Synthesis of FCUR procedure
FCUR was synthesized according to our previously reported procedure and characterized by 1HNMR, 13CNMR, and HRMS spectra [38].
Animals
Female Six-to-eight-week-old C57BL/6J mice were purchased from Pasteur Institute (Tehran, Iran). Mice were weighed about 17 ± 2 g and those who were less than 15 g were not included in the study. Also, we excluded mice that had any signs of illness before the intervention or died in the first 10 days after EAE induction. Animals were maintained in a controlled environment (22 ± 2 °C, ~78 % humidity, 12 h light/dark cycle), with standard animal diet and free access to water. All experiments were performed in accordance with the policies of the Animal Care and Use Protocol of the Shahid Beheshti University of Medical Science. To avoid bias, mice were randomly divided into experimental groups, and the investigation was carried out in a blinded manner. All mice were housed in separate cages to reduce animal stress.
EAE induction and clinical assesment
Mice were randomly assigned to EAE induced or naïve groups. EAE was induced to the first group as described in the literature. The mice were injected subcutaneously on the back with 200 μg of myelin oligodendrocyte glycoprotein (MOG35-55) (Sigma-Aldrich, St. Louis, MO, USA) emulsified in complete Freund’s adjuvant (Sigma-Aldrich, St. Louis, MO, USA) containing 400 μg of heat inactivated Mycobacterium tuberculosis H37Ra . At 0 and again 48 h after immunization, 200 ng of pertussis toxin was administered intraperitoneally.
Every day, animals were evaluated blindly by two independent investigators according to their motor symptoms based on the following 0-5 scale: 0, normal; 1, flaccid tail or awkward gait with tail tonicity; 2, flaccid tail plus hind limb weakness; 3, flaccid tail plus hind limb paraplegia 4, flaccid tail plus hind limb and fore limb quadriplegia; 5, dead.
Drug Treatment
The mice of EAE group were divided into three subgroups: control group, CUR group, and FCUR group, which were treated with 25 ϻL DMSO, 25 ϻL DMSO+ 4 mg/Kg CUR, and 25 ϻL DMSO+ 4mg/kg FCUR, retrospectively. Placebo and drugs were injected intraperitoneally to the animals 10 days after immunization. Treatment was continued for 20 days with similar time intervals.
Isolation of serum, splenic leukocytes and B cells (splenocytes), and spinal cord lymphocytes
On the 30th day following immunization, mice were euthanized by cervical dislocation and blood collection was performed through the abdominal cavity. The blood was centrifuged (2,500 rpm, 15 min) and the serum specimen was taken.
The animals’ spleen was obtained aseptically. Each spleen was mechanically disrupted and filtered through a sterile 70 µm filter and centrifuged at 2000 rpm for 4 min. Then red blood cells were lysed using 0.9% ammonium chloride and the suspension was re-centrifuged. The remaining pellet was suspended in culture media and preserved in liquid nitrogen.
The spinal cord was removed from the spinal column of the sacrificed mice using sterile PBS (Sigma-Aldrich, St. Louis, MO, USA). To yield single-cell suspension, the tissue was passed through a sterile 70 µm filter, digested with collagenase (2.5 mg/mL) and DNase I (50 µg/mL) and incubated in 37°C for 45 min. then, it was subjected to the ex vivo examination.
Histology analysis
While a part of spinal cord tissue was used for leukocyte isolation, the remaining was fixed with 4% paraformaldehyde. Then the tissue was embedded in paraffin and 5ϻm sections were prepared and stained with hematoxylin and eosin (H&E) to assess inflammation. Sections were evaluated with a light microscope in a blinded manner.
Enzyme-linked immunosorbent assay (ELISA)
The levels of IL-6, IL-17, and TGF-β in the serum and splenocytes were detected in triplicate using ELISA commercial kit (Sigma-Aldrich, St. Louis, MO, USA) according to instructions from the manufacturer. The absorbance was measured at 570 nm by an ELISA plate reader (PerkinElmer, USA).
Western blot
Protein was obtained from spinal cord tissue using mixture of RIPA buffer (Thomas Scientific Inc., USA) and protease inhibitors. MBP protein production level was measured by western blot analysis. To evaluate protein concentration, BCA protein assay kit (Thermo Fisher Scientific, UK) was employed. The protein sample was loaded onto 10 % SDS-polyacrylamide gel electrophoresis and then separated and transferred onto a PVDF membrane (Millipore, USA) by electro-blotting. The membranes were blocked in Tween 20 Tris‐buffered saline (TBST) containing 5% non-fat milk for 1 h at room temperature and then incubated overnight with 1:1000 dilution of MBP antibody. After washing with TBS for three times, the membrane was incubated with secondary antibody (Santa Cruz Biotechnology, UK) in the dark for 1.5 h at room temperature and washed in the same buffer. To visualize the protein chemiluminescent kit (SuperSignal, Thermo Fisher Scientific, UK) was employed. Finally, the density of the protein bands was quantified by ImageJ software (NIH, Bethesda, USA).
Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis
mRNA levels in the spinal cord were measured by qRT-PCR. Total RNA was extracted from the homogenate tissues using RNeasy mini kit (Qiagen, Germany), according to the manufacturer’s protocol. This procedure was performed under RNase-free conditions. After quantifying the RNA using Nanodrop 2000c spectrophotometer (Thermo Scientific, USA), the total 1 μg/ml single-stranded RNA was reverse transcribed by the cDNA Synthesis Kit (Bio FACT, Daejeon, South Korea) in compliance with the instructions manual. The specific genes were quantified by qRT-PCR applying SYBR Green PCR Master Mix (Ampliqon, Denmark) with proper primers (Table 1) and analyzed with ABI PRISM 7900HT system (Applied Biosystems, USA). Primers were designed by Primer Express software v1.5 (Applied Biosystems). Releative gene expression was analysed by the 2-ΔΔCt method.
Statistical analysis
Statistical analyses were performed using GraphPad Prism v6.07 software (GraphPad, San Diego, CA). All data were expressed as mean ± SD. One-way ANOVA followed by a post-hoc Tukey test was conducted to analyze differences among groups. P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***) are considered statistically significant.