Mediation Of The SPHK1/S1P Pathway Following Vitamin D Treatment Of MS/EAE

Background:Sphingosine-1-phosphate (SIP) is a bioactive lipid that is predominantly distributed in body, It’s mainly produced by hematopoietic cells, it exists in serum and other tissues, especially inflammatory tissues. Experimental allergic encephalomyelitis (EAE) is multiple sclerosis (MS) animal model. The study found that cerebrospinal fluid from MS patients and Serum, Spinal cord from EAE rats, SIP content is increased. It showed that SIP may be associated with the pathogenesis of MS/EAE. In recent years, a large number of studies have shown that SIP in combination with S1PR can result in the generation of reactive astrocytes, reactive astrocytes and neurons apoptosis are the two major characteristics of MS. Vitamin D can regulate the expression of protein in nerve cells, this has the effect of delaying disease course in MS/EAE. Consequently, immune regulatory mechanisms associated with Vitamin D adjuvant therapy used for the treatment of MS, may be involved in the regulation of S1P.Objective: This report focuses on the relationship between SPHK/S1P and Vitamin D and provides a potentially novel approach for the treatment of MS.Methods:We analysed and compared the expressions of SPHK1 detected by western blot. We observed for the first time that Vitamin D results in a reduction in EAE rats SIP levels. We adopt western blot test the change of SPHK expression in EAE rats (E) and normal rat tissues(C).In order to study the relationship between vitamin D and SIP synthesis, using western blot test the change of SPHK expression in EAE rat (E) and EAE rats feed with vitamin D (VE).Through consulting relevant literature, synthesis and decomposition of SIP control by a variety of enzymes. Sphingosine in both kinase-sphingosine kinase 1 (SPHK1) and sphingosine kinase 2(SPHK2) under the action of formation, and decomposition of SIP-SIP specificity lyase (SGPL1) and two kinds of phosphatase (SGPP1,2) work together. Using Real-time PCR, at the cellular level, before and after the detection of 1,25-(OH)2D3 stimulates, detection the quantity of the five kinds of enzyme gene expression can illustrate the Vitamin D have direct control on several enzymes or not.PC 12, a common nerve cell line derived from Rattus norvegicus, was obtained from ATCC. PC 12 Cells were cultured in 6-well Cell culture plates. Separated into six groups based on different concentration of 1,25-(OH)2D3. Cultures were maintained at 37 ℃ in a humidified incubator with 5% CO2(Thermo). Collected RNA, protein, SPH and S1P at 24h,48h,72h.We analysed SPH and S1P expression in rat serum and cells using enzyme-linked immunosorbent assay (ELISA)Many of the biological functions that are traditionally associated with Vitamin D are mediated through the Vitamin D receptor (VDR). As part of this analysis, we revealed the relationship between VDR and SPHK1.This was performed using over-expression experiments and observations relating to the interference effects of SPHK1 on VDR. we used ELISA and western blot to test SIP and SPHK1Results:The results suggest an increase in SPHK1 in the tissues of EAE and a reduction in SPHK1 in the tissues of EAE rats whose diets were supplemented with Vitamin D.Used Quantitative Real-Time PCR and western blot to test five different enzymes related to S1P, we observed significant changes in SPHK1 mRNA levels. The expression levels associated with the lyase (SGPL1, SGPP1 and 2) did not change significantly. SPHK1 levels were significantly altered at different concentrations of 1,25-(OH)2D3.ELISA was used to analyze rat serum and cell extracts following incubation with different concentrations of 1,25-(OH)2D3. As part of this study we observed changes in SIP and found that when high concentrations of 1,25-(OH)2D3 were applied, S1P levels decreased, but SPH did not change significantly.This was performed using over-expression experiments and observations relating to the interference effects of SPHK1 on VDR,We observed the levels of SPHK1 are reduced in PC 12 Cells stimulated with 1,25-(OH)2D3, the levels of SPHK1 are not change significantly in PC 12 Cells stimulated without 1,25-(OH)2D3 in the over-expression experiments. We observed the RNA interference effectively reduced VDR protein levels compared to the control and found that the levels of SPHK.1 did not change significantly in PC 12 Cells stimulated with 1,25-(OH)2D3 when reduced VDR protein levels.Conclusions:1. Vitamin D regulates the expression of the inflammatory factor SIP in EAE is likely caused by a change in SPHK1 levels.2. Vitamin D may regulates the expression of SPHK1 following binding of VDR, S1P synthesis is changed.3. It is likely that if Vitamin D is administered in conjunction with other anti-SIP drugs, there is an increased chance of MS/EAE prevention and treatment.