| Objective: Multiple sclerosis is not only an autoimmune disease of the central nervous system, but also a kind of neurodegenerative disease. There is an obvious relevance between the incidence of multiple sclerosis and global latitude, the farther from the equator, the higher of the incidence. We aim to study the effects of 1,25(OH) D23 on the progression of experimental autoimmune encephalomyelitis(EAE), both concentrate on the changes of myelin in white matter and the neurons in the gray matter of the central nervous system. Further we plan to explore the potential mechanisms of 1,25(OH) 2D3.Methods: 27 female C57BL/6 mice, which were 8-10 weeks and weighed 18-20 g were randomly divided into three groups: Control group, EAE group, and VD3 group(it represents 1,25(OH) 2D3 treated group), and each group contains 9 mice. The last two groups were immunized with MOG peptide to establish EAE mice models. 250μg MOG35-55 synthetic peptides were dissolved by 500μl saline, and then mixed with an equal volume of complete Freund’s adjuvant(CFA) supplemented with 1 mg/m L of heat-killed mycobacterium tuberculosis H37 Ra. Then mixed well, mice were injected subcutaneously into four flank sites. Subsequently they were injected intraperitoneally with 500 ng pertussis toxin at 0 and 48 h post-immunization. Mice of VD3 group were injected intraperitoneally with 0.1ml 1,25(OH)2D3(diluted in normal castor oil in the concentration of 1 μg/ml 1,25(OH)2D3) every three days since the immunized day, mice of other two groups were injected with same dose of vehicle on the same day,and they were weighed and examined daily(8:00 and 16:00) for clinical signs of EAE since immunized day. On day 20 post-immunization, mice were directly euthanized, and the brain tissue surrounding the lateral ventricle was removed, some tissues were immediately frozen via submersion in liquid nitrogen,then placed in a-80℃ refrigerator, these tissues were used to detect the expressions of Beclin1 and LC3 by Western Blot. The lumbosacral enlargements of spinal cords were removed and fixed with 4%(w/v) paraformaldehyde, some of them were embedded in paraffin for HE, LFB and Nissl staining to evaluate the degree of inflammatory infiltration, demyelination and changes of neurons; other brain tissues(surrounding the lateral ventricle) and lumbosacral enlargements were made into crystal sections for immunofluorescence assay, they were stained with Neu N, Neu N and Beclin1, Neu N and LC3 respectively, to observe the changes of neurons and the autophagic level in neurons. Data are presented as mean ± SD. The onset rates of EAE between EAE and VD3 [referred to as 1,25(OH)2D3] groups were analyzed by Fisher’s exact test. The difference in clinical scores between groups was examined by Mann-Whitney U-test. The differences in inflammation and demyelination between the two groups were examined by Student’s t-test, All other statistical comparisons among groups were tested by One-Way ANOVA, followed by the SNK-q test or the Dunnett’s Multiple Comparison test. A P-value less than 0.05 was considered statistically significant.Results: 1 1,25(OH)2D3 significantly alleviates clinical symptoms in EAE mice Mice in control group exhibited normal activity and appetite. The incidence in EAE group mice was 100%, mice had different degrees of weight loss since the immunized day, they showed reduced activity, tail weakness, insensitive reaction and rough fur. The incidence in VD3 group was 11.1%(P<0.01). By comparing the clinical scores of the last two groups, we found that the scores in VD3 group were significantly lower than EAE group since the 11 day post-immunization(11th day, P<0.05; after 11 th day P<0.01). 2 1,25(OH)2D3 mitigates inflammation and demyelination in EAE mice2.1 HE staining of lumbosacral enlargements in mice showed: Control mice had no inflammatory infiltration and “blood vessel muffs”. On the peak stage of EAE, mice showed diffuse inflammatory infiltrations, some were with “blood vessel muffs”. VD3 mice had little inflammatory cell infiltrations. The pathological scores of HE staining were significantly different. 2.2 LFB staining of lumbosacral enlargement in mices showed: Control mice showed no demyelination. There were amount of demyelination areas in the sections of peak stage EAE mice. VD3 mice had no significant demyelination area. The pathological scores of LFB staining were significantly different. 3 1,25(OH)2D3 prevents neurodegeneration in GM of EAE 3.1 Nissl staining of lumbosacral enlargements in mice showed: EAE mice had significantly reduced number of neurons in the gray matter comparing with the control mice(P<0.01), and neurons showed varying degrees of damage, such as nuclear shrinkage, reduced number of Nissl bodies and so on. The number of neurons in VD3 mice was increased compared with EAE mice(P<0.01). 3.2 Immunofluorescence staining showed: compared with the control group, the number of neurons in the gray matter of the EAE mice spinal cord was significantly decreased, and the neuron number was increased in VD3 group. 4 Autophagic flux is impaired in EAE mice and 1,25(OH)2D3 influences autophagy in vivo. 4.1 Western Blot showed: EAE mice on the peak stage of disease showed decreased expression of Beclin1 compared to control mice(P<0.05), while the expression of Beclin1 in VD3 mice were significantly higher than the EAE mice(P<0.05). The LC3-II expression in each group showed the opposite trend, the expression of LC3-II was significantly increased in EAE mice compared with control ones(P<0.01), while VD3 mice showed significantly lower expression of LC3-II than the EAE mice(P<0.05). We added another EAE group treated with chloroquine, on the peak stage of disease, the expression of LC3-II showed no difference between chloroquine group and EAE group.4.2 Immunofluorescence showed: a certain expression of Beclin1 was detected in the control mice neurons, the expression of Beclin1 in the EAE mice neurons was significantly lower. In VD3 mice, Beclin1 was diffused expressed. LC3 in neurons of control mice and VD3 mice was barely detectable, which was higher expressed in EAE mice.Conclusions: 1 Autophagy played a role in the development of multiple sclerosis. On the peak stage of disease, EAE mice showed blocked autophagy flux. 2 1,25(OH) 2D3 EAE mice could not only reduce the inflammatory demyelination in white matter, but also could alleviate neuronal damage and loss by regulating neuronal autophagy. In summary, this study proved that 1,25(OH) 2D3 and its analogs, or other neuronal autophagic regulator could be expected to become the new direction for the treatment of multiple sclerosis. |
PDF Full Text Request