| Background:Multiple sclerosis is a neurological demyelinating disease, which tends to attack the optic nerve, brain stem and spinal cord. Though MS pathogenesis is ill defined, the hypothesis that MS is an immune response to autologous antigen of the axon myelin sheath cell under specific genetic and environmental conditions has generally been accepted. Secondary progressive multiple sclerosis (SPMS) is one type of MS, often accompanying with varying degrees of physical disabilities.Objective:Comparative proteomics research was carried out in the cerebrospinal fluid (CSF) between the patients with SPMS and controls to investigate the potential biomarkers; and the relationship between these biomarkers and the disease was explored using the experimental autoimmune encephalomyelitis (EAE) animal model, in order to providing the foundation for clinical diagnosis and treatment.Methods:CSF was extracted from patients by the method of lumbar puncture and pooled from patients with SPMS and patients with other neurological disease. The proteins were precipitated with ice-cold acetone. Then the two-dimensional difference gel electrophoresis (2-D DIGE) was applied and the differential expression protein spots were selected by the DeCyder software. The peptides mass fingerprint (PMF) was got by the matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and searched in SWISSPROT database to identify each proteins. Then vitamin D-binding protein was validated by western blot.The EAE model was established in Lewis rats by immunizing rats with myelin basic protein (MBP), complete Freund’s adjuvant (CFA) and pertussis vaccine (PV). These rats were examined for the clinical signs and weight daily. Tissues of central nervous system (CNS) were dissected after12days from induction, and the pathological changes as well as the distribution of DBP in spinal cord were observed with histopathological, immunohistochemistry, western blot, and RT-PCR techniques.20Lewis rats were randomly divided into four groups;+DC group:the control group with vitamin D supplements,-DC group:the control group without vitamin D supplements,+DE group:the EAE group with vitamin D supplements,-DE group:the EAE group without vitamin D supplements. Each rat of+DC and+DE group was fed agar containing3μg vitamin D once every two days. Then the clinical signs and weight were examined daily. The spinal cords were dissected after12days from induction. To investigate the influence of DBP and vitamin D on EAE model, the concentration of DBP, vitamin D metabolites and DBP mRNA were compared by western blot, ELISA and real time-PCR among the four groups.Results:2-D DIGE maps of CSF from SPMS and control group were got.13protein spots exhibited significant difference, corresponding to8distinct proteins. DBP was up-regulated in the experimental group, which was validated with western blot analysis.EAE model was established successfully. HE staining showed pathological changes in the spinal cords of EAE rats. The result of immunohistochemistry showed the distribution of DBP in neurons of spinal cord, and RT-PCR proved the synthesis of DBP mRNA in spinal cord. The concentration of DBP was elevated in spinal cord of EAE rat; however, the spinal cord DBP mRNA did not varied obviously.After supplementing vitamin D, the serum concentration of25-(OH)D3was higher in+DC group compared with-DC group. The serum levels of1,25-(OH)2D3increased in both the+DC and+DE groups. There was no significant difference of spinal cord1,25-(OH)2D3concentration between+DE and-DE group, however, the DBP concentration was higher in-DE group than+DE group. In summary, compared with-DE group, the rats of+DE group displayed delayed onset of EAE and a decrease in disease severity.Conclusions:DBP in the cerebrospinal fluid could serve as a specific diagnostic biomarker for SPMS. We demonstrate the vital function of increased levels of free vitamin D metabolites for multiple sclerosis treatment, especially for SPMS patients. |
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