Mechanism Involved In Matrine Treatment For Astrogliosis In Experimental Autoimmune Encephalomyelitis Rat Brains

Objective:To study the effect of matrine(MAT)on clinical symptoms and histopathological characteristics of experimental autoimmune encephalomyelitis(EAE),the animal model of multiple sclerosis(MS);To explore the effect of MAT on EAE astrogliosis in rats by detecting the changes of astrocytic surface markers-glial fiber acidic protein(GFAP)and chondroitin sulfate proteoglycan(CSPG);To examine the alterations of sphingosine(SPH),sphingosine 1 phosphate(S1P)and plasma gelsolin(pGSN)level,sphingosine kinase(SPHK)and sphingosine 1 phosphate receptor 1(S1P1)expression on astrocytes;To explore the mechanisms inloved in MAT reducing astrocyte proliferation phenomenon in EAE rats and thus providing experimental and theoretical basis for clinical treatment of MS.Methods:1.Establishment of EAE rat model under aseptic conditions: Emulsify the same volume of guinea pig spinal cord homogenate(GPSCH)as well as complete Freund’s adjuvant(CFA)which contains 6mg/ml of Bacillus Calmette-Guérin(BCG)to produce stable water-in-oil antigen emulsion.Use the mixture to inject 6-8 weeks old female Wistar rats(0.5ml/ mouse)subcutaneously in four foot pads to establish GPSCH-induced EAE model.Since the first day of immunization,daily neurological scores of the experimental animals were evaluated by two researchers independently.2.Animal grouping and drug delivery: Randomly divide thirty immunized rats into three groups(n = 10 per group)using random number table,which are: 1)EAE+Saline: daily intraperitoneal(i.p.)injection of normal saline(6.7ml/kg)was performed from the first day post immunization(p.i.);2)Matrine treatment group(EAE+MAT): rats received 6.7ml /kg(250mg/kg)of matrine i.p.injection every day since the 11 th day p.i.;3)Matrine prevention group(EAE+ MAT-P): rats received the same treatment as the matrine-treatment group from day 1 p.i..All of the animals were treated until the 17 th day after immunization.3.Samples collection: On day 18 p.i.,heart perfusion was performed after animals were anesthetized.Brains were harvested and washed with phosphate buffer solution(PBS).Then left brains were immediately put into liquid nitrogen and then in 80 ℃ refrigerator for storage,the right brains were stored in paraformaldehyde for fixation.4.Detection of related indicators: Hematoxylin and eosin(HE)staining as well as Luxol fast blue(LFB)staining were used to evaluate the infiltration and demyelination condition of brain tissues of experimental animals;The expression of GFAP,CSPG and the co-expression of GFAP as well as S1P1 were detected by immunofluorescence(IF)assay;The expression of GFAP and pGSN were detected by western blot(WB);Enzyme-linked immunosorbent assay(ELISA)was used to detect SPH and S1 P concentration;Quantitative real-time polymerase chain reaction technique(qRT-PCR)was performed to detect SPHK1 and SPHK2 mRNA expression.GraphPad Prism 6.0 software was used for statistical analysis of the experimental data.Results:1.Clinical neurofunctional score: statistical analysis showed that at the peak of EAE development(the 18 th day after immunization),the mean neurofunctional score of the MAT-T group was lower than that of the EAE+Saline group(P<0.001).Significant differences exist between the MAT-T group and the MAT-P group(P <0.05).2.Histopathological alterations: Compared with the EAE-Saline group,the inflammatory cell infiltration and demyelination condition in rats of the MAT-T group were significantly alleviated(P <0.001,P <0.05),while the situation in MAT-P group was significantly alleviated compared with the control group(P <0.05,P <0.01).3.Changes in astrocyte proliferation in brain tissue: Western blot and immunofluorescence staining results showed that compared with the MAT-T group,GFAP protein expression and fluorescence intensity were significantly higher in the EAE+Saline group(P <0.001,P <0.01),while the expression was lower in the MATP group than in the MAT-T group(P <0.001,P <0.01).Compared with the EAE+Saline group,the fluorescence intensity of CSPG in the brain of the MAT-T group was also significantly decreased(P <0.01),while the MAT-P group showed a lower trend(P <0.05).4.Levels of S1 P,SPH,pGSN and expression of SPHK1 and SPHK2 mRNA in brain tissue: ELISA results showed that compared with the EAE+Saline group,the consentrations of S1 P and SPH in the brains of MAT-T group were significantly reduced(P <0.001,P <0.001),the MAT-P group was also significantly reduced compared with MAT-T group(P <0.001,P <0.001).qRT-PCR results showed that the EAE model group showed significantly reduction in SPHK1 and SPHK2 mRNA expression in the brains after matrine treatment(P <0.05,P <0.001).Compared with the matrine treatment group,significant decreasement of the e SPHK1 mRNA expression was observed in the brains of the MAT-P group(P <0.001),while there was no significant difference in the expression of SPHK2 mRNA between the two groups.In addition,western blot was used to detect the expression of pGSN protein in brain tissue,and results showed that compared with the EAE+Saline group,matrine treatment significantly increased the expression of pGSN protein in the brain of rats(P <0.001),while the amount of pGSN protein in the MAT-P group was also significantly lower than that in the MAT-T group(P <0.001).5.Expression level of S1P1 in brain and astrocytes: qRT-PCR data showed that in comparison to the EAE+Saline group,the level of S1P1 mRNA expression in the brains of the MAT-T group was significantly reduced(P <0.001),while there is no significant difference exists between the MAT-P group and the EAE+Saline group.Correspondingly,immunofluorescence double staining results showed that compared with the MAT-T group,the EAE+Saline group showed higher expression of S1P1 in the astrocytes(P <0.001),while the MAT-P group showed no statistical difference from the EAE+Saline group.Conclusion:In summary,our study demonstrates that matrine has a favorable therapeutic and preventive effect on EAE rats and can significantly relieve astrogliosis in the brains of EAE rats.Matrine can significantly reduces the content of S1 P in the EAE rat brain,possibly by inhibiting the generation of SPH and the expression of SPHK1&2,upregulating pGSN,and inhibiting the expression of S1P1 in astrocytes,thus playing a preventive and therapeutic role in EAE rats.