, Nissl staining and IF analysis were used to detect the living neuron cells (Figure 2D,F). The number of living neurons in the anterior horn of the zinc group was higher than that in the vehicle group (Figure 2E,G).

3.3 Zinc promotes the expression of autophagy-related proteins after SCI

To investigate the potential mechanism of the neuroprotective effects of zinc, we examined the levels of the autophagy-related proteins P62, LC3B, and Beclin1 by using Western blot analysis (Figure 3A). It is worth clarifying that the protein expression level of LC3BII/I and Beclin1 reflects the activity of autophagy. The level of P62, unlike LC3BII/LC3BI and Beclin1, is negatively correlated with autophagy. The results showed that P62 expression was suppressed in the zinc-treated group compared to the vehicle group (Figure 3B). The vehicle group showed a decrease in autophagy compared to that the sham group, while zinc treatment caused a significant rise in the protein expression of LC3BII and Beclin1 compared to that in the vehicle group (Figure 3C,D), indicating that zinc has the potential ability to promote the autophagy after SCI. At the same time, we performed transmission electron microscopy on injured spinal cord tissues (Figure 3E), and the results showed that the number of autophagosomes increased significantly after zinc treatment (Figure 3F).

3.4 Treatment with zinc suppresses the NLRP3 inflammasome after SCI

Further study showed that zinc treatment inhibited NLRP3 inflammasome activation in microglia. WB was used to detect the NLRP3 inflammasome-related proteins NLRP3, ASC, IL-1β, and Caspase-1 (Figure 4A). Compared with sham group mice, mice with spinal cord injury expressed high levels of NLRP3, ASC, IL1, and Caspase-1, and this increase in protein expression was suppressed by zinc treatment (Figure 4B-E). IF analysis of NLRP3 was carried out to confirm these results (Figure 4F). The proportion of NLRP3-positive IBA-1 cell in the zinc group tended to be decreased compared to that in the SCI group (Figure 4G). ELISA for NLRP3 showed that zinc treatment significantly inhibited the expression of NLRP3 after SCI (Figure 4H).

3.5 Zinc inhibits the NLRP3 inflammasome in LPS-treated BV2 cells by activating autophagy

To investigate the complex relationship between autophagy and NLRP3 after zinc treatment, we successfully induced an inflammatory phenotype involving the NLRP3 inflammasome in BV2 cells with LPS and ATP for 6 hours, as detected by WB and ELISA (Figure S1). The expression of NLRP3 and ASC did not change significantly when only LPS was applied, and NLRP3 was successfully induced after additional treated with ATP (Figure S1B-D). The MTT assay was used to confirm the protective effect of zinc in LPS-primed BV2 cells (Figure S2A,B). As mentioned earlier, treatment with excess zinc causes some cytotoxicity.²⁶ When the concentration of zinc was greater than 120 µmol, the survival of BV2 cells was significantly inhibited (Figure S2A). In addition, we used JC-1 staining to evaluate the mitochondrial membrane potential of BV2 cells under different treatment conditions (Figure S2C). The results showed that the zinc treatment group can significantly enhance the aggregation of JC-1 and mitochondrial matrix, indicating that the administration of zinc significantly improves the mitochondrial function of the cells and reduces the level of apoptosis (Figure S2D). The levels of NLRP3 inflammasome- and autophagy-related proteins were examined by WB (Figure 5A). 3-MA is a highly effective autophagy inhibitor in BV2 cells. In the zinc-treated group, the expression level of P62 was lower than that in the LPS and ATP-treated group, and the effect of 3-MA increased the level of P62 (Figure 5B). The same trend was also observed for the protein expression levels of NLRP3, ASC, IL-1, and Caspase-1. The effect of zinc also enhanced the expression of autophagy-related proteins (Figure 5E-H). The levels of LC3B and Beclin1 in the zinc-treated group were significantly higher than those in the LPS and ATP-treated group (Figure 5C,D). We also used cell confocal microscopy to observe the expression of NLRP3 in BV2 cells (Figure 5I). After zinc application, the average fluorescence intensity was significantly lower than that in the LPS-treated BV2 cells. Additionally, 3-MA significantly increased NLRP3 levels (Figure 5J). NLRP3 levels in the BV2 cell culture medium were detected by ELISA to confirm the above changes. As expected, the level of NLRP3 was lower in the zinc-treated group than in the LPS and ATP-treated group, and 3-MA application caused a trend compared to that in the zinc-treated group (Figure 5K). Bafilomycin A1 (BafA1) blocks autophagosome and lysosome fusion to inhibit autophagy and is often used to detect autophagic flux, which was detected by WB in BV2 cells (Figure 5L). The expression of p62 in the zinc-treated group was lower than that in the Baf A1-treated group and the 3-MA-treated group (Figure 5M). The effect of BafA1 increased the proportion of LC3BII, unlike 3-MA, which reduced the expression of LC3BII (Figure 5N). All the data showed that after 3-MA application, the inhibition of the NLRP3 inflammasome was abolished after the zinc-induced increase in autophagy was blocked. This result indicates that zinc inhibits the NLRP3 inflammasome in LPS-treated BV2 cells by activating autophagy.

3.6 Zinc-induced ubiquitination facilitates the degradation of the NLRP3 inflammasome

To determine how zinc exerts its anti-inflammatory effects, we used co-IP to detect changes in the protein expression of ubiquitin during the process of autophagy. We found that zinc treatment increased the protein level of ubiquitin compared with that in the LPS + ATP-treated group (Figure 6A). MG-132, which is a ubiquitin inhibitor, was applied to confirm the effect of zinc (Figure 6B) by WB analysis. Compared with BV2 cells not treated with MG-132, BV2 cells treated with MG-132 showed higher NLRP3 protein levels, while LC3B levels did not change significantly (Figure 6C,D). Thus, we used co-IP to demonstrate the complicated relationship (Figure 6E). The results showed that NLRP3 can bind to ubiquitin, which suggests that zinc treatment can recruit ubiquitin to bind to NLRP3 and cause the degradation of NLRP3 via the ubiquitination modification system. We used ELISA analysis to confirm these results. The level of NLRP3 in LPS-primed BV2 cells treated with MG-132 was higher than that in the zinc-treated group (Figure 6F). All these results indicate that the promotion of ubiquitination by zinc facilitates the degradation of the NLRP3 inflammasome.