| Multiple sclerosis is an autoimmune inflammatory demyelinating disease of the central nervous system,characterized by oligodendrocyte(OLs)loss,demyelination,inflammation and neurodegeneration.Clinically,there are multiple spatial and temporal lesions.It is widely distributed all over the world.It can lead to severe physical or cognitive impairment and neurological deficits.The course of the disease is repeated,the disability rate is high,and the burden of family and society is aggravated.However,the etiology and pathogenesis of multiple sclerosis remain unclear.At present,it is believed that genetic susceptibility,environmental factors,viral infection,autoimmune abnormalities,oxidative stress and other factors play a comprehensive role.Oligodendrocyte is the only myelin-forming cell.At present,it is believed that myelin sheath is formed by a flat process formed by oligodendrocyte membrane.The number and function of OLs play an irreplaceable role in the integrity of myelin sheath.Protecting oligodendrocyte from damage in NS also provides a new idea for the treatment of multiple sclerosis.Mitochondria are cell energy processing plants.Mitochondrial damage leads to cell energy metabolism disorder and cell energy exhaustion.At the same time,when mitochondria stimulate endogenous apoptotic pathways mediated by mitochondria due to noxious factors,they lead to apoptosis.Racemic-3-n-butylphthalide(NBP),a racemic substance extracted from celery,is a new drug with independent intellectual property rights in China.It is mainly used in patients with cerebral infarction.It was found that NBP could stabilize the structure of mitochondria,protect the function of mitochondria,improve the blood flow of brain tissue,anticoagulation and protect the blood-brain barrier,and achieve good benefits in the treatment of patients with cerebral infarction.EB is an inlaid DNA dye.When EB is injected into brain tissue,localized demyelination of white matter can be seen.It can induce demyelination at specific sites.It is widely used in demyelinating model of central nervous system.objectiveThe aim of this study was to establish an EB model by directional injection of EB into the right ventricle to induce demyelination of periventricular white matter.To explore the effect of NBP on EB model and its mechanism,NBP was used as an intervention drug.MethodThirty-six healthy SD rats were randomly divided into three groups: EB group(12 rats),NBP group(12 rats)and sham operation group(12 rats).The central nervous system(CNS)demyelination model was established in EB group and NBP group by injecting EB into the right ventricle.The sham-operated group received saline injection into the right ventricle as the control group.Rats in NBP group were given intraperitoneal injection of 20 mg/(kg.d)NBP intervention,while rats in EB group and sham-operated group were injected with the same amount of saline in the same way.After 14 days,LFB staining was used to detect the degree of demyelination;HE staining was used to detect the infiltration of inflammatory cells in the white matter of rats in each group;Tunel method was used to detect the apoptosis of oligodendrocytes in each group;immunohistochemical staining was used to detect the expression of MBP and caspase 9;Western blot was used to detect the expression of MBP and Cyt C.ResultFourteen days after modeling,LFB myelin staining showed that compared with sham-operated group,severe myelin sheath loss in periventricular white matter and large areas of vacuolar degeneration were observed in EB group.Compared with EB group,the myelin sheath around white matter in NBP group was more complete and the demyelination was less.Demyelination scores of EB group,NBP group and sham-operated group were 2.5±0.522,1.333±0.492 and0.167±0.389,respectively.The demyelination score of peripheral white matter in EB group was significantly higher than that in sham-operated group(P < 0.05),and that in NBP group was significantly lower than that in EB group(P < 0.05).It was confirmed that CNS demyelination was successfully induced by directional lateral ventricular injection.HE staining showed that the periventricular white matter tissue of EB group was loose and inflammatory cells infiltrated.Compared with EB group,the periventricular white matter tissue of NBP group showed less inflammatory cells and relatively dense tissue.Tunel staining showed that apoptotic cells were diffusely distributed in the periventricular white matter of EB group,some apoptotic cells were found in NBP group,and few apoptotic cells were found in sham operation group.The percentage of apoptotic cells in EB group,NBP group and sham operation group were 0.397±0.015,0.188±0.019 and 0.164±0.012,respectively.Compared with sham-operated group,there were a lot of OLs apoptosis in the periwhite matter tissue of EB group(P < 0.05).Compared with EB group,the apoptosis of OLs in the periwhite matter tissue of NBP group decreased(P < 0.05).Immunohistochemical staining of MBP showed that the expression of MBP decreased in EB group,and a large vacuole area could be seen.Compared with EB group,MBP expression increased in NBP group,but some plaque loss was observed.The average optical densities of MBP in EB group,NBP group and sham operation group were 0.204±0.014,0.368±0.021 and 0.392±0.030,respectively.Compared with sham-operated group,MBP expression decreased(P < 0.05).Compared with EB group,MBP expression increased,but some plaque MBP was lost(P < 0.05).Caspase 9 immunohistochemical staining showed that caspase 9 was widely expressed in periventricular white matter of EB group,and decreased in NBP group compared with EB group.The average optical densities of caspase 9in EB group,NBP group and sham operation group were 0.221±0.030,0.129±0.034 and 0.104±0.021,respectively.Compared with sham-operated group,caspase 9 expression decreased(P < 0.05),while caspase 9 expression decreased(P < 0.05)compared with EB group.Western blot was used to detect the expression of MBP.The gray values of MBP in EB group,NBP group and sham operation group were0.600±0.190、1.076±0.162 、 1.083±0.188 。 Compared with sham operation group,MBP expression decreased(P < 0.05),and increased(P < 0.05)compared with EB group.Western blot was used to detect the expression of Cyt C.The gray values of Cyt C in EB group,NBP group and sham operation group were 1.203±0.243,0.835±0.138 and 1.076±0.162,respectively.Compared with sham operation group,Cyt C expression decreased(P < 0.05),and compared with EB group,Cyt C expression increased(P < 0.05).conclusionProtective effect of NBP on EB-induced demyelination of CNS.It may be related to inhibition of mitochondria-mediated endogenous apoptosis in OLs. |
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