EAE in Rats
All experimental protocols involving animals were approved by the Animal Care Committee of Shandong University . Eight-week-old male Lewis rats were injected subcutaneously in two hind foot pads with 100 μl immunization emulsion, and 50 μl per pad. Control animals were injected with parallel emulsion without the MBP component. Details have been described previously by Zhu Y. et al(2014) [30]. The rats were first separated into two groups based on their EAE status: control (C) and EAE (E). The rats from EAE groups were fed with Vitamin D (V) or without Vitamin D. This resulted in four groups of rats: C, VC, E and VE. The synthetic diet contains all essential nutrients, except Vitamin D. The diet containing Vitamin D supplementation was prepared with 3 μg/ day added for each rat. Fresh food was provided to rats every other day for 2 weeks prior to euthanasia.
Cell Culture
PC12, a common nerve cell line derived from Rattus norvegicus, was obtained from ATCC. PC12 cells were cultured in 75 cm2 cell culture flasks or 6-well cell culture plates (Fisher Scientific) using Dulbecco’s Modified Eagle’s Medium (DMEM) (Hyclone) supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) penicillin and streptomycin. The cultures were stimulated with different concentrations of 1,25-(OH)2D3. Cultures were maintained at 37°C in a humidified incubator with 5% CO2 (Thermo).
Measurement of S1P
Tissue homogenates were prepared from 30 mg of excised spinal cord using a homogenizer in 100 μl of 25 mM HCl/1 M NaCl, and the S1P concentrations in the spinal cord homogenates were measured using HPLC.
Cells (1 × 106) were diluted with PBS (pH 7.2–7.4). The cells were lysed following repeated freeze/thaw cycles to release intracellular components. The S1P concentrations in the PC12 cells supernatant were measured with enzyme-linked immunosorbent assay (ELISA) kits (ShangHai QiaoDu Biotechnology) according to the manufacturer’s instructions.
Quantitative Real-Time PCR
Total RNA was isolated from cultured cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Complementary DNA (cDNA) synthesis was performed using EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TRANS). These reactions were performed in accordance with the manufacturer’s instructions. Quantitative Real-time PCR amplification was performed on the CFX96 Real-Time PCR System (Bio-Rad) using the SYBR Green PCR Master Mix (Roche, Germany). The Real-Time PCR used the following conditions: initial denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 15 s, 55°C for 15 s, and 72°C for 30 s. GAPDH, a housekeeping gene, was used as an internal control. The relative concentration of SPHK1 was calculated using the formula x = 2 - ΔΔCT.
Western Blotting
To assess SPHK1 expression in EAE rats, we collected spinal cord samples from both EAE and normal rats to investigate the difference in protein expression levels between the different groups. PC12 Cells and spinal cords were lysed in RIPA Lysis Buffer (Beyotime, Shenzhen, Guangdong, China) followed by homogenization and centrifugation steps. Protein concentrations were measured using the BCA protein assay (Beyotime). Equal amounts of protein were electrophoresed using 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membrane was subsequently blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBS-T) at room temperature for 1.5 h. The membranes were incubated overnight with SPHK1 primary antibody (1:1,000) at 4°C. Next, the membranes were washed with TBS-T and incubated with horseradish peroxidase labeled anti-rabbit IgG (1:5,000, ZSGB-BIO, Beijing, China). The membranes were subsequently washed three times with TBS-T buffer, and the immunoreactive complexes were visualized using the Lumina Enhanced Chemiluminescent Kit (Millipore, Billerica, MA, USA). Finally, luminescent products were visualized using photographic film. Actin was used as an internal standard.
RNA expression
A VDR expression plasmid (GV230, Genechem, Shanghai, China) was injected into PC12 cells facilitating stable expression of VDR. Alternatively, a control plasmid (NC) was injected into PC12 cells. PC12 cells were cultured in 6-well plates and transfected when 70–80% confluence was reached. Transfections were performed using the Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. VDR expression, facilitated by the introduction of the plasmid, was verified by Western blotting.
RNA Interference
In order to facilitate VDR expression interference, 19-mer siRNA oligos (GGAAGTACAGGGAGCTATT)were designed and synthesized (Vdr-RNAi, Genechem, Shanghai, China). A Lentiviral expression vector (GV118, Genechem, Shanghai, China) and control Lentiviral expression vector were used to ensure transfection efficiency. The Lentiviral expression vector was injected into PC12 cells, thereby stably interfering with VDR expression. The PC12 cells used in this experiment were grown in 6-well plates and transfected at 30–50% confluence. Transfections were performed according to the manufacturer’s instructions. VDR interference, facilitated by introduction of the Lentiviral vector, was verified by Western blotting.