The Influence Of VD To Ceramide In PC12 And The Proteomic Study In Serum Of EAE Treated With VD Or Not

Background:Multiple sclerosis(MS)is a autoimmune disease associated with neuronal cell death and demyelinating in central nervous system.The patients often suffer from cognitive disorder,visual vison and so on.The etiology of multiple sclerosis is not yet clear.The main argument focuses on the combined effects of the environment and the genes.The drugs that are used in MS are still foucus on immune regulator and immunosuppressant,while they have limited effect and serious side effects.Vitamin D(VD)as a necessary trace material in the human body,the main function is that VD can regulate the calcium homeostasis.Recently,VD is concern because of its function on treament of cancers and immune system diseases.Epidemiology shows that MS incidence and light are significantly correlated.Light can catalyze VD synthesis in vivo,and scientists speculate that MS is associated with VD.The follow studies show that vitamin D content is significantly decreased in MS patients compared with normal,the lack of vitamin D can increase the risk of MS.Further studys show that in EAE(Experimental Allergic Encephalomyelitis),the animal model of MS,Vitamin D supplements can relieve the symptoms of MS but can not cure EAE.There are many researches on the mechanism of vitamin D relieve MS/EAE,but the etiology is not clear.Therefore,the in-depth study of VD relieve MS/EAE can prove the necessary experimental basis for the clinical application of vitamin D,which has important theoretical and practical significance.The influence of VD to Ceramide in PC12Sphingolipids are a class of lipids involved in a variety of biological functions and pathologies.Ceramide(Ceramide,Cer)as a central metabolite of sphingolipids participate a variety of cellular activities,such as cell migration,apoptosis and inflammatory response.Short-chain ceramide(C2-Cer)and natural ceramide can promote PC12 cell apoptosis,mainly for the destruction of the nucleus and nuclear DNA damage.Recent studies have shown that the concentration of C16:0-Cer in the spinal cord of EAE is significantly increased and the levels of mRNA and protein levels of ceramide synthase 6 are significantly increased.The preliminary work of our group shows that vitamin D can sigjnificantly reduce the concentration of S IP that is a Cer metabolite in spinal cord of EAE and alleviate the pathogenesis of EAE,but the mechanism is not clear.Therefore,we use PC 12 cells in this experiment to study whether vitamin D has an effect on ceramide and explore the mechanism,to provide the basis of cell experiment for the study of the mechanism of vitamin D alleviating MS/EAE.Objective:To investigate the effect of vitamin D on ceramide in PC 12 cells to provide the basis of cell experiments for the mechanism research of VD treatment MS.Methods:1,MTT assay was performed to verify the pro-apoptosis function of C2-Cer(20,10,5,2.5,1.25,0.62,0 ?M)on PC12 cells.2,The concentration of C16:0-Cer in PC12 cells stimulated with VD(50 nM or 0 nM)was determined by high performance liquid chromatography tandem mass spectrometry.3,The mRNA levels of enzymes related to ceramide in PC 12 cells stimulated with VD(50nM or OnM)was measured by RT-PCR.The enzymes includes the synthesis enzymes of ceramide present in the spinal cord,SPT(SPTLCl,SPTLC2),Cers(Cersl,Cers2,Cers3,Cers5,Cers6),and metabolites Cerk,Smpdl-3,Sgsml,Asah,Gba,Sms.4,The protein levels of SPTLCI and Cers6 in PC 12 cells that were stimulated with vitamin D(200,100,50,25,12.5,0 nM)was measured by Western Blot.Results:1.The vitality of PC 12 cells decrease during the increase of the concentration of VD and VD does-depented(P***<0.05).2,Content of C16:0-Cer was significantly reduced(P*<0.05)in PC 12 cells that treated with VD compared with PC 12 cells,indicating that vitamin D has the effect of reducing the content of C16:0-Cer.3,In the PC12 cells stimulated by 50nM vitamin D compared with PC12 cells stimulaed by OnM vitamin D,the mRNA levels of SPTLC1 was significantly increased(P***<0.05)and the mRNA levels of CerS6 was also significantly increased(P***<0.05).4,The protein levels of SPTLC1 in PC 12 cells stimulated with vitamin D was significantly decreased.When vitamin D concentration was 50nM,the relative protein levels of SPTLC1 reached the lowest level(P***<0.05).The protein levels of Cers6 in PC 12 cells stimulated with vitamin D was significantly increased.When the concentration of vitamin D was 200nM,the levels of Cers6 was the highest(P<0.05).Conclusions:Vitamin D can reduce the concentration of C16:0-Cer by reducing the rate-limiting enzyme(SPTLCl)to reduce the pro-apoptosis effect of ceramide on PC 12 cells.So,we speculate that VD reduce the effect of ceramide in neuronal apoptosis to relieve MS.we provide a new idea for the study of vitamin D in the treatment of MS.The different proteins in serum of EAE rats before and after vitamin D supplementation are detected by iTRAQThe relative and absolute quantitative isotope labeling(iTRAQ)is a relative and absolute quantification technique based on equal isotope labeling and a rapid development technology used for proteomics high throughput screening.Objective:To detect the different levels of protein in serum of EAE compared with normal and EAE supplemented with vitamin D compared with EAE didn't supplement with vitamin D by iTRAQ technique,which provided new ideas for the treatment of MS/EAE by vitamin D.Methods:Prepare the EAE rat and collect serum.The serum of control rat(C),EAE(EAE)and EAE supplemented with vitamin D(VE)were analyzed by iTRAQ technique in proteomics analysis.1,Construction of EAE rats model and VD supplemented.There are 18 rats divide into 3 groups:group control(C,6 rats),group EAE(E,6 rats)and group EAE and VD supplemented(VD,6 rats).The myelin basic protein solution(MBP,lmg/ml)was mixed with an equal volume of complete Freund's adjuvant(CFA,containing 5mg/ml pertussis vaccine),and Lewis rats were immunized with two subcutaneous injection in hind foot to induce EAE by them.The weight and score of the rats were recorded from the date of immunization.After the onset of immunization,the rats were sacrificed and the blood from the heart of the rats was taken.2,We used iTRAQ to detect the different levels of protein from the scrum of group E compared group C and group VE compared group E.Each 3 rats from one group mixed into one sample cell,the protein was quantitative after removing the high abundance of protein in the serum.Protein was hydrolyzed by enzymatic to get peptides.We used the isotope to labeled the peptides,the SCX fraction was followed by gradient Q-exactive analysis for 15 LC-MS/MS analyzes.After obtaining the original mass spectrometry data,the proteins were retrieved by Proteome Discoverer analysis software to obtain differentially levels proteins.3,The different levels of protein from group E compared with group C and group VE compared with group E were screened.4,The functional properties and function of differential proteins were analyzed by bioinformatics methods such as GO function annotation and KEGG pathway analysis.Results:1,We induce of EAE rat sucessfully.2,The selection criteria is that the P value is less than 0.05 and the expression difference ratio should greater than 1.2 times.There were 74 proteins with significant differences,including 42 uptake proteins 32 down-regulated proteins in group E compared with group C.There were 80 proteins with significant difference in group VE compared with group E,among which 40 kinds of up-regulated proteins and 40 kinds of down-regulated proteins.There are 20 kinds of coincident proteins.3,The functions and the pathway of the proteins were analysis.4,We select Protein S100-A8 and Interleukin 1 receptor antagonist as candidate proteins.They are not only associated with the pathogenesis of EAE but also can be decreased under the action of vitamin D.Conclusions:In this study,proteomics was first applied in EAE rat serum to detect differnt proteins.We found 74 kinds of different proteins in group E compared with group C,and we provide the proteomic date to pathogenesis of EAE.We also found 80 kinds of different protein compared EAE rat supplied with VD with EAE rat,Which provides a new breakthrough point for the study of the mechanism of MS treatmed with VD.Through the analysis of the target protein,we select the protein Proterin S100-A8,which is related to spinal cord injury,and the Interleukin 1 receptor antagonist,which is related to the pathogenesis of MS,as target proteins,it provided a new target for the study of the mechanism of VD treatment of MS.