The Research Of Matrine Preventive Effect On Experimental Allergic Encephalomyelitis In Rats

Objective:The preventive effect was observed of oxymatrine (MAT)on experimental allergic encephalomyelitis (EAE).Discuss the therapeuticeffect of MAT on EAE in rats and its mechanism,finding a theoretical basis forthe treatment of MS.Method:The6-7weeks old, weighing180-220g of50female Wistar rats arbitrarily assigned into five groups. In guinea pig spinalcord homogenate (GPSCH) as a sensitizing antigen, mixed with completeFreund’s adjuvant (CFA) established EAE models.4groups of rats wererandomly selected to establish EAE model after sensitization, and set to EAEcontrol group, low, middle, high dose MAT group (MAT-L, MAT-M, MAT-Hgroup), the remaining1groups as normal control group.Since the date ofmodeling the normal control group and EAE group received no treatment forregular feeding, MAT-L, MAT-M, MAT-H group since the beginning of theimmune daily respectively application Kushensu50mg/kg· d,100mg/kg· d,200mg/kg· d dose gavage once for15days. After modeling requires regulardaily observation of the clinical manifestations of Wistar rats in each group,such as mental diet, weight changes, physical activities to determine the onsetof incubation period, development period,and the peak of neurologicaldysfunction score.No incidence of rats were killed after1months,the incidenceof EAE rats for3consecutive days was observed neurological dysfunctionscore did not increase, or severe ataxia, limb completely paralyzed, dying time as the end of the experiment time.At the same time in the termination of ratsduring the experiment to take the peripheral vein blood, brain tissue to bedetected. By conventional pathology, ELISA and other assay methods toobserve morphological changes of the diseased tissue,Changes of Th1, Th2,Th17represents the immunity factor concentrations in brain homogenate andmyelin basic protein (MBP) concentration, T lymphocyte subsets (CD4+、CD8+)proportional change in blood.Based on the immune mechanism of EAE and MSselected interferon gamma (IFN-γ), interleukin-4(IL-4), interleukin-17(IL-17)as the Th1, Th2, Th17represents the immunity factor, through the SPSS17.0software for data analysis to investigate the prevention effect of MAT onEAE.Results:1. Incidence of rats:(1) clinical features: the rats’ spirit, diet,sleep and activity with the usual in the normal control group, there is noonset.EAE control group and MAT-L group, MAT-M group, MAT-H group ofratsafter different degrees of hair color is dim, poor spirit, diet sleepdecreased,reduced activity, tail weakness, paralysis, incontinence, and even convulsionsand death phenomenon.The clinical manifestations of the rats in MAT group islight than in EAE control group, the MAT-H group is the most light.(2) theincidence of occult features: EAE control group was the shortest time in eachEAE rats group,was9.30±0.94days; MAT-H group the longest, was16.60±0.97days. Compared with the EAE control group of period of time occult, eachMAT group was significantly prolonged (P <0.01); MAT-L group, MAT-Mgroup, MAT-H group of rats hiding in order to extend the time, MAT group compare statistically significance difference (P <0.01).(3) the development ofdiseases characterized: EAE control group was longest in each EAE ratsgroup,was7.20±1.14days; MAT-H group shortest was2.90±0.74days.Compared with the EAE control group of development time, each MAT groupwas significantly shorter (P <0.01); MAT-L group, MAT-M group, MAT-Hgroup in order to shorten the development time of rats, MAT group comparestatistically significance difference(P <0.01).(4) the peak incidence of neuralsystem features: EAE control group was highest in each EAE rats group, was3.70±0.95points; MAT-H group was the lowest, was2±0.82points. And thegroup of nervous system function damage score compared with EAE controlgroup, the MAT group was significantly decreased (P<0.01, P<0.05); MAT-Lgroup, MAT-M group, MAT-H group the peak of the nervous system followedby functional impairment score decreased，between the MAT group scoreswere statistically significance difference (P<0.05).(5) the onset of symptomseverity and mortality characteristics: the EAE control group was the highest ineach EAE rats group, lowest in MAT-H group; compared with the EAE controlgroup’s symptoms and mortality,the MAT group reduce(P<0.01,P<0.05).MAT-L group, MAT-M group, MAT-H group rats’ symptoms and mortalitydecreased, MAT group compare statistically significancedifference(P<0.05).2.CNS pathological changes characteristic:(1) in rat braintissue biopsy features: the brain tissue of EAE rats in each group were seenvarying amounts of inflammatory cells infiltrating the brain tissue and blood vessels on different degree, the EAE control group most obviously, MAT-Hgroup lightest, normal control group was no exception.Pathological section ofbrain tissue in EAE control group change the typical, showed diffuse damage inthe white matter demyelination and oligodendrocytes, visible axonal damage,vacuolar deletion.MAT-L group, MAT-M group, MAT-H group of brain tissuein rats inflammatorypathological changes degree in order to reduce.(2) braininflammation pathological features: the pathological score based on the EAEcontrol group was the highest, being2.9±0.32;MAT-H group was the lowest,being1.2±0.63points. Compared with the score and inflammationpathological brain tissue in EAE control group, each MAT group pathologyscore decreased obviously (P<0.01, P<0.05); MAT-L group, MAT-M group,MAT-H group of brain tissue in rats inflammatory pathological score weredecreased, the MAT group has statistically significance difference of pathologicscore (P<0.01, P<0.05).3.Concentration characteristics of EAE rat brainhomogenates immune factors: Compared with IFN-γ, IL17-concentration andIFN-γ/IL-4value in the normal brain homogenates, EAE control group, theMAT group and its concentration increased significantly (P<0.01); with EAErat brain homogenates IFN-γ, IL-17concentration and IFN-γ/IL-4valuecompared to the concentration level of each MAT group decreased significantly(P<0.01).MAT-L group, MAT-M group, MAT-H group rat brain homogenatesIFN-γ, IL17-concentration and IFN-γ/IL-4value followed by decline, itsconcentration among MAT group was statistically significance difference(P<0.01, P<0.05). Compared with the normal control group of brainhomogenates of IL-4concentration, EAE control group, the MAT groupdecreased their concentration levels were significantly (P <0.01); comparedwith brain homogenates of EAE group, the concentration of IL-4, each MATgroup’s concentrations are significantly increased (P<0.01). MAT-L group,MAT-M group, MAT-H group rat brain homogenates in turn increases theconcentration of IL-4, its concentration among MAT groups was statisticallysignificance difference (P<0.01).4.The concentration of MBP in venous blood:compared with the normal control group,EAE control group and MAT groupincreased significantly (P<0.01);compared with the EAE control group, MBPlevel in venous blood of each MAT group decreased significantly (P<0.01).MBP concentration in MAT-L group, MAT-M group, MAT-H group rats invenous blood decreased, its concentration among MAT groups was statisticallysignificance difference (P<0.01,P<0.05).5. T lymphocyte subsets (CD4+、CD8+)characteristic of proportionalchanges: the ratio of T lymphocyte subsets innormal control group ratscompared with EAE control group, MAT group, theblood CD4+、CD8+lymphocyte decreased (P<0.01, P<0.05), CD4+/CD8+increased (P<0.05).T lymphocyte subsets change compared with the EAEmodel rats, each MAT group venous blood CD4+、CD8+lymphocyte ratioincreased (P <0.01), CD4+/CD8+value decreased (P<0.01). Compared withthe MAT-L group T lymphocyte subsets change, MAT-M group, MAT-H groupvenous blood CD4+、CD8+lymphocyte ratio increased (P <0.01, P <0.05), CD4+/CD8+value lower (P <0.01, P <0.05). Compared with the MAT-Mgroup T lymphocyte subsets change, MAT-H group venous blood CD4+、CD8+lymphocyte ratio increased (P<0.01, P<0.05),CD4+/CD8+value decreased (P<0.05).6.Biochemical markers of immune correlation with clinical andpathological features of peak incidence in rats:(1) EAE control group andMAT group of rat brain homogenates IFN-γ levels and the incidence of occultwas negatively correlated with statistically significance difference (P <0.01);and disease development period, the peak of the nervous system dysfunction,inflammation and pathological changes in brain tissue were positivelycorrelated with statistically significance difference (P <0.01).(2) EAE controlgroup and MAT group of rat brain homogenates of IL-4levels and theincidence of occult was positively correlated with statistical significancedifference (P <0.01, P <0.05); and disease development period, the peak of thenervous system dysfunction, inflammation and pathological changes in braintissue was negatively correlated with statistically significance difference (P<0.01, P<0.05).(3) EAE control group and MAT group of rat brainhomogenates of IL-17levels and the incidence of occult was negativelycorrelated with statistically significance difference (P <0.01, P <0.05), anddisease development period, the peak of the nervous system dysfunction,inflammation and pathological changes in brain tissue were positivelycorrelated with statistically significance difference (P<0.01, P<0.05).Conclusions:1. the peak incidence of EAE rats immune imbalance, CD4+、CD8+lymphocyte ratio decreased, the ratio increased; proinflammatorycytokines IFN-γ,IL-17secretion increased,anti-inflammatory cytokine IL-4secretion decreased;MBP concentration increased.2.MAT has extended EAErats onset latency, shortening disease progression, reduced the peak of diseaseof the nervous system dysfunction, reduce the damage effect of inflammatorycell infiltration in the brain tissue, its impact and MAT dose size was positivelycorrelated.3.MAT control EAE rats and possible mechanisms of CD4+、CD8+lymphocyte ratio rises, both ratio decreased; reduce pro-inflammatorycytokines IFN-γ, IL-17and increased secretion of anti-inflammatory cytokineIL-4; make Th1cells decreased expression, Th2cells express elevated,maintenance Th1/Th2balance shaft, which play a role in the regulation of theimmune function of EAE rats and adjust the degree of positive correlation MATdose size.