2.1 Animals
The C57BL/6 clean-grade female mice were purchased from the Animal center of Chinese Academy of Science (Shanghai, China), and housed in a humidity and temperature-controlled environment with free access to food and water (20-25°C, 65% humidity, 12h light/dark cycle). All animal experiments involved in this study followed the guidelines established by the Animal Ethical and Welfare Committee of Huashan Hospital, Fudan University.
2.2 Mouse model of SCI
The SCI mice model was prepared following the protocol as described in our previous research[5]. In brief, C57BL/6 female mice were weighed and anesthetized with intraperitoneal injection of 4% pentobarbital sodium (35mg/kg). The T8-T9 vertebrae were removed by laminectomy and the spinal cord was exposed. The mice in SCI group were inflicted by laterally compressing the cord using Dumont-type forceps with a spacer of 0.2mm for 20s. The mice in Sham group received laminectomy alone without injury to the spinal cord. All the wounds were washed with sterile saline and sutured layer by layer. Following surgery, Penicillin (20,000 U) was injected intramuscularly once a day for 3 consecutive days and bladder massage was conducted manually 3 times a day to assist urination.
After operation, the mice in SCI-Melatonin group received daily intraperitoneal injections of 10mg/kg melatonin (Sigma-Aldrich, St. Louis, MO, USA) for 3 days (for histological analysis) or 28 days (for BMS score analysis). In SCI-C176 group, the STING antagonist C-176 (MedChemExpress, 750nmol dissolved in 200μL corn oil) was firstly injected intraperitoneally at 1h post-injury [38], and then be used as the same way as melatonin.
2.3 Assessment for motor function of mice
The Basso Mouse Scale (BMS) score (0 to 9 points) was recorded at 1, 3, 5, 7, 14, 21 and 28 days after surgery to assess the locomotion function of the mice[39]. Briefly, the mice were allowed to move freely for 5 minutes in an open field, and the hindlimb movements, stepping and coordination points were assessed by two investigators who were blinded to treatment.
2.4 Cell culture and drug treatments
The murine BV2 microglia cell line was purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China) and cultured in DMEM medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and an antibiotic mixture (1% penicillin/streptomycin) (Invitrogen, Carlsbad, CA, USA) at 37 °C and 5% CO2.
The cells were randomly assigned into the control and the LPS/ATP treatment groups. In LPS/ATP group, BV2 cells were exposed to the 1000 ng/ml LPS (Sigma-Aldrich, St. Louis, MO, USA) for 4h, followed by 5mM ATP (Sigma-Aldrich, St. Louis, MO, USA) for 2h to induce pyroptosis. In Melatonin group, the cells were pre-treated with melatonin at the concentration of 5μM or 10 μM for 24h before LPS/ATP stimulation.
The adenoviral infection was adopted to overexpress STING in BV2 cells according to the manufacturers' instructions. Adv-control or Adv-STING were designed and synthesized by Han Heng (Shanghai, China). Briefly, when cells reached 60% confluence, they were infected with Adv-control or Adv-STING at different multiplicity of infection (MOI), followed by incubation for 6 h. Then, the culture medium was replaced with fresh completed DMEM.
2.5 Cell viability assay
The cell viability of BV2 cells after melatonin treatment was assessed by CCK-8 assay (Sigma-Aldrich, MO, USA) according to the manufacture’s protocol. Briefly, BV2 cells were seeded in the 96-well plates and cultured for 24h, followed by a series concentration of melatonin (5, 10, 100, 500, 1000μM) treatment for 24h. Then the culture medium was removed and 110μL CCK8 work solution (containing 10μL CCK8 reagent) was added in each well. After incubation for 2h, the absorbance of each well was detected at 450nm.
2.6 Cell Morphological observation
Following drug treatment (LPS/ATP, melatonin), the medium was removed and the 4% formaldehyde was added for 30min. After that, cells washed with PBS for 3 times (10min per time), then the morphology of BV2 cells was photographed under light microscope.
2.7 Propidium iodide staining
After corresponding treatment, BV2 cells were stained with propidium iodide staining (PI) and Hoechst for 15min and the PI positive cells were observed under the fluorescence microscope (Leica, Germany).
2.8 Cell death assay
The secreted levels of lactate dehydrogenase (LDH) were detected using the LDH Cytotoxicity Assay Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions.
2.9 Enzyme-linked immunosorbent assay (ELISA)
The levels of IL-1β, IL-18 and TNF-α in cell culture supernatants and mouse serum were measured using commercial ELISA kits (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s protocol. The absorbance was detected by microplate reader (Thermo Scientific, Waltham, MA, USA) at 450 nm wavelength.
2.10 Measurement of cGAMP
cGAMP in spinal cord tissue and cell lysate was measured by chemiluminescent enzyme immunoassay (2’3’-cGAMP ELISA kit, Cayman Chemical, USA) according to manufacturers’ protocols.
2.11 RNA extraction and quantitative real-time PCR
Total RNA was extracted from the spinal cord tissue or the harvested BV2 cells using Trizol reagent (Invitrogen, San Diego, CA, USA) and reverse-transcribed, then cDNAs were amplified according to the manufacturer’s instruction. The SYBR Green PCR Master Mix Kit (Applied Biosystems, Shanghai, CHINA) was used to quantify the relative mRNA levels. β-actin served as the internal controls and the 2-ΔΔCT method was adopted to analyze the relative expression levels of mRNAs. Primer sequences are listed in Table.1.
2.12 Western blotting (WB) analysis
Total protein was extracted from the spinal cord tissues and BV2 cells using RIPA buffer containing phosphatase and protease inhibitor cocktail, and then quantified by the BCA Protein Assay kit. Protein samples were separated by 10% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After blocked with 5% non-fat milk at room temperature for 1 h , the membranes were incubated overnight at 4℃ with primary antibodies: NLRP3 (1:1000, CST, 15101s), IL-1β (1:1000, ABclonal, A16288), CASP-1 (1:1000, ABclonal, A18646), p65 (1:1000, CST, 8242), P-p65 (1:1000, CST, 3033), GSDMD (1:1000, Abcam, ab209845), ASC (1:1000, ABclonal, A11433), STING (1:1000, CST, 13647), TBK1 (1:2000, abcam, ab40676), P-TBK1 (1:1000, CST, 5483), IRF3 (1:1000, abcam, ab68481), P-IRF3 (1:1000, CST, 4947) and β-actin (1:2000, Abcam, ab8245). Following washed with TBST for 3 times, the HRP-conjugated secondary antibody (1:2000) was added and incubated at room temperature for 1 h. The protein bands were visualized using an enhanced chemiluminescence (ECL) kit, and blots were quantified by the gel imaging system (UVP LLC, Upland, CA, USA).
2.13 Histological analysis and immunofluorescence
On the third day after SCI, the mice (n=10) were sacrificed and the spinal cord segments near the lesion epicenter were harvested. The samples were then fixed with 4% paraformaldehyde overnight followed by embedded in paraffin. Hematoxylin and eosin (H&E) staining was performed according to the manufacturer’s instructions. For Nissl staining, the sections were stained with toluidine blue following a standard protocol.
Immunofluorescence analysis was performed as previously described[5]. The sections of spinal cord tissue or BV2 cells in each group were fixed with 4% paraformaldehyde for 30 min and then blocked in 5% bovine serum albumin (BSA) containing 0.3% Triton X-100. Then the samples were incubated with primary antibodies overnight at 4℃: GSDMD (1:100, Abcam, ab209845), NLRP3 (1:100, ABclonal, A12694), STING (1:100, Abcam, ab210070), IRF-3(1:100, ABclonal, A0816), A. On the second day, the samples were incubated with appropriate fluorescently labeled secondary antibodies (1:250, Molecular Probes) and counterstained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich). All images were observed with an optical or fluorescence microscope (Leica, Germany).
2.14 Measurement of ROS production
Total ROS production was detected by commercial fluorescence probe. Briefly, BV2 cells pretreated with or without melatonin were stimulated by LPS/ATP and then incubated in the dark with 10 μM DCFH-DA(MedChemExpress, USA)at 37℃ for 30 min. Then cells were washed with PBS to remove the extracellular DCFH-DA followed by observing under the fluorescence microscope (Leica, Germany). Relative ROS production was quantified by the change of fluorescence intensity comparing to the control cells at isosmolar condition.
2.15 mtDNA DNA isolation and mtDNA copy number analysis
mtDNA was evaluated by RT-qPCR as previously described[40]. In brief, BV2 cells were harvested after corresponding treatment in different groups. Thereafter, cytosolic fractions were isolated by centrifugation, and the DNA was isolated from the whole cell extracts and cytosolic fractions respectively (Qiaquick nucleotide removal columns, Qiagen). The whole cell extracts served as control for total mtDNA.
2.16 Statistical analysis
All results are presented as means ± standard deviation. Student’s unpaired t tests and one-way ANOVA analysis of variance (ANOVA) followed by Dunnett’s test were used to analyze all data. P <0.05 was considered to be statistically significant. All statistical analysis was performed with the SPSS 22.0 software (IBM, NY).