| Objective:In this study,a central nervous system demyelinating model was established in C57BL/6 mice induced by Cuprizone(CPZ).After the model was established,bilobalide(BB)was intraperitoneally injected to observe behavior of the mice,the changes of the myelin sheath,peripheral immune regulation and microglia polarization,elucidating the underlying cellular and molecular mechanisms.The clarification of the targets and pathways of BB in the treatment of central demyelinating diseases will provide scientific and in-depth theoretical basis for the application of BB in the treatment of MS,and furthermore to provide new ideas for the neuroprotection and myelin regeneration of BB.Methods:In this study,C57BL/6 male mice were used to establish CPZ-induced demyelinating model.The mice were divided into normal group,model group and BB treatment group.Eight mice in normal group were fed with normal diet for 6 weeks,and mice in model group and BB treatment group were fed with 0.2%cuprizone for 6 weeks.On day 28 after feeding,BB was administered by intraperitoneal injection of the treatment group consecutively until day 42.And the mice of normal group and model group were given daily intraperitoneal injection of saline.The behavioral tests and the weight of the animal were recorded from the time of modeling.At the end of the sixth week,the animals were sacrificed.Blood was taken from the eyes,spleen and brain tissue were taken.The splenic mononuclear cells(MNC)were separated.The brain tissue was divided into two parts:protein extraction and fixation.Luxol Fast Blue(LFB)staining,Black Gold II staining and MBP immunofluorescence staining were used to observe the myelin morphology and loss.Western blot was used to detect the expression of MBP.ELASA assay was used to determine the release of cytokines in serum,supernatant and brain.The changes of CD4~+T cells and B cells were detected by flow cytometry.The changes of glial cells were observed by immunofluorescence staining.Each group of experimental data was statistically analyzed using GraphPad Prism 5.0 statistical software.Results:1.During the first week of CPZ feeding,the weight loss of CPZ-fed mice was significant and maintained in a stable and lower weight for the next three weeks compared to normal mice.In the following two weeks,intraperitoneal injection of saline or BB every day was performed.Compared with the model group,the weight of mice in the BB treatment group was generally increased,and the behavioral performance was significantly improved.In Elevated Plus Maze test,the percentage of the open arm entry times in the model group was(6.05±0.76)%,and the BB treatment group was(10.4±1.97%,p<0.05).In the Pole test,the walking time of the model group was 15.29±1.24 second,and the BB treatment group was 12.05±1.15 second(p<0.05).2.Luxol Fast Blue staining,Black Gold II staining and MBP staining of the corpus callosum showed that the myelin loss was severe in the model group,and that of BB treatment group was significantly reduced compared with the model group.In LFB staining,the colouring area of corpus callosum in the model group was(3.17±0.65)*10~4,and the BB group was(4.41±3.33)*10~4,the difference between this two groups was statistically significant(p<0.05).In Black Gold II staining,the colouring area of corpus callosum in the model group was(11.31±0.61)*10~4,and that of BB group was(29.56±5.17)*10~4,the difference between the two groups was statistically significant(p<0.05).In MBP staining,the colouring area of corpus callosum in the model group was(9.67±2.13)*10~2,and that of BB group was(19.86±4.22)*10~2,the difference between two groups was statistically significant(p<0.05).3.The expression of MBP was detected by Western blot.Compared with the model group,the expression of MBP was significantly increased after BB administration(p<0.05).4.ELISA was used to detect the production of MOG antibody and the concentration of IL-1?,IL-6 and TNF-?in the cultured supernatant.The results showed that,compared with the model group,the BB treatment significantly inhibited the expression of IL-1?(the model group was 120.5±1.04,the BB treatment group 28.8±0.85,p<0.001)and IL-6(the model group was 298.3±2.75,the BB treatment group 114.5±3.19,p<0.001),however,the expression of TNF-?was not significantly different(p>0.05).5.Flow cytometry revealed no significant differences in CD4~+IL-17~+and CD4~+IFN-?~+of splenic MNCs.Immunofluorescence staining showed that T cells,B cells and macrophages were infiltrated in the cerebral cortex of the model group,and the infiltration of T cells(the model group was 3.8±0.37,the BB treatment group 1.2±0.2,p<0.05),B cells(the model group was 5.2±0.86,the BB treatment group 1.8±0.21,p<0.05)and macrophages(the model group was 6.2±0.66,the BB treatment group 2.4±0.51,p<0.05)was suppressed after BB administration.6.The expressions of Iba1~+iNOS~+and Iba1~+NF-?B~+in the brain were detected by immunofluorescence double staining.The results showed that in the model group,the expressions of Iba1~+iNOS~+and Iba1~+NF-?B~+were significantly activated,and the expressions of Iba1~+iNOS~+(the model group was 13.4±1.7,the BB treatment group3.8±0.58,p<0.01)and Iba1~+NF-?B~+(the model group was 9.6±0.81,the BB treatment group 3.6±0.87,p<0.01)were inhibited after BB treatment.7.Immunofluorescence double staining of Iba-1~+and O4~+showed that in the model group,Iba-1~+microglia migrated and aggregated in corpus callosum,medial septum nucleus and striatum,resulting in the death of O4~+oligodendrocytes,while BB prevented the damage of O4~+oligodendrocytes caused by migration of Iba-1~+microglia.Conclusion:1.BB can effectively attenuate the symptoms and pathological injury induced by the feeding of CPZ,which is related to increasing the expression of MBP and preventing the loss of myelin sheath.2.BB may reduce inflammatory injury by inhibiting the activation of Iba1~+NF-?B~+inflammatory signaling pathway,reducing the infiltration of T cells,B cells,macrophages and the secretion of inflammatory cytokines IL-1?and IL-6,inhibiting the production of MOG antibody and the activation of microglias,therefore,reduce the damage of oligodendrocytes,and promote the formation of oligodendrocytes. |
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