Effect Of Bu Shen Jie Du Tong Luo Formula On The Microglia In Experimental Autoimmune Encephalomyelitis Mice

Objective: To observe the effect of Bushen Jie Du Tong Luo Formula on experimental autoimmune encephalomyelitis(EAE)in mice,including the impact of changes in weight,clinical neurological scores and CNS microglia change,to study the effects of different doses of medicine on mice with EAE about clinical manifestations and pathological changes,meanwhile,by observing the relationship between microglia and EAE,explore possible intervention mechanism of Bushen Jiedu Tongluo Formula on the pathogenesis of MS,to further promote applications of Bushen Jiedu Tongluo Formula in the clinic.Methods:(1)the establishment of C57BL/6 mice with EAE model:Select female C57BL/6 mice as experimental animals,joint MOG35-55,complete Freund’s adjuvant(CFA)as an antigen to stimulate C57BL/6 mice,Establishment of C57BL/6 EAE model mice.Immune start the day,Detailed records of the incidence of C57BL/6 mice,including its weight,mobility,appetite,and regularly neurological function scores.(2)Animal grouping and Drug administration: 50 female C57BL/6 mice(8 to 10 weeks)were randomly divided into two groups,namely the blank group and the model group,selecting 10 for the blank group randomly,the rest of the mice were assigned to the non-blank group.The day when C57BL/6 mice were Immunized is day 0,40 mice of non-blank group were injected subcutaneously with a total of 0.2ml MOG35-55 antigen emulsion by four points.After 48 hours,C57BL/6 mice were injected into the abdomined cavity with 0.1ml(containing 400 ng PTX)PV once.The control group of mice were not given any drug intervention.The C57BL/6 model mice were divided into four groups according to random number table method.10 mice in each group,the four group is:high-dose group of Chinese medicine,middle-dose group,low-dose group and EAE model group.Since the seventh day,the Chinese medicine oral liquid were given to the Chinese medicine group.High dose group were feed with 200 ul by 10ml/kg,the middle dose group were feed with 100 ul by 5ml/kg,low dose groupwere feed with 50 ul by 2.5ml/kg.While 10ml/kg(200ul)physiological saline was used in EAE model group and blank group for 2 weeks.In the process of feeding mice,removed the mice of death,and added new mice randomly to ensure the number of animals in each group.(3)Clinical signs were observed in mice : the day after immune,observe the incidence of C57BL/6 mice twice a day,and record clinical indications related indexes in mice,containing the disease cycle,number,body weight and neurological function changes.(4)Sample collection: the mice were executed in the 21 days after immunization.Remove the mice cerebral cortex of the brain-basal ganglia and lumbar myeloid tissue(lumbar intumesence part),fixed in the fluid of the formalin,and embedded in paraffin.(5)Pathological changes: Hematoxylin and eosin(HE)staining to observe the brain and spinal cord tissue changes.(6)Immunohistological study: Immunohistochemical method were used to observe microglia in CNS changes of mice,and detect the central system calcium protein-1(namely Iba1)content.(7)Statistical analysis: Using SPSS20.0 software for statistical analysis.Results:(1)Incidence of mice in each group:Normal group of C57BL/6 mice have no disease,in the incidence of C57BL/6 mice,between three doses of Chinese medicine group and EAE model group,there is no obvious significant difference(P>0.05);(2)The onset time:between high-dose group and EAE model group,the former delayed obviously,which showed significant difference(P<0.05),low-dose group compared with EAE model group has no meanning.(3)The weight change:The weight of normal group C57BL/6 mice increased gradually,the rest group mice increased at first and then decreased.On day 21 after immunization,the drops of high dose group and middle dose group were significantly lower than EAE model group.Compared with EAE model group,low dose group of C57BL/6 mice have no significant change(P>0.05).(4)Neural function evaluation:At the early stage,before immunation,there was no significant difference in the neurological function scores(P>0.05),There was no significant difference in the neurological function scores between the traditional Chinese medicine group and the EAE group(P>0.05)for seventh days and fourteenth days;on day 21 after immunization,nerve function score of the high and middle dose group of C57 BL / 6 mice significantly lower than that of EAE group(P < 0.01).(5)Pathological changes: inflammatory lesions were found in mice of the Blank group,small blood vessels expand in the brain and spinal cord of EAE mice,surrounded by a large number of inflammatory cell infiltration,presented the "cuff like change.The traditional Chinese medicine group showed a similar model group,but the degree of infiltration of inflammatory cells decreased,and the amount of inflammatory lesions in the brain tissue and lumbar spinal cord tissue of traditional Chinese medicine group was significantly lower than that of EAE group(P <0.05).(6)Microglia:the microglia number of the Blank group relatively little in C57 BL / 6 mice,the nucleus became thin and the content of Iba-1 reduced;the microglia number of the EAE model group increased sharply,the nucleus becomes large,and Iba-1 increased.The number of high and middle dose group of MG relatively less than the EAE model group both in brain and lumbar spinal cord,Iba-1 content decreased significantly(P<0.05).Conclusion:(1)Using MOG35-55 to induce C57BL/6 mice to establish EAE model,the incidence rate is high.(2)Bushen Jie Du Tong Luo Formula can relieve the clinical symptoms of C57BL/6 mice and prolong the incubation period of the disease.(3)The intervention mechanism of Bushen Jie Du Tong Luo Formula on MS may be through inhibiting the activation of MG,to inhibit the occurrence of CNS inflammatory reaction,So as to delay the onset of EAE,to play a certain control effect on C57BL/6 EAE mice.(4)The high dose group and the low dose group were compared with the high dose group,the former has obvious advantages.