Reagents and antibodies
The Resveratrol (RES) and microglial activator lipopolysaccharide (LPS) were all purchased from Sigma-Aldrich (USA). The primary antibodies used in this study included mouse anti-Sirt1 (Abcam, USA); mouse anti-Neurofilament (Novus, USA) ;rabbit anti-iNOS, Arginase (Santa Cruze Biotechnology); rabbit anti-GFAP,CD206 and β-actin (Abcam, USA); rabbit anti-p65, p-p65, STAT3, p-STAT3 (Cell Signaling Technology, USA) ;Rat anti-F4/80 (Abcam, USA).The secondary antibodies used in this study included Alexa-488 and Alexa-594 conjugated-goat anti-rabbit IgG (H+L) or goat anti-rabbit IgG(H+L) (Jackson ImmunoResearch, USA); horseradish peroxidase–conjugated goat anti-rabbit IgG (H+L) and horseradish peroxidase–conjugated goat anti-Mouse IgG (H + L) (Invitrogen, USA); Nuclei was stained with DAPI dihydrochloride (Thermo Fisher Scientific, USA).
SCI mouse model
All procedures strictly complied with the recommended National Institute of Health Guidelines for Laboratory Animals, which were approved by the Ethics Committee of Wannan Medical College, Wuhu, China. All 6 weeks old male C57BL/6 mice were purchased from the animal testing center, which were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg of body weight) and underwent a laminectomy at vertebral level T10. After exposing the dorsal surface of spinal cords, a rod in the weight 5g was dropped from a height of 6 cm to hit the spinal cord tissues. After the operation accomplished, the skin was sutured. Auxiliary urination was performed once daily until bladder function recovery. Mice fulfilling SCI models were randomly divided into two groups, SCI group and Resveratrol (RES)group (n = 15 / group). The SCI-only group served as the control. RES was intraperitoneally injected at a dose of 200mg/kg daily for 7 days in the treatment group for subsequent analysis.
Tissue processing
The mice after SCI were sacrificed with an overdose of pentobarbital sodium. After opening the chest, 10ml pre-cooled normal saline and 10ml 4% paraformaldehyde were perfused through heart successively. The spines were exposed, and the spinal cord tissues containing the lesion site were carefully extracted. The tissues were fixed in 4% paraformaldehyde overnight, followed by gradient dehydration with sucrose solution. The spinal cord tissues were embedded with OCT (SAKURA, Japan) and cut into 18µm thick sections, which were stored at -40℃.
Basso Mouse Scale (BMS) scores
Basso Mouse Scale (BMS) was used to evaluate the motor function after SCI. The motor function was quantified by scores ranged from 0 to 9 according to the rules.
Footprint analysis
The front and rear paws of mice were dyed with different colors of ink. Mice of different groups were placed into a narrow passage 28 days post injury. After crawling through the passage, marked track was left on the paper. Taken photos of the track to analysis.
Swimming test
Swimming test was used to evaluate the motor function recovery of mice after SCI. It is easy to operate, repeatable and non-invasive. Mice of different groups were placed into a transparent tank filled with water 28 days post injury. Photos were taken during swimming.
Nissl staining
The spinal cord tissues were stained with Nissl staining solution on the 28th day post injury. Spinal cord slices were fixed with 4% paraformaldehyde at room temperature for 10 min. After washing by ddH2O, the slices were stained with Nissl staining solution (Beyotime, China) for 10 min. After staining, the sections were dehydrated in 95% alcohol for 2 minutes twice, then cleared in xylene for 5 minutes twice. The images were captured by microscope.
BV2 Cell culture and treatment
The BV2 microglial cell line was obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China). The complete cell culture medium was mixed with Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific, USA), 10% fetal bovine serum (FBS, Thermo Fisher Scientific, USA),and 1% PS(DMEM, Thermo Fisher Scientific, USA), which was refreshed every 2 days. The BV2 cells were cultured at 37°C with 5% CO2. When the BV2 cells grew to a confluence of about 60%, the medium was replaced with serum-free DMEM, and incubated with LPS (100ng/ml) with or without RES for 24 h.Then, the cells were used for further experiments.
Western blot analysis
Total protein was extracted from cells by RIPA lysis (Beyotime, China). Protein concentration was examined by Bradford Protein Assay Kit (Beyotime, China). Protein samples were separated by SDS-PAGE through electrophoresis. The protein samples were then transferred to PVDF membrane (Millipore, USA).C was then blocked in 5% bovine serum albumin at room temperature for 1.5 h. After blocking, ncubated overnight at 4 °C with primary antibodies. The next day, membrane were incubated in at room temperature for 2 h. Reacting bands were exposed by chemiluminescence kit (Beyotime), and the density of protein bands was quantified by Image J (National Institutes of Health, USA).
Immunofluorescence staining
Spinal cord tissues or cultured BV2 cells were fixed with 4% paraformaldehyde at room temperature for 10 min. After washing by PBS, the samples were permeabilized with 0.2% Triton X-100 for 5 min, and then blocked with 10% donkey serum for 1 h. After blocking, the samples were incubated at 4°C overnight with primary antibodies. Fluor 488– and Alexa Flour 594–conjugated goat secondary antibodies were applied to label primary antibodies respectively at room temperature for 1 h. After washing by PBS, nuclei were stained with DAPI (Beyotime). The images were captured by fluorescence microscope (Biotek, USA).
Real-time reverse transcription polymerase chain reaction (RT-qPCR)
Total RNA was extracted from the cells after different treatments using Trizol Reagent (Invitrogen, USA) and the cDNA was amplified using the HiScript II Q RT SuperMix for qPCR (Vazyme, China) according to the manufacturer’s instructions. The qPCR was performed with SYBR Green PCR Master Mix (Vazyme, China) by ABI steponeplus real-time PCR system (Applied Biosystems, USA). The level of expression was standardized to GAPDH or U6, and the relative expression level was evaluated using the 2-ΔΔCT approach. The primers (GENEbay, China) were synthesized by the following sequence:
IL-1β:5’-GCAACTGTTCCTGAACTCAACT-3’(forward),5’-ATCTTTTGGGGTCCGTCAACT-3’ (reverse).
iNOS:5’-GTTCTCAGCCCAACAATACAAGA-3’(forward),5’GTGGACGGGTCGATGTCAC-3’ (reverse)
Arg-1: 5’-CTCCAAGCCAAAGTCCTTAGAG-3’ (forward) 5’-AGGAGCTGTCATTAGGGACATC-3’ (reverse).
CD206:5’-CTGCAGATGGGTGGGTTATT-3’(forward),5’-GGCATTGATGCTGCTGTTATG-3’ (reverse).
GAPDH: 5’-AGGTCGGTGTGAACGGATTTG-3’ (forward),5’-TGTAGACCATGTAGTTGAGGTCA-3’ (reverse).
Statistical analyses
SPSS and GraphPad Prism were used for statistical analysis. All quantitative data results were presented in the form of mean ± standard deviation. T-test was used to compare the differences between two groups, and one-way ANOVA was used to compare the data between multiple groups. The differences were considered significant at P value < 0.05. All experimental data were repeated three times.