Mice
Female C57BL/6 mice (6-8 weeks old, 20 ± 2 g) were purchased from Vitallihua Experimental Animal Co., Ltd. (Beijing, China). All mice were housed in a specific pathogen-free facility. All experiments in this study were approved and performed in accordance with the guidelines of the Animal Ethics Committee of Xiamen University (Approval ID: XDYX2015008).
ATO
Arsenious acid and sodium chloride injection was purchased from Harbin Medical University Pharmaceutical Co., Ltd. (Harbin, China) with an ATO concentration of is 1 mg/mL. A working solution was prepared for in vitro and in vivo experiments by diluting the injection in normal saline.
EAE induction and ATO treatment
To induce EAE, female C57BL/6 mice were immunized with 200 μg of MOG35-55 peptide (BAM Biotech Co., Ltd., Xiamen, China) in complete Freund's adjuvant (Sigma, MO, USA) supplemented with 2.5 mg/mL H37RA (Cohesion Biosciences, CA, England). Pertussis toxin (500 ng; List Biological Laboratories Inc., CA, USA) was administered intraperitoneally on the day of immunization and 48 h later. The clinical symptoms were scored as follows: 0, normal; 1, tail paralysis; 2, partial hindlimbs paralysis; 3, complete hindlimbs paralysis; and 4, complete hindlimbs paralysis and partial forelimbs weakness. Animals were randomly divided into three groups (10 mice/group): control, no treatment; EAE, MOG treatment; and EAE + ATO (MOG combined ATO treatment). Starting at 10 days post-immunization, mice in the EAE + ATO group were intraperitoneally injected with ATO (0.5 mg/kg/day) for 8 days. On day 22 post-immunization, mice were sacrificed and peripheral blood, spinal cord, and spleen were collected and used for further experiments.
Histopathology, Immunohistochemistry, and Immunofluorescence
The spinal cord was dissected and fixed with ice-cold 4% paraformaldehyde overnight at 4 ℃, embedded in paraffin, cut into 5 μm slices, and stained with luxol fast blue (LFB) and hematoxylin and eosin (HE). LFB-stained sections were scored for demyelination as follows: 0, none; 1, rare foci; 2, a few areas of demyelination; 3, one to two large areas of demyelination; and 4, extensive demyelination. Representative examples of LFB stained histological sections illustrating the different demyelination scores were presented in supplementary figure 1. HE-stained sections were also scored for inflammation as follows: 0, none; 1, a few scattered inflammatory cells; 2, perivascular infiltrates; 3, extensive perivascular cuffing with extension into adjacent parenchyma; and 4, extensive cell infiltration in the white matter [23]. Additionally, the sections were subjected to indirect immunostaining. The primary antibodies were used as follows: anti-CD4 (1:70, Servicebio, Wuhan, China), anti-MBP (1:50, Boster, Wuhan, China), and anti-Iba-1 (1:200, Servicebio). Staining was quantified using the HALO™ image analysis software (Indica Labs, NM, USA). Briefly, in the HALO analysis software, we set the measurement target area for each slice. For analysis of CD4+ cells in the spinal cord, Indica Labs-Multiplex IHC module was used to set Stain 1 as negative cells (blue nuclei) and Stain 2 as positive cells (brown granule). For analysis of Iba-1+ cells in the spinal cord, Indica Labs-HighPlex FL module was used to identify positive cells (red cytoplasm) and negative cells (blue nuclei). After the setup was completed, the software automatically calculated the number of positive and total cells on the section. The percentage of positive cells in total cells was calculated from nine different sections of the spinal cord.
Proinflammatory cytokine detection
Blood was collected from the cavernous sinus in the posterior eye orbit and kept at room temperature for 30 min. After centrifugation at 4,000 rpm for 20 min, the serum was transferred into a new tube and stored at -80 ℃. The concentration of IFN-γ in serum was measured using a commercial V-PLEX proinflammatory panel 1 kit (MSD, NJ, USA).
Flow cytometry
Red blood cells were removed to obtain peripheral and spleen lymphocytes. The cells were incubated in anti-CD4-FITC (Biolegend, CA, USA) at 4 °C for 30 min. The IgG-FITC isotype antibody (Biolegend) was used as the negative control. The stained cells were examined by flow cytometry with Beckman Cytoflex S (CA, USA). All data from flow cytometry were processed using FlowJo software V.10.
Assessment of liver and kidney function parameters
ALT (alanine aminotransferase), AST (aspartic aminotransferase), creatinine, and urea in serum were measured through mindray automated biochemical analyzer (Mindray Bio-medical Electronics Co., Ltd., Shenzhen, China) using standard diagnostic kits and analytical grade reagent (Mindray, Shenzhen, China) according to the manufacturer’s instructions.
Apoptosis
Apoptosis of CD4+ T cells was detected with the Annexin V-FITC/PI Detection Kit (Meilunbio, Dalian, China). Briefly, after washing with PBS, cells were stained with Annexin V-FITC and PI for 15 min at room temperature. Subsequently, cells were detected by flow cytometry with Beckman Cytoflex S. Apoptosis signals of spinal cords were examined with the Apoptosis Detection Kit (Servicebio).
JC-1 staining
CD4+ T cells were sorted from the spleen of naïve C57BL/6 mice with the Mouse CD4+ T cell Isolation Kit (Miltenyi, Bergisch Gladbach, Germany), seeded in 12-well plates, and treated with 5 μg/mL ConA alone or 5 μg/mL ConA + ATO (1, and 2 μM) for 24 h. JC-1 staining was performed to monitor the change of mitochondrial membrane potential. Briefly, the CD4+ T cells were incubated with 1640 RPMI medium contained 10 mg/mL JC-1 probe (Sigma) for 30 min at 37 °C. After washing three times with PBS, the stained cells were examined by flow cytometry with Beckman Cytoflex S.
Transmission electron microscope (TEM)
CD4+ T cells were cultured and treated as described above. After washing with PBS, the cells were fixed with 2.5% glutaraldehyde overnight at 4 °C. The following day they were fixed again with 1% osmium tetroxide for 2.5 h at room temperature. Subsequently, the cells were embedded after dehydration. Ultra-thin sections were counterstained with uranyl acetate for 30 min and lead citrate for 30 s and observed by a TEM (HT7800, Hitachi).
Caspase 3 Activity Assays
Total protein was extracted from CD4+ T cells to measure Caspase 3 activity with the Caspase 3 Activity Assay kit (Applygen, Beijing, China). Briefly, BCA assay was used to determine the protein concentrations; then, 10 μL of Caspase 3 substrate was incubated with 10 μL of the total protein (30 μg) in a final volume of 100 μL for 3 h at 37 °C. The absorbance of p-nitroanilide was measured using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 405 nm in turn to calculate the Caspase 3 activity.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from spinal cords or spleen with Trizol (TansGen, Beijing, China). Reverse transcription was performed using the cDNA Synthesis SuperMix for qPCR kit (TansGen). The mRNA expression was quantified using qPCR SuperMix kit (TansGen) and normalized to β-actin. The relative mRNA expression of genes was calculated using the 2-ΔΔCt method. ΔΔCt = (CT, target – mean CT, β-actin) treated sample - (mean CT, target – mean CT, β-actin) control sample. The control sample is the spinal cord from naïve C57BL/6 mice and used as the calibrator. The sequences of primers are as follows: IL-1β, forward 5′-TCGCAGCAGCACATCAACAAGAG-3′, reverse 5′-TGCTCATGTCCTCATCCTGGAAGG-3′; IL-2, forward 5′-GGAGCAGCTGTTGATGGACCTAC-3, reverse 5′-AATCCAGAACATGCCGCAGAG-3′; IL-6, forward 5′-TGGGACTGATGCTGGTGACA-3′, reverse 5′-ACAGGTCTGTTGGGAGTGGT-3′; IFN-γ, forward 5′-CGGCACAGTCATTGAAAGCCTA-3′, reverse 5′-GTTGCTGATGGCCTGATTGTC-3′; TNF-α, forward 5′-GCCTCTTCTCATTCCTGCTTGTGG-3′, reverse 5′-GTGGTTTGTGAGTGTGAGGGTCTG-3′; MBP, forward 5′-GCTCTGGCAAGGACTCACACAC-3′, reverse 5′-TGGAGGTGGTGTTCGAGGTGTC-3′; β-actin, forward 5′-CATCCGTAAAGACCTCTATGCCAAC-3′, reverse 5′-ATGGAGCCACCGATCCACA-3′.
Western blot
Spinal cords were homogenized and CD4+ T cells were lysed with cold RIPA buffer (Proteintech, IL, USA) supplemented with a protease inhibitor cocktail (Sigma). After centrifugation at 4 °C for 15 min, the supernatants were collected and used for western blot analysis. The following antibodies were used: anti-Bax (1:1,000, Proteintech, IL, USA), anti-MBP (1:1,000, Proteintech), anti-cleaved Caspase 3 (1:1,000, Affinity, OH, USA), anti-cleaved Caspase 9 (1:1,000, Affinity), anti-Bcl-2 (1:1,000, Proteintech), anti-cleaved PARP (1:1,000, Affinity), anti-β-actin (1:5,000, Bioworld, MN, USA).
Statistical analysis
Data were analyzed with GraphPad Prism 6 software (La Jolla, CA, USA) and represented as means ± SD of three separate experiments. Clinical scores, demyelination scores, and inflammation scores were compared using the Kruskal–Wallis test. One-way ANOVA was used for multiple comparison in the rest of assays. A p value<0.05 was considered to be statistically significant.