The Effectiveness And Mechanisms Of DL-3-N-Butylphthalide In The Cuprizone Model Of Demyelination

Multiple sclerosis?MS?is an autoimmune disease characterized by inflammation and demyelination of white matter of the central nervous system?CNS?.Most of drugs are used for inhibiting demyelinating progression through regulating immune response,and rarely show straight-effects on myelin repair.More results have suggested that this disease may be relieved by increasing remyelination and offering protections for the CNS.DL-3-n-butylphthalide?NBP?,a chiral compound from Chinese celery seed extract,has shown improvements to growth and apoptosis in neurons,astrocytes cells and microglia cells by ameliorating mitochondrion dysfunctions.Our previous study showed that NBP improved symptoms,electroencephalogram?EEG?features,and brain magnetic resonance imaging?MRI?in a patient with demyelination and showed an anti-apoptotic effect in the ethidium bromide model of demyelination.However,the mechanisms are still not well understood.In MS studies,the cuprizone?CPZ?model of demyelination is a common tool characterized by mitochondrion dysfunctions resulting in oligodendrocyte apoptosis.Therefore,to identify a new candidate for treatment of MS,we conducted this study to explore the effectiveness and mechanisms of NBP in MS using the CPZ model of demyelination.Part ? Construction of Cuprizone Model of Demyelination,Behavioral Experiments and Analysis of DemyelinationPurpose:Using the CPZ model of demyelination created by feeding CPZ,we explored behaviors for mice with demyelination and the changes in myelin and axons after NBP treatment.Methods:Male C57BL/6 mice with body weights ranging between 15 and16 g?6-7 weeks old?were assigned to the model group and the treatment group.The model group consisted of 2 subgroups:1)the first subgroup included 3 mice who were fed with normal powdered chow every day;2)the second group included 15 mice who received powdered chow mixed with 0.2%CPZ every day,which were documented their body weights every 2 weeks.At the end of the first week,we started collecting tissue samples from 3 mice every week for 5 weeks.The treatment group included 4 subgroups?the control subgroup,the CPZ subgroup,the 10 mg/kg NBP subgroup,and the 20 mg/kg NBP subgroup?:1)each subgroup consisted of 10 mice.The control group received normal powdered chow every day,other subgroups were fed with powdered chow mixed with 0.2%CPZ every day;2)the 10 mg/kg NBP subgroup and the 20 mg/kg NBP subgroup started intraperitoneal injection of 10 or 20 mg/kg of NBP every day at the second week.The control subgroup and the CPZ subgroup also were treated with 0.1 ml0.9%saline by intraperitoneal injection.Each mouse was recorded body weight at the every weekend for 5 weeks and was further treated at the end of the 5th week;3)each mouse was tested learning and memory capacity using Morris water maze test at the end of the 4th week.4 subgroups were recorded balance and motor function using rotarod test and open field test at the beginning of the 5th week;4)we examined integrality of myelin and axons using luxol fast blue staining and NF68 immunohistochemistry staining,and observed the microstructure with electron microscopy,and measured the expression levels of proteolipid protein?PLP?and peripheral myelin basic protein?MBP?by western blot analysis amongst 4 subgroups.Results:Using LFB staining,demyelinations were observed in the mice fed with CPZ at the 2nd week and the peaks were at the 4th week?P<0.05?.The CPZ model of demyelination was successfully constructed at the end of 5th week.The body weights significantly decreased in the CPZ subgroup over time as compared with that in the control subgroup.The mice mental conditions and body weights were improved after NBP treatment 4 weeks?P ₁<0.01,P ₂<0.01?.The results of rotarod test showed the latencies in the rotarod test for the CPZ subgroup were shorter than that for the control subgroup?P<0.05?,which meant that the mice in the CPZ group had worse balance capacity.The two NBP subgroups showed better balance capacity than that the CPZ subgroup showed?P₁<0.05,P ₂<0.05?.However,there were no significantly different movement speeds and distances amongst the 4 subgroups?P>0.05?.The escape latencies for the 4 subgroups were decreased for the first 5 days,but the cumulative distances in the target areas and the frequencies across the platforms were no different amongst the 4 subgroups at the 6th day?P>0.05?.The axon mitochondria and the microstructures of myelin sheath were damaged in the CPZ subgroup,but the symptoms were relieved with dose manner through keeping the integrality of myelin lamellar structure and protecting the axon mitochondria in the two NBP subgroups.The expression levels of PLP and MBP were significantly decreased in the CPZ subgroup as compared with that in the control subgroup?P<0.05 and P<0.01?.There were no differences in the expression levels of PLP and MBP between the CPZ subgroup and the 10 mg/kg NBP subgroup?P>0.05?,but the 20 mg/kg NBP treatment increased the expression levels of PLP and MBP as compared with mice treated with CPZ alone?P<0.05 and P<0.01?.In the CPZ subgroup,the NF68 expression was reduced as well as the positive stains were not continually arranged as compared with that in the control subgroup.As compared with the CPZ subgroup,the NF68 expressions were increased in the two NBP subgroups after NBP treatment?P ₁<0.001,P ₂<0.001?.Especially,the high-dose NBP subgroup showed more NF68 expression than that was observed in the low-dose NBP subgroup?P<0.05?.Conclusions:1)Decreased body weights and balance dysfunctions were observed in the CPZ group as well as NBP treatment increased body weights and relieved balance dysfunctions.2)The mice were almost complete demyelination at the end of the 5th week.NBP treatment ameliorated demyelination with dose manner.3)High-dose NBP treatment promoted remyelination by increasing the expression levels of PLP and MBP.4)NBP protected the axon mitochondria and kept the integrality of myelin lamellar structure.Part ? NBP treatment reduced inflammatory response via suppressing activity of NF-?B signaling pathwayPurpose:To explore vary the influences of NBP treatment or CPZ treatment to inflammatory cell infiltration,and further to illustrate the effects of NBP on activated NF-?B signaling pathway.Methods:The animals and subgroups used for this research were described above.The inflammatory cell infiltration and the levels of astrocyte and microglia in the callosum area were assessed using H&E staining,GFAP and Ibal-1immunohistochemistry?IHC?for the 4 subgroups.Western-blot?WB?was used to evaluate the expression levels of NF-?B?p65??I?B??TNF-?amongst the 4 subgroups.Results:The inflammatory cells were risen in the CPZ subgroup over time,the peaks were from the 4th week to the 5th week?P<0.05?,and the number of activated astrocyte and microglia were significantly increased at the 5th week as compared with that in the CPZ subgroup?P<0.001 and P<0.001?.The inflammatory cells were reduced with dose manner after NBP treatment 4 weeks?P ₁<0.001,P ₂<0.001?.Meanwhile,NBP suppressed astrocyte proliferation?P ₁<0.001,P ₂<0.001?and decreased the number of activated microglia?P₁<0.001,P ₂<0.001?.The levels of NF-?B?P ₁<0.05,P₂<0.001?and cytoplasmic inhibitory protein I?B??P ₁<0.05,P₂<0.001?were increased as well as tumor necrosis factor alpha?TNF-???P ₁<0.05,P₂<0.01?was further releases in the CPZ subgroup.NBP treatment relieved these with dose manner?P<0.05?.Conclusions:1)The inflammatory cells were risen in the CPZ subgroup over time,and were decreased after NBP treatment.NBP treatment inhibited astrocyte proliferation and decrease the number of activated microglia.2)NBP treatment suppressed inflammatory response by decrease of activity of NF-?B signaling pathway and TNF-?expression.Part ? NBP protected oligodendrocyte via inhibitions of activity of mitochondrial apoptotic signaling pathwayPurpose:To assess the number of Oligodendrocyte?OL?and Oligodendrocyte precursor cell?OPC?amongst the 4 subgroups and the effects of NBP on OL profile in the CPZ model of demyelination.Methods:The animals and subgroups used for this research were described above.The number of OL and OPC were evaluated by CC1,Olig2 and NG2 IHC.OL apoptosis was assessed using TUNEL test and immunofluorescent staining.WB was used for evaluation of expression levels of Bax,Bcl-2,CytC,and Cleave Caspase-3.Results:In callosum area,the number of OLs started decreasing at the 2nd week?P<0.05?,the valley was observed at the 4th week followed by increase of OLs at the end of the 5th week?P<0.05?.The expression level of NG2 started increasing at the 2nd week,and summit was observed at the end of the 5th week.At the end of the 5th week,the number of OLs was significantly increased in the CPZ subgroup as compared with that in the control subgroup.However,the number of OLs was reduced with dose manner after NBP treatment?P ₁<0.001,P ₂<0.001?.High-dose NBP treatment decreased OL apoptosis by reducing the expression levels of Bax/Bcl-2?P<0.01?,CytC?P<0.01?and Cleaved Caspase-3?P<0.05?as compared with that in the CPZ subgroup.Increased NG2expression was observed in the mice treated with CPZ alone,as compared with that in the mice treated with saline?P<0.001?.However,the NG2 expression was significantly decreased in the mice treated with NBP plus CPZ?P ₁<0.001,P ₂<0.001?.The similar phenomenon were observed on the Olig2 expression?P₁<0.001,P ₂<0.001?.Conclusions:1)The OLs damage and apoptosis in callosum area were observed after CPZ treatment from 2 to 4 weeks,but its reactive hyperplasia started at the 5th week.In the early stage,OPC proliferation and migration were observed in the CPZ subgroup.OPC proliferation achieved summit at the end of the 5th week followed by differentiation to mature OL.2)High-dose NBP treatment promoted remyelination by inhibition of the expression levels of Bax/Bcl-2,CytC and Cleaved Caspase-3.Conclusion of all textIn this study,we examine the effect of NBP in the cuprizone model of demyelination by evaluating the pathologic,functional and behavioral consequences of treatment with NBP.The results showed that:1.NBP can alleviate demyelination and axonal injury in CPZ demyelinated mice;2.NBP can reduce inflammatory response by inhibiting NF-?B signaling pathway activation;3.NBP inhibits mitochondrial apoptosis signaling pathway to protect the OL.These data suggest that NBP may not only have anti-inflammatory properties but also promote the survival of OL in a mouse cuprizone model of demyelination.