| Multiple sclerosis(MS)is a a long-lasting autoimmune inflammatory and demyelinating disease of the central nervous system(CNS).The pathological hallmarks of MS are white matter demyelination,inflammation,axon damage,and blood-brain barrier(BBB)disruption.The etiology of MS is still not clear,the pathogenesis of MS is complicated,it is known to be attributed to viral infections,genetic background,and environment factors.Therefore,MS may be not one disease,but rather a collection of different syndromes presenting themselves with inflammation and demyelination and multiple mechanisms underlying the etiology of the disease.Because of the complex etiology of MS,in recent years,several studies have shown that necroptosis is involved in the pathogenesis of multiple sclerosis.Regulated cell death has essential functions in development and in a tissue homeostasis.Necroptosis is a newly discovered pathway of regulated necrosis,it is a programmed cell death.For many years apoptosis was considered to be the only form of regulated cell death,whereas necrosis was seen as an unregulated accidental cell death process.Recently years,a lot of evidence has been shown that necroptosis is one of the best characterized forms of regulated necrosis.Much of our knowledge of necroptosis comes from studies of tumour necrosis factor TNF-? signalling.TNF-? is binding with TNFR1,then the necrosome is activated.At last,necroptosis is achieved by mitochondrial PGAM5-DRP axis.NBP is a synthesized compound based on the pure component,originally extracted from the seeds of Apium graveolens Linn.NBP has protective effects that reduce ischemia-induced brain damage and neuronal cell death,improve cerebral blood flow,decrease brain edema,and preserve the bloodbrain barrier.In addition,NBP has antiapoptotic,antithrombotic,protection of mitochondria and anti-inflammatory properties.Based on its protective effect on mitochondria,we think that NBP may be beneficial for EAE treatment.There are more than one model of multiple sclerosis.Several animal models have been developed in different animal species to focus on different aspects of the disease.Experimental autoimmune encephalomyelitis(EAE)is the most commonly used model for MS.In our study,we used the MOG35-55 immunized C57BL/6 mice to prepare the EAE model,and focused on the neuroprotective effect on EAE.Silence of PGAM5 in spinal cord,observed mechanisms of ameliorating the disease severity in the mice with EAE.In vitro,BV2 microglia cells were cultured,and the mechanism of neuroprotective effect of butylphthalide on EAE mice was explored,finding a potential target for NBP.Part one The effect of NBP on prevetion of experimental autoimmuneencephalomyelitis and necroptosisObjective:The most common and classic mouse model of multiple sclerosis was established,We used NBP to assess the impact on EAE and necroptosis by HE staining,TEM and Western Blot.Methods:1.The selection of experimental mice and the establishment of animal models.C57BL/6 female mice,about 8-10 weeks old,weighed about 18-20 g.The EAE model was induced in mice by subcutaneously injecting into the hindquarters with MOG35-55(250?g for four sites)emulsified in an equivalent volume of Complete Freund's Adjuvant,containing additional 4mg/ml of heat-killed mycobacterium tuberculosis H37 Ra,immunized mice also received two intraperitoneal(i.p.)injections with 500 ng of pertussis toxin.2.The experimental mice were randomly divided into normal control group,EAE group and butyphthalide group.Since the first day after onset,butyphthalide group mice were intraperitoneally injected with butyphthalide 80 mg / kg daily,and normal control group and EAE model group were given intraperitoneal injection of the same amount of DMSO and normal saline solution daily from the first day after the onset of the disease.3.From day 1 after immunization,the mice were scored twice daily(8 : 00 and 16:00)for two time periods and weighed.The neurological score was used to Weaver's(15 score)method.4.Histopathological observation observing the infiltration of inflammatory cells in the lumbar spinal cord of experimental mice by HE staining.5.The changes of mitochondria and myelin sheath in mice were observed by transmission electron microscope.6.The expression of protein in necroptosis pathway and inflammatory related protein were detected by Western Blot with experimental mice.7.SPSS19.0 statistical software was used for statistical analysis.The measurement data are expressed as "mean ±standard deviation"(x ±s).The data are tested for normality and homogeneity of variance.Kruskal-Wallis test and LSD test were used to compare the mean of multi-group measurement data.The difference was statistically significant(P < 0.05).Results:1.Using butyphthalide to treat EAE mice could relieve the clinical symptoms of EAE mice and decrease the neurological function score of EAE mice.2 Butylphthalide could reduce the infiltration of inflammatory cells in the central nervous system(CNS).2.Butyrophthalide could protect mitochondria and alleviate demyelination in EAE mice.4 Butyphthalide down-regulated the expression of proteinin in the necroptosis pathway and inflammatory related protein: TNF-?and TNFR1 were down-regulated comparing with the model group,the expression of RIPK1,RIPK3,MLKL,PGAM5 and DRP1 were down-regulated in drug intervention group compared with the model group.The expression of IL-1? in drug intervention group was lower than that in model group.Part two Effect of silencing PGAM5 gene expression on experimentalautoimmune encephalomyelitis in miceObjective: The model of EAE was established,silencing the PGAM5 gene to abserve the changes of inflammatory cell infiltration in spinal cord in PGAM5 gene silencing group and EAE mice group.The expression of PGAM5,DRP1,p-DRP1 protein and the transcription level of PGAM5,TNF-?,IL-1? in spinal cord were observed.The changes of mitochondria and myelin sheath were observed in this process.Methods:1.The selection of experimental mice and the establishment of animal models.C57BL/6 female mice,about 8-10 weeks old,weighed about 18-20 g.The EAE model was induced in mice by subcutaneously injecting into the hindquarters with MOG35-55(250??g for four sites)emulsified in an equivalent volume of Complete Freund's Adjuvant,containing additional 4?mg/ml of heat-killed mycobacterium tuberculosis H37 Ra,Immunized mice also received two intraperitoneal(i.p.)injections with 500?ng of pertussis toxin.2.Histopathology observation silencing PGAM5 gene to interfere with the EAE mice.At the peak of the disease,the spinal cord lumbar was taken from the experimental mice,and the infiltration of the spinal cord inflammatory cells in the experimental mice was observed by HE staining.3.Western Blot silencing PGAM5 gene to interfere the EAE mice.The spinal cord of the mice with peak period was taken and the protein expression level of PGAM5,DRP1,p-DRP1 were detected.4.qRT-PCR Silencing the PGAM5 gene,interfering with EAE mice,taking the spinal cord of the experimental mice with peak period,the transcription level ofPGAM5,TNF-?,IL-1?were measured.5.TEM Silencing PGAM5 gene,taking the spinal cord of the experimental mice with peak period the changes of mitochondria and myelin sheath in mice were observed by transmission electron microscope(TEM).6.SPSS19.0 statistical software was used for statistical analysis.The measurement data are expressed as "mean ±standard deviation"(x ±s).The data are tested for normality and homogeneity of variance.Kruskal-Wallis test and LSD test were used to compare the mean of multi-group measurement data.The difference was statistically significant(P < 0.05).Results:1.In the EAE group,observing diffuse inflammatory cells by with HE staining and the inflammatory cells in the EAE+ PGAM5 shRNA group decreased significantly.2.Western blot test :Expression of PGAM5 protein in EAE group is higher than that in normal control group The expression of PGAM5 in the silencing group was significantly lower than that in the EAE group.The expression of DRP1 was up-regulated in the EAE group compared with the normal control group,and down-regulated in the PGAM5 silencing group by western blot.The expression of P-DRP1 in EAE group was lower than that in normal control group,and in PGAM5 silencing group was higher than that in EAE group.3.There were higher mRNA levels of PGAM5,TNF?,IL-1? in EAE mice than those in normal control mice.And There were lower mRNA levels of PGAM5,TNF?,IL-1? in silencing PGAM5 of EA Emice than those EAE mice.4.The demyelinating degree of silencing PGAM5 group was reduced,and the structure of mitochondria was relatively intact in the silencing PGAM5 group.Part three Butylphthalide inhibits necroptosis of microglia by modu-lating PGAM5Objective : A model of necroptosis was established by using BV2 microglia to study the effect of butylphthalide on PGAM5-DRP1 axis in necroptosis pathway and to explore the mechanism of butylphthalide in treating EAE mice.Methods:1.The murine microglial cells BV-2 were obtained from the National Infrastructure of Cell Line Resource(Beijing,China)and cultured in the DMEM supplemented with 10% fetal bovine serum,100 U/mL penicillin and 100 ug/mL streptomycin.Cultured in constant humidity incubator(37 ?,containing 5% CO2).2.Building of necroptosis model,drug or virus intervention.BV2 microglia cells were divided into 7 groups.When the cells reached 50% to 60%,the silencing PGAM5 gene carried by adenovirus was added in group 4,and the empty adenovirus was added to group 5.In the 6th group,the overexpression PGAM5 gene carried by adenovirus was added,the overexpression PGAM5 gene of empty virus was added to the seventh group,and in the 21 st hour,the butylphthalide(10uM)was added in groups 3,6 and 7 respectively.The 24 th hour,TNF-?(50 ng / ml)and Z-VAD-FMK(50 ?mol/L)were added into the groups except group 1,and in the 48 th hour,necroptosis model was established.7 groups of cells were collected and the targets were detected.3.The mRNA expression of IL-1?and IL-1?were detected by qRT-PCR in different BV2 cell groups.4.Detection of the protein expression of PGAM5,DRP1,p-DRP1 in different BV2 cells by Western Blot.Results:1.The transcription mRNA level of IL-1?mRNA was up-regulated in the model group compared with that in the normal control group.The transcription level of IL-1 ? in shPGAM5 group was lower than that in model group.The transcription mRNA level of IL-1? in shPGAM5 empty virus control group was not significantly different from that in model group.The transcription level of IL-1? in butyphthalide group was significantly lower than that in model group.There was no significant difference in the transcription level of IL-1 ? between the NBP+ overexpression PGAM5 gene group and the model group.The transcription level of IL-1?in NBP+ overexpression PGAM5 gene of empty virus control group was lower than that of model group.2.The relative mRNA level of IL-1? was significantly higher in the model group than that in normal control group.The mRNA level of IL-1? in model + shPGAM5 group was significantly lower than that in model group.There were no significant differences in mRNA level of IL-1? between shPGAM5 empty virus control group and model group.The transcription level of IL-1? mRNA in butyphthalide group was lower than that in model group.There were no significant differences in the mRNA level of IL-1? between the NBP +PGAM5 overexpression group and the model group.The mRNA level of IL-1? in NBP+ overexpression PGAM5 gene of empty virus control group was lower than that of model group.3.The expression of PGAM5 in 7 groups were detected by Western Blot.The expression of PGAM5 in model group was significantly higher than that in normal control group.The expression of PGAM5 in shPGAM5 group was lower than that in model group.There were no significant differences in the expression of PGAM5 between the shPGAM5 empty virus control group and the model group.The protein expression of PGAM5 in butyphthalide group was lower than that in model group.There were no significant differences in the expression of PGAM5 protein between the NBP +PGAM5 overexpression group and the model group.The expression of PGAM5 in NBP+ overexpression PGAM5 gene of empty virus control group was lower than that in model group.4 The expression of DRP1 protein in 7 groups were detected by Western Blot.The expression level of DRP1 in model group was significantly higher than that in normal control group.The expression of DRP1 in sh PGAM5 group was lower than that in model group.There were no significant differences in the expression of DRP1 between the shPGAM5 empty virus control group and the model group.The protein expression of DRP1 in butyphthalide group was lower than that in model group.There were no significant differences in the expression of DRP1 protein between the NBP +PGAM5 overexpression group and the model group.The expression of DRP1 in NBP+ overexpression PGAM5 gene of empty virus control group was lower than that in model group.5 The expression of p-DRP1 protein in 7 groups were detected by Western Blot.The expression level of p-DRP1 in model group was significantly lower than that in normal control group.The expression of p-DRP1 in shPGAM5 group was higher than that in model group.There were no significant differences in the expression of p-DRP1 between the shPGAM5 empty virus control group and the model group.The protein expression of p-DRP1 in butyphthalide group was higher than that in model group.There were no significant differences in the expression of p-DRP1 protein between the NBP +PGAM5 overexpression group and the model group.The expression of p-DRP1 in NBP+ overexpression PGAM5 gene of empty virus control group was higher than that in model group.Conclusion:1.Butyphthalide alleviated the clinical symptoms of EAE mice,inhibited inflammatory reaction,down-regulated the expression of TNF-? and IL-1 ?,and protected mitochondria of EAE mice.The expression of protein in necroptosis pathway was inhibited by butyphthalide.2.Silencing PGAM5 gene also played a neuroprotective role in EAE mice.PGAM5-mediated necroptosis was involved in the pathogenesis of multiple sclerosis/EAE.3.In vitro experiments of BV2 microglial cells,by modulating PGAM5,butylphthalide inhibited IL-1?,IL-1? expression,and inhibited the necroptosis in microglial cells.It was proved that PGAM5 is the target of butylphthalide in the pathogenesis of necroptosis in multiple sclerosis/EAE. |
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