| Objective: This study is designed to investigate the potential mechanism of matrine(MAT)on treating classical multiple sclerosis(MS)animal model in acute experimental autoimmune encephalomyelitis(EAE)rats or chronic progressive EAE mice.We observed the clinical symptoms and pathological characteristics,and analysised the proliferation of oligodendrocyte progenitor cells(OPCs NG2)and maturation of myelin sheath cell surface markers(MBP)expression in the pathogenesis of rat,and checked the coexpression of CD68+i NOS+ in pro-inflammatory M1 macrophages/ microglia and CD68+Arg-1+ in anti-inflammatory M2 macrophages/microglia,in addition of the changes of NT3,LIF,IGF-1 and JAK2/STAT3 signaling pathway.We also checked the expressions of NT3,i NOS,Arg1,NT3+CD68+,NT3+NG2+,NT3+GFAP+,NT3+i NOS+ and NT3+Arg1+ in the spinal cord of EAE mice.These data were used to investigate the immunomodulatory effects of MAT on macrophages/microglia in acute EAE rats and chronic progressive EAE mice,and try to find an effective,safe and affordable therapy for MS.Methods: 1 1.1 EAE rats induction: 30 Wistar female rats were immunized after anesthesia with a mixture of guinea-pig spinal cord homogenate and an equal volume of complete freund’s adjuvant(CFA)containing 6 mg/ml Bacillus Calmette-Guérin vaccine.Rats were monitored and weighed daily to evaluate the body weight,the actions and clinical scores of EAE after immunization.1.2 Animal groups and the treatment of EAE rats:Immunized rats were randomly divided into three groups(n = 10 each group)for different treatments.Briefly,MAT was dissolved in normal saline and injected intraperitoneally(i.p.)daily at the dose of 200 mg/kg from daye 1(MAT-P)or day 11(MAT-T)to day 17 p.i.,the dosage calculated at 6.7 ml/kg.Immunized rats that received the same amount of normal saline only i.p.served as a vehicle control,and 10 nonimmunized naive rats that received the same amount of normal saline i.p.served as the normal group.2 2.1 The establishment of chronic progressive EAE mice model : MOG(10 mg)dissolving in 2 ml normal saline which containing 2.5 mg/ml of Mycobacterium tuberculosis in complete freund’s adjuvant,then the liquid were completely mixed with glass syringe on the ice,made of water in oil emulsion white emulsion,static the 10 min layer is qualified.C57BL/6 mice were anesthetized with 2% pentobarbital and were immunized subcutaneously at the dorsal midline of the spine at a total of four points(0.2 ml).On the 0 day and the 2 day of immunization,mice were injected intraperitoneally with 200 ng pertussis toxin(PBS)solution to establish EAE mice model.The changes of body weight,diet,activity,clinical symptoms and neurological function of mice were observed and recorded on the day of immunization.2.2 Animal groups and the treatment of EAE mice:Immunized mice were randomly divided into two groups(n = 30 each group)for different treatments.Briefly,MAT was dissolved in normal saline and injected intraperitoneally(i.p.)daily at the dose of 200 mg/kg from daye 13 to day 22 p.i..Immunized mice that received the same amount of normal saline only i.p.served as a vehicle group.3 Sample collections:All the animals were sacrificed at disease peak(day 18 p.i.for rats or day 23 p.i.for mice)after extensive perfusion with saline.The lumbar region of the spinal cords and the left hemispheres were removed and fixed in 4% paraformaldehyde solution.The serum,spinal cords and right hemispheres of all the rats and mice were also harvested at disease peak(day 18 p.i.for rats or day 23 p.i.for mice).4 The detection of indexes:The degree of histopathologic changes in spinal cord was assessed directly by hematoxylin-eosin(HE)、Luxol Fast Blue(LFB)and Bielschowsky staining.The expressions of MBP and NT3 were determined by single immunofluorescence staining(IF).In addition,methods of reverse transcription polymerase chain reaction(RT-PCR)analysis of the content of NT3,IGF-1,LIF and JAK2;Western Blot analysis the expression of i NOS and Arg-1;doubleimmunofluorescence staining to analysis the coexpression of NG2+PCNA+,CD68+i NOS+,CD68+Arg1+,NG2+NT3+,GFAP+NT3+,CD68+NT3+,NT3+Arg1+ and NT3+i NOS+;enzyme linked immunosorbent(ELISA)to detect STAT3 level.The main results are divided into the following three parts: Part one: The effect of MAT on the clinical symptoms and pathological symptoms of acute EAE rats and chronic progressive EAE mice.1 EAE incidence:The incidence of Normal group was not observed,Vehicle group had 9 cases,MAT-T group had 6 cases,MAT-P group had only 5 cases.The results of Fisher analysis showed that the incidence of normal group was markedly lower than that of vehicle group(p < 0.01).The incidence of MAT-T group and MAT-P group was significantly lower than that of vehicle group,the difference was statistically significant(p < 0.05).There was no significant difference between the two MAT groups(p > 0.05).There were 30 EAE mice in each group,vehicle group had 26 cases,MAT group had 22 cases,the incidence in MAT-treated mice was not statistically significant compared with the incidence in vehicle group(p > 0.05).2 Body weight trend : The body weight in the vehicle-treated rats was significantly reduced compared to normal group and MAT-treated groups rats(p < 0.01),while body weight in MAT-treated groups were slightly decreased(p < 0.01),with the lesser reduction in the MAT-P group(p < 0.05).The body weight in the vehicle-treated mice was also significantly reduced compared with MAT-treated mice(p < 0.01).3 Clinical score trend:In this study,the average neurological score of the control group was 0.On the 18 day after immunization,the average neurological function score of the model group was significantly higher than that of the group(p < 0.05).The neurological score of MAT-T group was significantly higher than that of group MAT-P(p < 0.05).On the 23 day after immunization in EAE mice,the average neurological function score of the model group was significantly higher than MAT-treated group(p < 0.05).4 CNS histopathology:Average scores of inflammatory,demyelination and axonal injury in EAE groups rats was increased significantly compared with normal group rats(all p < 0.01).The average scores of infiltrating inflammatory demyelination and axonal injury in MAT-T group were significantly lower than that Vehicle group(all p < 0.05),and MAT-P group were further decreased compared with MAT-T group(all p < 0.05).The average scores of inflammatory,demyelination and axonal injury in vehicle group were increased significantly compared with MAT group(all p < 0.05).The second part: The effect of MAT on the proliferation of oligodendrocyte progenitor cells,neurotrophic factor IGF-1,LIF,NT3 and JAK2/STAT3 pathway in acute EAE rats.1 The expression of MBP and NG2+PCNA+ in the corpus callosum of EAE rats: Compared with the normal group,the content of MBP and NG2+PCNA+ in Vehicle group were significantly lower(p < 0.01).The expression of MBP and NG2+PCNA+ in MAT-T group and MAT-P group were significantly higher than those in modelgroup(p < 0.01),and MAT-P group was significantly higher than MAT-T group(p < 0.05).2 The contents of IGF-1,LIF and NT3 in the spinal cord: IGF-1,LIF and NT3 in vehicle group were markedly lower than in the normal group(p < 0.01).The levels of IGF-1,LIF and NT3 in the two MAT-treated groups were significantly higher than those in the vehicle group(p < 0.01),and the MAT-P group increased more significantly than MAT-T group(p < 0.05).3 The contents of JAK2/STAT3 in the spinal cord and brain of rats: Compared with the normal group,the content of JAK2 in spinal cord and STAT3 in brain in vehicle group were significantly increased(p < 0.01).The levels of JAK2 and STAT3 in two MAT-treated groups were significantly lower compared with vehicle group(p < 0.05).The ability of MAT-P to reduce the levels of JAK2 and STAT3 in EAE rats was significantly higher than that MAT-T(p < 0.01).The third part: The effect of MAT on glial cell line derived NT3 and macrophage / microglia in EAE acut EAE mice and chronic progressive EAE mice.1 The expression of CD68+i NOS+ and CD68+Arg1+ in the corpus callosum of EAE rats: Compared with the normal group,the content of CD68+i NOS+ in vehicle group was higher and the i NOS level checked by WB increased significantly(p < 0.01).The levels of CD68+i NOS+ in two MAT-treated groups was lower than that in the vehicle group and WB showed i NOS level decreased significantly(p < 0.01),and the MAT-P group decreased more significantly(p < 0.01).The content of CD68+Arg1+ in vehicle group was higher than normal group but the Arg1 level checked by WB decreased significantly(p < 0.01).The expression level of CD68+Arg1+ in MAT-T group and MAT-P group were significantly higher than vehicle group and WB showed Arg1 level increased significantly(p < 0.01),and the MAT-P group was higher than MAT-T group,the difference was statistically significant(p < 0.01).2 The expression of NT3,i NOS and Arg1 in spinal cord of EAE mice: Compared with vehicle group,the content of i NOS in MAT group was significantly decreased(p < 0.001),however the levels of Arg1 and NT3 in MAT group was significantly increased(p < 0.01,p < 0.05,respectively).3 Coexpression of NT3+GFAP+,NT3+NG2+ and NT3+CD68+ in the spinal cord of EAE mice: Compared with vehicle group,MAT group significantly increased the coexpression of NT3+NG2+,NT3+CD68+,NT3+GFAP+(p < 0.05,p < 0.01,p < 0.001,respectively).4 Coexpression of NT3+i NOS+ and NT3+Arg1+ in the spinal cord of EAE mice: The coexpression level of NT3+i NOS+ was significantly higher in vehicle group than MAT group(p < 0.01),however the coexpression level of NT3+Arg1+ in vehicle group was significantly lower than that MAT group(p < 0.001).Conclusion: MAT possesses obvious preventive treatment on acute EAE model in wistar rats and chronic progressive EAE model in C57BL/6 mice,probably by upregulating of glial cells-secreted neurotrophic factors NT3,down-regulating the expression of JAK2/STAT3,and promoting the proliferation of OPCs and the transformation of macrophages/microglia cells from proinflammatory M1 type to anti-inflammatory M2 type,thus improve the survival of neurons microenvironment which contributes to the prevention and treatment effect on EAE mice. |
PDF Full Text Request