The Mechanism Research On Vitamin D Therapy In MS And EAE

Background:Multiple sclerosis (MS) is an autoimmune, inflammatory and demyelinating disease. T lymphocytes attack myelin, causing axonal damage and demyelination. Lesion sites mainly exist in brain and spinal cord, accompanied by inflammatory cell infiltration, causing visual, motor, cognitive disorders. The disease has high incidence in Europe and North America, and in recent years, the number of MS patients in the Asian region showed an increased trend. Although more and more people suffer from this disease, the etiology and pathogenesis is not clear. Moreover, existing drugs can only partially alleviate the disease, and there are no drugs to cure the disease. Therefore, it is become urgent to find more effective drugs. Epidemiological studies found that vitamin D could reduce the incidence of MS. However, experimental datas have shown that vitamin D can only delay disease course in multiple sclerosis (MS) animal model---experimental allergic encephalomyelitis (EAE), but could not prevent the disease process and cure disease. It is obviously that the treatment of vitamin D in MS and EAE is limited.Objective:To clarify the mechanism of vitamin D in treating EAE, and provide experimental evidence for searching better treatments.Methods:We collected cerebrospinal fluid (CSF) and blood from MS patients and patients with other neurological diseases (OND), and we analysed and compared GSN expression using western blot.The myelin basic protein solution (MBP,1mg/ml) was mixed with an equal volume of complete Freund’s adjuvant (CFA, containing5mg/ml pertussis vaccine), and Lewis rats were immunized with two subcutaneous injection in hind foot to induce EAE; Lewis rats in control group were injected with parallel emulsion lacking the MBP component. We evaluate EAE condition from weight, score and inflammatory cell infiltration. From the immunization day, rats were weighed and scored, until day25. At day13after immunization, three rats from EAE group and control group were taken to heart perfusion. The cerebral cortex and spinal cord tissue from rats were taken to histopathological staining (H&E) to observe the condition of inflammatory cell infiltration.Sixty Lewis rats were first separated into three groups based on the EAE status: control (C), pre-stage EAE (P) or acute stage EAE (E). The rats from each of these three groups were fed with a synthetic diet without vitamin D (-D) or with a diet supplemented with vitamin D (+D), to produce six groups designated as follows:-DC,+DC,-DP,+DP,-DE,+DE.-D group rats were fed with diet lacking vitamin D, but containing other nutrients;+D group rats were fed with diet containing vitamin D (3μg/day). The vitamin D was first dissolved in ethanol (1mg/ml), and then mixed with agar. Rats were fed every other day before euthanizing, two weeks in total.25-hydroxyvitamin D and1,25-dihydroxyvitamin D in rats serum were measured using ELISA, in order to determine whether vitamin D was effective absorbed. We also observed and recorded the onset time, inflammatory infiltration and score. We use high performance liquid chromatography (HPLC), western blot, real-time quantitative PCR techniques to analyze sphingosine1-phosphate (S1P) and gelsolin (GSN) concentration in serum and spinal cord from rats.We constructed small interfering RNA targeting GSN (siGSN) and control siRNA (NC), and transfected PC12cells. The apoptosis rate was detected by flow cytometry.Results:We have successfully induced EAE model in Lewis rats:The weight of control rats consistently increases, however, EAE rats weight begin to fall about day11, and the weight begin to recover about day15; The symptom emerged at day11, peaked on the day13, and disappeared at day19; H&E staining showed inflammatory infiltration was obvious in spinal cord of EAE rats.Vitamin D could suppress but could not completely prevent EAE:vitamin D supplementation significantly delayed the onset of EAE and lightened inflammation, but could not reduce disease score. Vitamin D downregulated S1P in serum and spinal cord of EAE rats:S1P level in serum and spinal cords of EAE rats was higher during the acute stage compared with control (C) and pre-stage rats (P), regardless of vitamin D intake; S1P level in serum and spinal cords in EAE rats supplemented with vitamin D were lower than EAE rats fed without vitamin D diet (-DE).Vitamin D downregulated GSN expression:In serum, pGSN level were markedly decrease during the acute stage of EAE rats compared with the pre-stage of EAE rats and control rats, regardless of vitamin D intake; EAE rats fed with vitamin D had reduced cGSN levels in the acute stage. Conversely, in the spinal cords, cGSN was slightly increased during the acute stage of EAE, whether or not they were supplemented with vitamin D. Furthermore, similar to the results from serum, EAE rats fed with vitamin D had reduced cGSN levels in the acute stage.cGSN play a protective role in neuronal apoptosis:Cell apoptosis was significantly higher in cells transfected with siGSN than NC group.Conclusions:1.Vitamin D lower SIP and simultaneously downregulated GSN expression, which is an important reason that vitamin D could not completely cure MS;2. Vitamin D together with human recombinant pGSN may gain more beneficial effect on treating MS;3. Oral FTY720and vitamin D in combination may also have a better therapeutic effect on treating MS.