The Mechanism Research Of GSN Mediated The Prevention And Treatment Of Vitamin D On MS/EAE

BackgroundMultiple Sclerosis(Multiple Sclerosis,MS)is a common autoimmune disease with unknown cause in central nervous system,accompanying with the occurrence of the inflammatory reaction and neural degenerative change,patients appear typical clinical symptom such as intermittent neurological disorders,and optic neuritis,limb paralysis and sensory disorder,balance and coordination difficult,cognitive and sleep disorders.The etiology of multiple sclerosis is still not clear.So far,no drug has been found could cure MS,the commonly used drugs mainly focused on two aspects.one is immune adjustment and the other one is immune therapy.Although these drugs can alleviate the disease to some extent,reduce the recurrence rate,but the treatment effect is not satisfactory,and almost all drugs have serious side effects.Therefore.looking for a more efficient,safe,convenient drugs,is the first problem needed to solve in the present study on MS treatment.Vitamin Dis well known for its calcium homeostasis regulating function;however,in recent years,the study of Vitamin Dbegan to shift to immune system diseases.An epidemiological survey has found that there is a close correlation between the incidence of MS and light,and since light is the catalyst of Vitamin Dsynthesis in body,people presume that light affects the levels of Vitamin Dand thus,affects the incidence of MS.Subsequent experiments present that 25-(OH)2D3 level in MS patients is significantly lower than normal,and animal experiments in EAE prove that dietary supplement of Vitamin Dduring the acute stage of EAE can effectively delay the onset time and alleviate the symptoms(our previous animal experiments also got the same results.These results all indicated that,there is a strong correlation between Vitamin Dand the occurrence and development of MS.Gelsolin is the most widely expressed actin-severiing protein in humans.It exists as 2 isoforms in the human body,a cytoplasmic(cytoplasmic gelsolin,cGSN)and a secreted isoform(plasma gelsolin,pGSN).The most extensively examined role of pGSN is scavenging extracellular actin filaments released from dead cells into the blood and binding to pro-inflammatory molecules.The major function of cGSN is being involved in cell motility and cell apoptosis regulation.It has been reported that GSN concentrations decreased significantly in the blood of MS patients and EAE model,our previously research has found that the concentrations of pGSN and cGSN both decreased significantly in EAE,and both of them plays a protective role in the development of EAE.However,our previous study also found that the level of GSN decreased in the rats with Vitamin Dsupplement(no matter EAE induced or not),these results suggest that the downregulate of GSN by Vitamin Dmight be the limitation of Vitamin Dtherapy on EAE.Based on the above assumptions,in this study,we injected GSN overexpression lentivirus(LV-GSN)into EAE rats to increase the level of GSN expression,in order to observe whether GSN could play a protective role during EAE disease.The results show that increase the content of GSN in EAE rats could obviously delay the onset of EAE and alleviate the disease.LV-GSN administration combined with Vitamin Dsupplementation does a better therapeutic effect in EAE comparing with LV-GSN administration or Vitamin Dsupplementation individually.These results suggest that the downregulate of GSN by Vitamin Dmight weaken the protective effect of Vitamin Don EAE.Though performing VDR gene interruption and overexpression in PC 12 cells(a rat cell line common used in the study of neurons),we find that the mediation of Vitamin Don GSN expression is through the Vitamin Dreceptor(VDR).Function experiment confirm that cGSN indeed play a protective role in the process of cell apoptosis.In addition,for the system research on the mechanism of the role Vitamin Dplays in prevention and treatment of EAE,we use iTRAQ(a quantitative proteomics approach)to detect the differentially expressed proteins the spinal cord between the rats with EAE induction and without EAE induction,or between the EAE rats with Vitamin Dsupplementation and without Vitamin Dsupplementation.The results show that there are 722 proteins have different expression level between the control group(normal group without EAE induction)and the acute EAE group(the selection criteria is that the expression difference ratio should greater than 1.2 times and the P value is less than 0.05),the number of different expression protein between the EAE rats with Vitamin Dsupplementation group and without Vitamin Dsupplementation group is 184(the selection criteria is that the expression difference ratio should greater than 1.2 times and the P value is less than 0.05).However,there are 44 proteins changed between the 3 groups.Bioinformatics analysis show that these proteins mainly involved in cell structure,cell growth,cell apoptosis process.The results of subsequently Western Blot experiments show that the concentration of Heat shock 27kDa protein 1(Hsp27)and Galectin-3(Gal-3)increased in the acute phase of EAE(compared with control group),and decreased after Vitamin Dsupplement.This is consistent with the iTRAQ results.In conclusion,this study mainly discusses the possible mechanism of GSN effect on the progress of Vitamin Dprevention and treatment on EAE,detects the proteins regulated by the Vitamin Dduring the onset of EAE process.Our results prove that the GSN play a protective role in the process of EAE disease,increase the expression of GSN while Vitamin Dsupplementary could enhance the effect of Vitamin Don the prevention and treatment of EAE,and the regulation of the expression of GSN by Vitamin Dmay through VDR.At the same time,we also provide candidate proteins such as Hsp27 and Gal-3 and new thought in the mechanism study of the prevention and treatment of Vitamin Don EAE,meantime,we also impelled the application process of Vitamin Din clinical treatment of MS.Part I The research of the effect of GSN on Vitamin Dtherapy on EAE/MSObjectiveTo clarify the influence of GSN on EAE course,confirm that increase the expression of GSN while Vitamin Dsupplementary could enhance the effect of Vitamin Don the prevention and treatment of EAE,to provide a new train of thought and experimental basis in the research of a better role Vitamin Dcould play in prevention and treatment of MS.Methods1.The cerebrospinal fluid(CSF)and blood from patients with MS and other neurological diseases(OND)were collected,and western blot was used to analysis and compare the expression of GSN.2.Construction of EAE rats model:The myelin basic protein which was dissolved in normal saline(MBP,1mg/ml)was mixed with complete Freund’s adjuvant(CFA,containing 5mg/ml pertussis vaccine)at a ratio of 1:1,and in EAE induced group Lewis rats were immunized with two subcutaneous injection in hind foot to induce EAE;the rats in control group were injected with parallel emulsion without MBP component.From the immunization day(denoted day 0),rats were weighed and scored,until the 25th day.At the 13rd day after immunization,three rats from each group were taken to heart perfusion.The spinal cord tissue was taken from rats for histopathological staining(H&E)to observe the condition of inflammatory cell infiltration.Weight,score and inflammatory cell infiltration was calculated to evaluate EAE condition.3.The injection of LV-GSN and the supplementation of’ Vitamin D:48 Lewis rats were first separated into two groups based on the EAE status:control group(C),EAE group(E).The rats from each of these two groups were divided into 4 groups based on the injection of LV-GSN or the supplementation of Vitamin Dor administration of both of them.In this way,48 rats used in this research were divided into 8 groups(group E,GE,VE,VGE,C,GC,VC,VGC)based on different treatments,every group contains 6 rats.We performed the LV-GSN injection into the ventricle and caudal vein of rats(groups GC,VGC,GE,VGE)3 days after the immunization considering it takes 5 days for the LV-GSN to increase the GSN concentration,and the other groups were injected by control lentivirus.The rats in groups with Vitamin Dsupplement were fed with diet containing Vitamin D(3μg/day)every other day before euthanizing,two weeks in total.Rats were weighed and scored,until day 12.At day 12 after immunization,rats were killed to observe the condition of inflammatory cell infiltration.4.ELISA was performed to measure the concentration of 25-hydroxy Vitamin Dand 1,25-dihydroxy Vitamin Din rats,in order to determine whether Vitamin Dwas effective absorbed.Western Blot was used to detect the gelsolin(GSN)concentration in rats with LV-GSN injection.5.Western Blot and qRT-PCR was used to measure the expression of GSN and VDR in the rats of every group.6.The expression of GSN and VDR was detected after stimulated by 1,25(OH)2D3 in gradient concentrations in PC 12 cells.7.The expression of GSN was measured by Western Blot after the gene of VDR was interfered and over-expressed in PC 12 cells.8.Nuclear was staining with Hoechst 33258 after the gene of-GSN was interfered and over-expressed in PC 12 cells.Results1.The level of GSN expression in the CSF of patients with MS was Significantly lower than in the patients with ONDs.2.EAE model in Lewis rats was successfully induced:The weight of EAE rats weight begin to fall at about day 11 and begin to recover at about dayl5,however,the weight of control rats increase consistently.The symptom of EAE emerged at dayl 1,peaked on the day13,and disappeared at day 19.The result of H&E staining showed that the inflammatory infiltration in spinal cord of EAE rats was obviously.3.LV-GSN administration delayed the onset of EAE and decrease disease severity of EAE.LV-GSN administration combined with Vitamin Dsupplementation enhanced Vitamin Dtherapy in EAE,VGE rats had a delayed start of the disease and lower severity score compared with GE and VE rats.4.In serum the concentrations of 25-(OH)D3 and 1,25-(OH)2D3 are higher in all of the groups of rats that had Vitamin Dsupplementation than that in rats without Vitamin Dsupplementation.Therefore,these results imply that additional dietary vitamin D3 is effectively absorbed by all 4 groups of rats.The levels of GSN expression in rats with GSN injection are apparently higher than that in rats with the control lentivirus injection,no matter if EAE was induced or not.The results indicate the LV-GSN could successfully increase the level of GSN expression in rats.5.VDR and GSN levels are significantly lower in EAE rats than the control rats,VDR was Upregulated after Vitamin DSupplementation while Both pGSN and cGSN were Downregulated.6.Western blot results show an obvious increase in the VDR level,but in contrast,the expression of GSN decreased significantly with the increase of 1,25-(OH)2D3 concentration,the most obvious effect occurred at 50nM concentration.7.The regulation of Vitamin Don the expression of GSN might rely on the binding of VDR:the levels of GSN were reduced in PC 12 cells transferred with VDR plasmid in comparison with the NC plasmid and in the PC 12 cells that were transferred with VDR interruption shRNA lentiviral expression vector(VDRi),the GSN levels were significantly increased while the VDR levels were downregulated by VDRi.8.cGSN plays a protective role in apoptosis:Following interference of GSN,significantly more PC 12 cells exhibited marginalization and nucleic chromatin condensation compared with the NC transfected cells.In contrast,overexpression of GSN resulted in a decreased number of PC 12 cells with nucleic chromatin condensation compared with the cells transfected with the NC inhibitor.Conclusion1.Overexpression of GSN could delay the onset of EAE and decrease disease severity of EAE.2.LV-GSN administration combined with Vitamin Dsupplementation enhanced Vitamin Dtherapy in EAE,the down-regulate of GSN by Vitamin Dmight be the limitation of Vitamin Dtherapy in EAE.3.The regulation of Vitamin Don the expression of GSN might rely on the binding of VDR.Part II The study of proteomics detect on proteins regulated by Vitamin Din EAE rats during the process of EAEObjectiveTo detect the differentially expressed proteins the spinal cord between the rats with EAE induction and without EAE induction.or between the EAE rats with Vitamin Dsupplementation and without Vitamin Dsupplementation.To provide candidate proteins and new thought in the mechanism study of the prevention and treatment of Vitamin Don EAE.Methods1.Construction of EAE rats model:30 Lewis rats were first separated into two groups based on the EAE status:control group(C,contains 10 rats),EAE group(E,contains 20 rats).The rats from group EAE were divided into 2 groups based on the supplementation of Vitamin D(VE,contains 10 rats)or not(E,contains 10 rats).The methods of EAE induction and Vitamin Dsupplementation were the same as mentioned in part 1.2.We used isotope labeled relative and absolute quantitative(iTRAQ)proteomics method to detect the differentially expressed proteins the spinal cord between the rats with EAE induction and without EAE induction,or between the EAE rats with Vitamin Dsupplementation and without Vitamin Dsupplementation.The EAE rats were sentenced to death at acute disease process(day 12),protein from the spinal cord was extracted,enzyme was used to get peptides after protein quantitative.ITRAQ labeling peptides,gradient Q-exactive analysis at total of 15 LC-MS/MS was performed after SCX classification,the original mass spectrum data was get,then differentially expressed proteins were achieved by using analyzing software Proteome Discoverer to search database.3.Detect the protein both changed between the 3 groups.4.Bioinformatics approach such as Gene Ontology function annotation,KEGG pathway analysis to indentify the protein function,attributes of the pathway,and interaction between and differentially expressed proteins other proteins.Results1.The original data we detected 4987 kinds of proteins by mass spectrum qualitative.The results show that there are 722 proteins have different expression level between the group E and group C(E/C)(the selection criteria is that the expression difference ratio should greater than 1.2 times and the P value is less than 0.05),among them 438 proteins were up-regulated and 284 proteins were down-regulated.The number of the different expression protein between group VE and group E(VE/E)is 184(the selection criteria is that the expression difference ratio should greater than 1.2 times and the P value is less than 0.05),among them 43 proteins were up-regulated and 141 proteins were down-regulated.2.There are 44 proteins changed between group E and C,group VE and E.3.The function and disease correlation of each protein and the network of differentially expressed proteins was obtained from bioinformatics analysis.4.The results of Western Blot showed that the concentration of Hsp27 and Gal-3increased in the acute phase of EAE(compared with control group),and decreased after Vitamin Dsupplement.This is consistent with the iTRAQ results.ConclusionOur study for the first time use iTRAQ(a quantitative proteomics approach)to detect the differentially expressed proteins the spinal cord between the rats with EAE induction and without EAE induction,or between the EAE rats with Vitamin Dsupplementation and without Vitamin Dsupplementation.As the result showed we obtained 44 kinds of proteins which might be associated with EAE course and may be regulated vitamin D.At the same time,we also provide candidate proteins and new thought in the mechanism study of the prevention and treatment of Vitamin Don EAE.