SCI model
Male Sprague–Dawley rats (6 weeks, 220–250 g) were purchased from Animal Experimental Center of Zhejiang University. All rats were housed at 22–23 °C, 55–60% humidness, 8:00–20:00 light/ dark cycle, and received standard rodent chow and access to water ad libitum. This study was approved by Ethical Committee of The First Affiliated Hospital of Zhejiang University.
Rats were fixed in the prone position and anesthetized by 35 mg/kg of pentobarbital. Skin was cut to 2–3 cm middle incision in the back, and vertebral T7–T9 were exposed. After stabilizing vertebral T7 and T9, a laminectomy was performed at the thoracic level T8. A syringe needle was used to induce the injury, which was released from a height of 12.5 mm above the surface of the cord, inflicting a moderate contusion. Hemostatic suture was performed layer by layer, and alcohol was then applied for disinfection.
BBB score, the water content of spinal cord and HE staining
After 24 h of induction SCI, BBB test was performed as locomotor rating scale of 0 (no observable hind-limb movements) to 21 (normal locomotion) [12]. After 24 h, rat was intraperitoneally anesthetized with 35 mg/kg pentobarbital sodium and sacrificed using decollation. Spinal cord tissue samples were collected and cut into two parts. One part tissue was weight as damp weight. Then, it was dried at 80 °C and weighed as dry weight. The water content of spinal cord was calculated (damp weight − dry weight) /damp weight × 100%.
Next, part tissue fixed with 4% paraformaldehyde for 24 h, dehydrated, and embedded in OCT compound. Tissues were cut in the sagittal or axial plane into 10-μm sections. Thick sections were stained with hematoxylineosin (H&E) for 15 min. Tissue sample sections were examined by a light microscope (Leica DFC280, UK).
Gene expression profiling
Total RNA was amplified into Affymetrix HG-U133 Plus 2.0 GeneChip arrays (Affymetrix, Santa Clara, CA, USA). Data were analyzed through Database for Annotation, Visualization, and Integrated Discovery (DAVID Database), and QIAGEN’s Ingenuity Pathway Analysis (IPA, QIAGEN, Redwood City, USA).
Real-time PCR analysis. Total RNA extracted from tissue samples and cell samples with Trizol (Invitrogen USA) and converted into cDNA by using M-MLV reverse transcriptase and cDNA Synthesis Kit (Invitrogen USA). Real-time PCR was conducted by using iCycler RealTime PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) with a SYBR ExScript RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China). The thermal cycling conditions were as follows: 95.0 °C for 10 min, 40 cycles of 95.0 °C for 15 s, 60.0 °C for 30 s and 72.0 °C for 5 min. The relative expression level was presented as 2−ΔΔCt [13].
ELISA kits. Tissue samples and cell samples were collected, and proteins were extracted by RIPA lysis buffer. 10 μg protein was used to analyze TNF-α, IL-1β, IL-6 and IL-18 levels using ELISA KITS.
Cell culture and transfection
PC12 cell was incubated using the Dulbecco’s Modifed Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) at an incubator with 5% CO2 at 37 °C. HIF-1α plasmid, miR-143, anti- miR-143 and negative mimics were transfected into cell using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA). After 48 h of transfection, Neuro-2a cell was induced with 200 ng/mL of LPS for 4 h. Next, after 4 h of transfection, Neuro-2a cell was treated with LC3 inhibitor (3-Methyladenine, 5 μM) or autophagy agonist (1 μM resveratrol) for 4 h, and Neuro-2a cell was induced with 100 ng/mL of LPS for 4 h.
Western blot analysis
Cells were harvested and proteins were extracted by RIPA lysis buffer. Proteins were separated on 10% polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were then blocked with 5% non-fat milk in Tris-buffered saline containing Tween-20 (TBST). at room temperature for 1 h and incubated with anti-HIF-1α, anti-p65, anti-LC3, anti-p62, anti-GAPDH (Cell signaling Technology, Beverly, MA, USA) at 4 °C overnight. Membranes were subsequently incubated with goat anti-rabbit peroxidase-conjugated secondary antibodies (Cell signaling Technology, Beverly, MA, USA) at 37 °C for 1 h. Membranes was detected using an enhanced chemiluminescence kit and Image Lab 3.0 (Bio-Rad Laboratories, Inc.).
Immunohistochemistry
Cell was washed with PBS and fixed with 4% paraformaldehyde for 15 min. Cell was washed with PBS and blocked with 5%-BSA supplemented with 0.25%-Tris-X100 in PBS for 1 h at room temperature. Cell was washed with PBS and incubated with anti-HIF-1α and anti-LC3 at 4 °C overnight. Cell was washed with PBS and incubated with 555- or 488- goat anti-rabbit peroxidase-conjugated secondary antibodies (1:100) room temperature for 1 h. Cell was washed with PBS and incubated with DAPI assay for 15 min at darkness.
Statistical analysis
All data are represented as mean ± SD for three independent experiments. Student’s t-tests or one-way analysis of variance (ANOVA) and Tukey’s post test were used for all comparisons involving continuous dependent variables. Results were considered statistically significant when the p-value was < 0.05.
