All experimental protocols and animal-handling procedures were approved by our institutional Animal Care Committee (No. IACUC-AEWC-F2010007, F2101010, F2105007) and were performed in accordance with the recommendations outlined in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.
Animals and grouping
A total of 210 adult male Sprague–Dawley rats (6–7 weeks, weigh 180–220 g) were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. Rats were housed with a fixed light/dark cycle (12/12 h), a room temperature of 21− 22 ℃, and ad libitum access to food and water. After adaptive feeding for 1 week, rats were randomly divided into different groups. All experiments were designed to minimize the number of animals used and their suffering.
In microarray and RNA-seq, a total of 14 rats were randomly divided into Sham and CCI groups, 7 rats per group. Among them, 3 rats were used for microarray analyses, and 4 rats for RNA sequencing. In addition, there were 12 rats randomly assigned to the two groups to verify the results of microarray and RNA-seq, 6 rats per group.
Moreover, a total of 100 rats were applied for experiments to explore and validate the potential mechanisms, including knockdown of Clec7a and C/EBPβ. There were 50 rats in each experiment randomly assigned to five groups: (1) Sham group; (2) Sham + si-NC group; (3) CCI group; (4) CCI + si-NC group; (5) CCI + si-Clec7a or si-Cebpb group, with 10 rats in each group.
In add-back rescue experiments, a total of 84 rats were randomly divided into six groups, including 9 rats in Sham group and 15 rats in other five groups (CCI group, CCI + pLV.1-NC + pLVSO5-NC group, CCI + pLV.1-shCebpb + pLVSO5-NC group, CCI + pLV.1-NC + pLVSO5-Clec7a-overexpress group and CCI + pLV.1-shCebpb + pLVSO5- Clec7a-overexpress group).
The CCI rat model was established as previously described . Rats were anaesthetised with isoflurane. Briefly, the left common sciatic nerve in each rat was exposed by blunt dissection. The area proximal to the sciatic nerve was free of adhering tissue, and four ligatures were tied loosely around it with approximately 1 mm interval. The sciatic nerve was exposed but not ligated in Sham group animals.
Mechanical allodynia analyses
Mechanical allodynia was measured using the von Frey test (ALMEMO 2390, IITC Life Science, USA) at 1 day before the surgical procedure at days 1, 3, 5, 7 and 9, respectively, after sciatic nerve injury. Rats were placed individually in a transparent plexiglass box on a 50 cm high shelf, and the top of the shelf is a wire mesh with small holes. Rats were acclimated to the test environment at least 20 min. After the rats adapt to being quiet, Von-Frey was applied to the plantar middle of the rat hind paw through the wire mesh for 4–6 s. It is measured once every 30 s for 5 times. The data of mechanical withdrawal threshold (MWT) were shown as the mean of five readings.
Gene expression profiling & data analysis
L4-L6 SC tissues collected from the rats in the Sham and CCI groups on postoperative day 10 were immersed in TRIzol (Invitrogen, CA, USA) and immediately frozen in liquid nitrogen for both the whole-genome microarray (n = 3 per group and mixed one sample for the detection) and RNA-seq (n = 4 per group) studies. Microarray detection was performed using the Affymetrix GeneChip® Rat Gene 2.0 ST Arrays (Affymetrix, Santa Clara, CA) by Shanghai GMINIX Biotechnology Corporation (Shanghai, China). RNA-sequencing was carried out by the Novogene Bioinformatics Technology Co.Ltd (Beijing, China). Sequencing libraries were generated using NEBNextUltra RNA Library Prep Set for Illumina (NEB, USA.) following manufacturer’s recommendations. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq SR Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq 2500/2000 platform. Gene expression profiling was analyzed using R (http://www.rproject.org/, version 3.1.1) with Bioconductor packages (http://www.bioconductor.org/). Raw intensities were normalized using Robust multi-array average (RMA method) . All raw data were be deposited in GEO datasets (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE186237 and https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE185278). Significant differentially expressed genes (DEGs) of the Sham vs. CCI groups were identified using the criteria of a |fold change|> 1.5 and P value < 0.05.
Small interfering RNA (siRNA) and transfection
Clec7a siRNA (si-Clec7a), C/EBPβ siRNA (si-Cebpb) and control siRNAs (si-NC) designed and synthesized by RiboBio (Guangzhou, China) were separately dissolved in Opti-MEM (Gibco, USA). After 5 min of equilibration at room temperature, each siRNA solution was combined with the respective volume of the transfection reagent, mixed gently and incubated for 20 min. BV2 cells were transfected with the transfection mixture in Opti-MEM for 6 h.The cells were then changed to antibiotic-free medium and continued culturing for 48 h before follow-up experiments.
To transfect siRNA, Lipofectamine 2000 (Invitrogen, USA) and in vivo RNA Advanced Transfection (Zeta Life, USA) were used for BV2 cells and rats, respectively. The sequences of siRNAs are provided in Additional file 2: Table S1.
siRNA solution was injected into the unilateral L4–L6 SC on postoperative day 4 at 2 µg in 10 µL, with the use of a glass micropipette connected to a Hamilton syringe. After each injection, a 5 min pipette retention was used before the glass pipette was removed. The expression levels of Clec7a and C/EBPβ proteins following the specific siRNA transfection were detected by Western blot.
Cell culture and simulation
Mouse microglial BV2 cell lines were maintained at 37 ℃ in a humidified atmosphere of 5% CO2 in DMEM medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 100 IU ml−1 penicillin, and 10 μg ml−1 streptomycin (Invitrogen, Waltham, MA).
The BV2 cells were adjusted to 5 × 10  cells well−1 and were seeded in a 6-well plate for 24 h, and then treated with 0.2 μg mL−1 LPS (Sigma, Germany) and 3 mM ATP (Sigma, Germany) for 4 h to establish pyroptosis model.
Generation of stable cell line inducing C/EBPβ knockdown
The Cebpb-shRNA was purchased from General Biosystems (Anhui, China). The pLV.1-shCebpb recombinant plasmid was constructed by HYY medical science Co.,Ltd (Guangdong, China). Briefly, the shRNA sequences and their antisense sequences were cloned separately into the pLV-CMV-GFP-Puro lentiviral vector, downstream of the CMV promoter. The empty vector was used as the control. BV2 cells were infected with pLV.1-Cebpb or control vectors with a multiplicity of infection (MOI) of 50 to 100. Then stable transfectants were screened using 2 μg ml−1 puroMycin (Gibco, USA) to generate BV2-pLV.1-shCtrl and BV2-pLV.1-shCebpb stable cell lines, respectively. The target sequences for shCebpb was provided in Additional file 2: Table S1.
Vector construction and lentivirus infection
shRNAs targeting C/EBPβ (shCebpb) as well as a negative control (shNC) were provided by HYY medical science Co.,Ltd (Guangdong, China). The shCebpb and shNC were amplified and cloned into LV.1-copGFP to generate pLV.1-shCebpb and pLV.1-shNC. The coding sequences of rat Clec7a were amplified and cloned into LVSO5-copGFP (lentivirus gene overexpression vector) to generate pLVSO5-Clec7a successfully. The empty pLVSO5 plasmid was used as a negative control. All constructs were verified by DNA sequencing. The packaging lentivirus and target vector were co-transfected into HEK293T cells and cultured for 48 h. The supernatant was then collected, and the lentivirus particles in the supernatant were filtered to detect virus titer. The 5 μL of concentrated lentivirus particles (1X109 transducing/mL) were administrated intrathecally following 1 week of CCI model construction.
Quantitative real-time PCR
Gene expression levels of Clec7a in BV2 cells in different groups were detected using quantitative real-time PCR with a TaqMan Gene Expression Assay by CFX96TM Real-Time Systems according to the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA). Total RNA was extracted using TRNzol Universal reagent (TIANGEN, DP424) according to the manufacturer’s protocol. RNA concentration was determined by a spectrophotometer at 260 nm and 280 nm. Identical amounts of RNA (2 µg) were reverse transcribed into complementary DNA (cDNA) using a FastKing cDNA synthesis kit (TIANGEN, KR118) according to the manufacturer’s instructions. Relative mRNA levels were calculated using the 2−ΔΔCT method . The sequences of primers are provided in Additional file 2: Table S1.
Western blot analysis
Total proteins were extracted from the L4–L6 spinal cord segment and BV2 cells in different groups using the RIPA lysis buffer(containing 1 mM phenyl-methylsulfonyl fluoride and protease inhibitor), and the protein concentration was measured using the bicinchoninic acid (BCA) protein concentration test kit (Beyotime, Shanghai, China). Equivalent protein amounts were analyzed on 11% and 8% SDS–PAGE and transferred onto polyvinylidene fluoride (PVDF, Millipore) membrane. The membranes were blocked in non-fat dried milk solution(5% in phosphate-buffer saline) at room temperature for 1.5 h, and then incubated at 4 ℃ overnight with primary antibodies. After washing with 1XTBST, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. Bands were visualized with electrochemiluminescence detection reagents. Band intensity was quantified using the Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA). The detailed information of antibodies used in this study is provided in Additional file 2: Table S2.
Following 10 or 30 days of CCI model construction, the L4–L6 spinal cord segments were obtained, then was fixed overnight in 4% paraformaldehyde, subjected to gradient dehydration, and paraffin-embedded. Sagittal serial sections with a slice thickness of 3 μm were generated. Antigen retrieval was performed by heating the slides at 96 ℃ in sodium citrate buffer (pH 6.0) for 15 min. After cooling to room temperature, slides were rinsed in 0.01 M phosphate-buffered saline (PBS), treated with 0.1% Triton X-100 PBS for 30 min, and washed with 0.01 M PBS three times for 3 min each. Subsequently, endogenous peroxidase was quenched using 3% hydrogen peroxide in PBS for 15 min. Sections were then washed with 0.01 M PBS three times for 8 min each and incubated with specific primary antibodies of Clec7a and C/EBPβ at 4 ℃ overnight. After washing in PBS, sections were incubated for 30 min with a secondary antibody at room temperature. Immunoreactivity was developed with PowerDAB for 7 min at room temperature. Subsequently, sections were counterstained with hematoxylin, rinsed in tap water, dehydrated in 100% ethanol and xylene, and mounted with Permount. The expression levels of Clec7a and C/EBPβ proteins among these sections were evaluated by mean optical density (integrated option density/area) using the Image-Pro Plus 6.0 (Media Cybernetics, Dallas, TX, USA). The optical intensity threshold was corrected to 0–250 in the process of analyses. The immunohistochemical image acquisition and quantitative analyses were performed by two observers, independently and blinded for the groups. The inter- and intra-examiner reliability was determined. The detailed information of antibodies used in this study is provided in Additional file 2: Table S2.
Following 10 or 30 days of CCI model construction, the L4–L6 spinal cord segments were prepared on a cryostat and processed for immunofluorescence as described previously . In brief, the sections were washed twice with PBS, treated with 0.2% Triton X-100 for 20 min, and washed twice with PBS. Then, sections were incubated with 10% goat serum for 2 h at room temperature, and then incubated with the primary antibody against Clec7a,NLRP3,Iba-1,GFAP or NeuN overnight at 4 ℃. In addition, the sections were washed three times with PBS and incubated with secondary antibodies for 1 h atroom temperature. The nuclei were stained with DAPI (S2110, Solarbio) After that, the stained sections were examined via laser scanning confocal microscopy (FluoView FV3000, Olympus Co. Tokyo, Japan). The Clec7a expression was analyzed and expressed as mean fluorescent intensity (integrated option density/area) using the Image-Pro Plus 6.0 (Media Cybernetics, Dallas, TX, USA). In the counting studies of double staining labeled cells (Clec7a/Iba-1, Clec7a/GFAP, Clec7a/NeuN, NLRP3/Iba-1, NLRP3/GFAP and NLRP3/NeuN), the co-labeled cells were counted in 3 representative capture views of the spinal dorsal horn (SDH) from 3 animals in each group. Cell counts on the examined sections were then averaged to provide a single value for the specific group. The detailed information of antibodies used in this study is provided in Additional file 2: Table S2.
Rat IL-1β and IL-18 levels were measured by using IL-1β ELISA kit (ml037361, mlbio, Shanghai, China) and IL-18 ELISA kit (ml002816, mlbio, Shanghai, China) according to the manufacturer’s instructions. In brief, samples were measured at 450 nm by spectrophotometer to determine absorbance. The concentrations of IL-1β and IL-18 were obtained by extrapolation from the standard curve.
Cell death in BV2 cells was assessed using LDH Cytotoxicity Assay Kit (Beyotime, Shanghai, China). The supernatants were collected and centrifuged (400 g, 5 min) for LDH activity determination. The absorbance at 495 nm was measured using a microplate reader.
Pyroptosis in the spinal cord and BV2 cells were detected by using a TUNEL Detection kit (ABP-Biosciences, Wuhan, China). The pyroptotic cells showed TUNEL-positive due to random breakage of chromatin DNA. The samples were incubated with a TdT detection mixture for 1 h at 37 ℃ in the dark, then stained with Hoechst 33,342 (Servicebio, Wuhan, China) for nuclear counterstaining and observed under an inverted microscope. TUNEL-positive nucleus was identified as nicks in the terminal end of nucleic acids. Images were randomly selected from three sections of each specimen. The TUNEL-positive rate was calculated as follows: TUNEL-positive rate % = TUNEL-positive nucleus /total nucleus ×100%.
BV2 cells were washed twice with cold PBS and resuspend cells in 1X Binding Buffer, then stained with FITC Annexin V and PI using the Annexin V-FITC Apoptosis Detection kit (BD, Franklin Lakes, NJ, USA). After incubation at RT in the dark for 15 min, the pyroptotic cells were analyzed by flow cytometry (ACEA Biosciences, Santiago, USA).
Caspase-1 activity detection
Caspase-1 activity was measured using a commercial Caspase-1 Assay Kit (ab39412, abcam). Briefly, cells were lysed and the lysates were removed by centrifugation at 14,000 rpm/min for 5 min. 100 μg of total protein was used to assess caspase-1 activity. Cell protein with YVAD-AFC were incubated at 37 ℃ for 2 h protected from light. The changes in fluorescence intensity (Ex/m = 400/505 nm) were monitored by a fluorescence spectrophotometer (Varioskan Flash 4FE, Thermo Scientific, Carlsbad, CA, USA).
Dual-luciferase reporter assay
The 1500-bp sequence upstream of the transcription start site (TSS) of C/EBPβ were amplified and cloned into the effector vector pcDNA3.1-GFP to generate pcDNA3.1-GFP-C/EBPβ. The 2000-bp promoter sequence of Clec7a was truncated in different lengths: p500, p1000, p1500, p2000. Those was respectively amplified and cloned into the reporter vector psiPRO to generate four reporters: psiPRO-Clec7a-: p500, p1000, p1500, p2000. The empty pcDNA3.1-GFP plasmid or psiPRO plasmid was used as a negative control. Based on psiPRO-Clec7a-p500, one mutated reporter was generated, which were mutated at binding site − 483 and − 467. HEK-293 T cells were transfected with these plasmids using Lipofectamine™ 2000 (Invitrogen).After 48 h, the activity of firefly luciferase (FL) and Renilla luciferase (RL) were respectively measured using the Luciferase Assay System protocol (Promega). Relative luciferase activity was represented by FL/RL.
Chromatin immunoprecipitation (ChIP) assay
ChIP was performed using a Pierce Magnetic ChIP kit (Cat.26157, Thermo Fisher, USA) according to the manufacturers’ instructions. In brief, 1 ×107 cells were used in the experiment. The BV2 cells were cross-linked with 1% paraformaldehyde, and the reaction was stopped with 125 mmol/L glycine. Then, they were lysed with lysis buffer, and chromatin was sonicated (amplitude, 120 w; process time, 8 min; ON time, 3 s; OFF time, 9 s) until crosslinked chromatin was broken into150- to 1000-bp fragments by MNase digestion. The chromatin was then immunoprecipitated with 2 μg (0.6 μg/μL, 3.4 μL) of rabbit anti-C/EBPβ antibody (ab32358, Abcam, Cambridge, MA, USA) or 2 μg of rabbit IgG with rotation overnight at 4 ℃. Then, 20 μL of magnetic beads were added into each tube, and the tubes were incubated overnight at 4 ℃ with mixing. The magnetic beads were washed with IP wash buffer five times and then eluted. After purification, the immunoprecipitated chromatin was analyzed by qRT-PCR. The sequences of primers used in this study are provided in Additional file 2: Table S1.
Data analysis were performed using SPSS 21.0 statistical software (SPSS, Inc, Chicago, IL, USA). Unless otherwise stated, values of n indicated in the Figure Legends refer to the number of independent biological replicates. The data obtained from this study were presented as the mean ± SEM. Significance (P-value) was evaluated using either unpaired Student’s t-test or one-way ANOVA followed by Tukey’s multiple comparisons post hoc test or Brown-Forsythe and Welch ANOVA tests followed by a Dunnett's T3 multiple comparisons test for multiple comparisons. Time-series data were analyzed with the two-way ANOVA. P < 0.05 was considered to be statistically significant.