The Mechanism Of Matrine On Treating EAE In Rats

Objective:This study is designed to investigate the potential mechanism of matrine (MAT)on treating experimental autoimmune encephalomyelitis (EAE) in rats. We observedthe clinical symptoms of EAE, the degree of histopathologic changes, the expressionsof Collagen IV、ZO-1、MMP-2、TIMP-2、Nogo-A、NgR in spinal cord, theconcentrations of MMP-9、TIMP-1in blood and the contents of Glu、GABA、GLT-1、GLAST、NMDAR and AMPAR in the brain. These data is used to find an effective,safe and affordable therapy for MS.Methods:1EAE Induction：40Wistar female rats were immunized under anesthesia with amixture of guinea-pigspinal cord homogenate and an equal volume of completeFreund’sadjuvant (CFA) containing6mg/ml Bacillus Calmette-Guérin vaccine. Ratswere monitored and weighed daily to evaluate the body weight，the actions andclinical scores of EAE after immunization.2Animal groups and the treatment of EAE：Immunized rats were randomlydivided into four groups (=10each group) for different treatments. Briefly, MATwas dissolved in normal saline and injected intraperitoneally (i.p.) daily at two doses:low (150mg/kg; MAT-L) and high (250mg/kg; MAT-H), the dosage calculated at6.7mL/kg, from day1until day17after immunization (p.i.). Dexamethasone (DEX) asthe positive control drug, was dissolved in normal saline (6.7mL/kg) and injected (i.p.) daily, from day1until day17p.i. at1mg/kg. Immunized rats that received thesame amount of normal saline only i.p. served as a vehicle control, and10nonimmunized naive rats that received the same amount of normal saline i.p. servedas the naive group.3Sample collections：All the animals were sacrificed at disease peak (day17p.i.)after extensive perfusion with saline. The lumbar region of the spinal cords and theleft hemispheres were removed and fixed in4%paraformaldehyde solution. Theserum, cervical spinal cords and right hemispheres of all the rats were also harvestedon day17p.i.4The detection of indexes：The degree of histopathologic changes in spinal cordwas assessed directly by hematoxylin-eosin (HE) and chromotrope-2R-brilliant green(C-2R-BG). The expressions of MMP-2、TIMP-2、NMDAR、AMPAR and Nogo-Awere determined by immunohisto-chemical techniques(IHC). In addition, the serumcollected on day17p.i. was assayed for concentrations of MMP-9and TIMP-1byELISA. Furthermore, the contents of Collagen IV、ZO-1、GLT-1、GLAST and NgRwere analysed using reverse transcription-polymerase chain reaction (RT-PCR).Results:1EAE incidence：The morbidity was significantly increased in vehicle groupcompared to na ve group (p <0.05). In addition, there weren’t significant differencesamong the vehicle group and drug-treated groups (p>0.05).2Body weight trend： The body weight in the vehicle-treated rats wassignificantly reduced compared to na ve control and MAT-treated rats (p <0.01),while body weight in MAT-treated groups was slightly decreased (p <0.01), with thelesser reduction in the MAT-H group (p <0.05). Significant difference for bodyweight had been observed between the vehicle group and DEX-treated group.(p <0.05).3Clinical score trend：In the vehicle-treated group, clinical decline typicallystarted on day10p.i., while EAE onset for MAT-treated rats occurred on day12p.i.(low dose) and13p.i.(high dose). Compared to the vehicle-treated group, bothMAT-treated groups exhibited significantly lower mean maximum clinical scores (p<0.05). Treatment with DEX also delayed disease progression and reduced clinical scores compared to the vehicle-treated group (p <0.05). There was no significantdifference in clinical score between animals treated with DEX and the two differentdoses of MAT (p>0.05).4CNS histopathology： Perivascular cuffing with mononuclear cells andinfiltration into CNS parenchyma were observed in the spinal cord of rats in thevehicle-treated group, while the extent of cellular infiltration was significantlydecreased in the MAT-treated groups (p <0.01). Treatment with DEX showed astronger inhibition in cellular infiltration than MAT-treated groups (p <0.01).Cellular infiltration was not observed in the naive group. Moreover, large areas ofdemyelination were observed in the vehicle-treated group, while MAT-andDEX-treated groups exhibited only a few areas of demyelination (p <0.01). Nosignificant difference in demyelination was observed between MAT-H and DEXgroups (p>0.05).5Collagen IV and ZO-1mRNA expression in cervical spinal cords：Thebrightest band was exhibited in the naive group, while the faintest band was in thevehicle-treated group (p <0.01). A dose-dependent increase in collagen IVexpression was observed in MAT-treated groups (p <0.01). While theMAT-L-treated group showed lower collagen IV expression than the DEX group (p <0.01), there was no significant difference between MAT-H and DEX groups (p>0.05). A similar pattern of mRNA expression of ZO-1was observed for collagen IV.6MMP-9and TIMP-1in Serum：The amount of MMP-9was dramaticallyincreased in the vehicle-treated group compared to the naive group (p <0.01). MATtreatment largely reduced MMP-9content compared to the vehicle-treated group (p<0.01), and the effect was dose dependent. Furthermore, a significantly lower amountof MMP-9was observed in the DEX-treated group than in the MAT-treated groups (p<0.01). A significant decrease in TIMP-1concentration in the vehicle-treated groupcompared to the naive group (p <0.01). TIMP-1serum levels in the MAT-treatedgroups were significantly higher than in the vehicle-treated group (p <0.01). While alow dose of MAT induced a lower serum TIMP-1level than DEX (p <0.01), a highdose of MAT increased the serum TIMP-1level to a greater extent than DEX (p <0.01). 7MMP-2and TIMP-2content in the spinal cords：MMP-2expression wassignificantly increased in the vehicle-treated group over the naive group (p <0.01).The differences in MMP-2expression between vehicle-treated and the twoMAT-treated groups were significant (p <0.01). Furthermore, the effect of DEX indecreasing MMP-2content was stronger than low-dose MAT (p <0.05), but therewas no significant difference compared with the MAT-H group (p>0.05). TIMP-2expression in the vehicle-treated group was significantly lower than in the na vegroup (p <0.01) and MAT-treated groups (p <0.05). A significantly lower amountwas obtained in the MAT-L group than in the MAT-H group (p <0.01). While theMAT-L-treated group showed lower TIMP-2expression than the DEX-treated group(p <0.05), there was no significant difference between MAT-H and DEX groups (p>0.05).8Glu and GABA level in cerebral cortex：The amount of Glu was significantlyincreased in the vehicle-treated rats vs. na ve control (p <0.01), and reduced Gluproduction was observed in high dose MAT-treated rats compared to vehicle-treatedEAE rats (p <0.05). The difference in decreased glutamate production betweenhigh dose MAT-treated and DEX-treated group was not significant (p>0.05).TheGABA level in the cerebral cortex of vehicle-treated EAE rats was significantly lowerthan in brains of na ve control rats (p <0.01). While the MAT-L-treated groupshowed lower GABA concentration than the DEX-treated group (p <0.05), there wasno significant difference between MAT-H and DEX groups (p>0.05).9mRNA expression of GLT-1, GLAST in cerebral cortex：EAE rats showed amarked down-regulation in mRNA levels of GLT-1and GLAST (p <0.01) incerebral cortex vs. naive control rats, and this expression was enhanced after MATand DEX treatment (p <0.01). Similar responses were found in the DEX-treated rats,and a more profound increase in GLAST mRNA expression was observed in ratstreated with high-dose-MAT than with DEX (p <0.05).10NMDAR, AMPAR production in frontoparietal cortex：NMDAR expressionincreased approximately twofold in the frontoparietal cortex of EAE rats vs. controlrats. Importantly, NMDAR expression was significantly decreased with MAT-treatedvs. vehicle-treated EAE rats (p <0.05). The NMDAR level was similar in rats treated with DEX and high-dose-MAT. AMPAR expression was largely increased in EAErats compared to control rats (p <0.01), and the upregulated AMPAR production wassignificantly suppressed by MAT treatment (p <0.05). Furthermore, the high doseMAT-treated group showed a lower AMPAR level than the DEX-treated group (p <0.05).11Nogo-A and NgR mRNA expression in the spinal cords：EAE rats showed amarked up-regulation in Nogo-A and NgR mRNA content (p <0.01) in cerebralcortex vs. naive control rats. The expression of Nogo-A and NgR mRNA in the spinalcord showed a marked reduction after MAT treatment(p <0.01)，particularly in ratstreated with higher doses of MAT(p <0.01). In additon, the MAT-treated groupshowed a higher Nogo-A and NgR mRNA level than the DEX-treated group (p <0.01).Conclusion:MAT possesses obvious preventive treatment to EAE model in Wistar rats，which were induced by being immuned with spinal cord extracts to together withcomplete Freund adjuvant. The possible mechanism of action is related to strengthenthe BBB integrity, regulate the balance between MMPs and TIMPs, reduceexpression of glutamate as well as its receptors, upregulate molecules that inhibitglutamate activities, including GABA and the glutamate transporters, and inhibit thecontents of Nogo-A and NgR.