| Objective: Multiple sclerosis(multiple sclerosis,MS) is a common chronic inflammatory demyelinating disease in the central nervous system with demyelination,axonal degeneration, inflammatory cell infiltrate,proliferation of glial cells and progressive neurological dysfunction as the main characteristic. MS morbidity is high,more frequent in young people and there is no effective treatment method now. The morphology and function of MS mitochondria damage research has become the focus of scholars at home and abroad. NBP(dl-3n-butlphalide, NBP) is the first with independent intellectual property rights of the national first class new drugs in the field of cerebrovascular disease treatment in our country,and it is the first innovation through the drugs’ effects on multiple targets and multiple links to treat ischemic cerebral apoplexy in the world. Clinical pharmacodynamics research show that the NBP has protection of mitochondrial function and increase the effect of ischemic perfusion,can also block multiple pathological link in ischemic brain injury caused by cerebral apoplexy,has strong anti cerebral ischemia action,can obviously reduce the infarction area,reduce the infarction cerebral edema,improve nerve function,protect the brain tissue. Based on NBP effect on ischemic encephalopathy research foundation, we hypothesized that NBP can play a role in the prevention and treatment of MS, but has not yet to see the relevant studies and reports. Therefore, this research making the human classic model of multiple sclerosis-experimental autoimmune encephalomyelitis(experimental autoimmune encephalomyelitis, EAE) mice as the research object, by intraperitoneal injection of different doses of butyl phthalide injection lead to drug intervention, through the dynamic observation clinical symptoms and the expression level of the extracellular signalregulated kinase 1 / 2(ERK1/2) and Potassium sodium atpase(Na+/K+ATPase)in spinal cord tissue of EAE mice at different stages,to explore whether NBP has therapeutic effect and possible mechanism of EAE, and provide a new idea and method for clinical treatment of MS, and provide more experimental basis for clinical application of the expansion of the scope of NBP.Methods: With complete freund’s adjuvant + low-dose MOG35-55 for immune antigen emulsions induce C57 BL / 6 mice to make the EAE model.Mices were randomly divided into four groups:,EAEgroup, NBP-L(low dose of NBP) group,NBP-H(high dose of NBP) group,and D(dexamethasone)group.The mice in NBP-L and NBP-H group were received NBP(80mg/kg/d and 120mg/kg/d) by peritoneal injection, the mice in D(dexamethasone)group were received dexamethasone( 0.07mg/kg) by peritoneal injection.Three groups were injection for 14 days in a row, EAE group do not do special intervention, at the same time take nine normal mice as normal control group(N group). Observe each group of mice infection situation and weight change and nerve dysfunction, and adopt the internationally used Benson(5 points)score standards for neurological function score.Mices would be put to death and remove the organization of the lumbar spinal cord parts on the onset day(0d),7 days(7 d) and 14 days(14 d) after drug intervention. In a real-time quantitative PCR(rt-pcr) method to observe ERK1/2 mRNA and Na + /K +ATPase mRNA expression in the mice lumbar spinal cord swelled,with immunohistochemical method to observe the expression of Na + /K +ATPase.The data of each group were deal with by SPSS13.0 statistical software.Each measurement data present by mean ±standard deviation(x±s),Multiple sets were compared by the single factor analysis of variance,comparison between count data set with chi-square test,Comparison between the two groups with the Student ’s t – test,with P < 0.05 for statistical significance.Result:1 Observation of clinical symptoms in each group of mice: 7 days after oneset,NBP-L group,NBP-H group, D group respectively compared withEAE group,the mice nerve function scores were smaller than that of EAE group(P < 0.05);NBP-L group,NBP-H group,D group mutual comparison, the mice nerve function scores didn,t have statistically significant difference(P >0.05). 14 days after oneset,NBP-L group,NBP-H group, D group respectively compared with EAE group,the mice nerve function scores were smaller than that of EAE group,the difference was statistically significant(P<0.05);NBP-L group,NBP-H group,D group mutual comparison, the mice nerve function scores didn,t have statistically significant difference(P> 0.05).2 The correlation analysis results of the nerve function score and the expression levels of ERK1/2 in the spinal cord tissue at 0, 7, 14 days in EAE mice: r = 0.9914,ν=1,we can conclude 0.025 ﹤ P ﹤ 0.05 by checking t boundary value table. According to the level ofα= 0.05,between the nerve function score and the expression level in spinal cord tissue of EAE mice has a negative linear relationship.3 The expression of ERK1/2mRNA in different groups and different stages: In the day EAE model mice onset 0 days, the expression of ERK1/2mRNA in EAE0 group was lower than the Control group,the difference was statistically significant(P<0.05).In the day EAE model mice onset 7 days, the expression of ERK1/2 in EAE7 group compare with EAE0,there was no statistically significant difference(P>0.05),the expression of ERK1/2 in NBP-H7 group was higher than the EAE7 group,the difference was statistically significant(P<0.05),the expression of ERK1/2 in NBP-L7 group or D7 group compared with EAE7 group, there was no statistically significant difference(P>0.05). In the day EAE model mice onset 14 days, the expression of ERK1/2 in EAE14 group were lower than the EAE0 group or EAE7 group(P<0.05); the expression of ERK1/2 both in D14 group and NBP-H group were higher than that in the EAE14 group,the difference was statistically significant(P<0.05);the expression of ERK1/2 in NBP-H14 group was higher than that in D14 group, the difference was statistically significant(P<0.05).4 the number of Na+/K+ATPase positive cells in spinal cord of the mice:EAE group, the NBP group and group D each subgroup Na + /K +ATPase positive cells expression are lower than normal control group(P <0.05), but the NBP group and group D all the Na + / K + ATPase number of positive cells were higher than the same period EAE group(P<0.05). The expressions of the Na + / K + ATPase Various in subgroups in group D are higher than those of the NBP group each subgroup(P< 0.05), The expression of the Na + / K + ATPase in NBP14 day is higher than NBP7 day group(P<0.05), but between different dose groups in terms of NBP Na + / K + ATPase expressed no obvious difference(P> 0.05).5 The expression of Na+/K+ATPase mRNA in different groups(real-time PCR): Mouse spinal cord Na + / K + ATPase expression level in EAE0, EAE7,EAE14, NBP – L7, NBP- H7, NBP L14, NBP H14 and D14 are lower than in normal control group(P<0.05), NBP- L14 and NBP- H14 group are higher than EAE7, EAE14 group(P<0.05), D group 14 day, expression is higher than EAE14 group day,(P<0.05), NBP –L7 and NBP- H7 express no significant difference between each other(P> 0.05), NBP-L14 and NBP- H14 express no significant difference between each other(P> 0.05).Conclusions:1 In the process of EAE pathogenesis associated with mitochondrial damage, the content of Na + / K + atpase decreased; Butyl phthalide can lessen the role of mitochondrial damage.2 There is a negative linear correlation between illness severity of EAE and the expression of ERK1/2 level.3 Butyl phthalide can improve ERK1/2 expression level to inhibition the permeability transition pore opening of the mitochondrial, thereby increasing the content of Na + / K + atpase to protect mitochondrial function, and the protection of nerve tissue.4 Dexamethasone through anti-inflammatory effects can significantly reduce mitochondrial damage,but because of its side effects larger,thus reducing its therapeutic effect,and the comprehensive consideration of safety and effectiveness,butyl phthalide of EAE therapy may be more benefical,especially long-term oral may have more benefits. |
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