| Objective: To observe the effect of Total Glucosides of Paeony (TGP)on the incidence、brain and spinal tissue’s pathological changesperipheral blood mononuclear cells’c tissue’s apbilty to cecretINF-γ and IL-4,contents of IL-2、TNF-α in brain tissue and subsets ofperipheral blood ymphocytes of female Wistar rats, to assure thetherapeutic effect of TGP on EAE and possible mechanisms, the aimis to explore new therapeutic approach on the multiple sclerosis (MS)and to provide a more meaningful experimental basis on medicationMethods:50reasonable middle-aged female Wistar rats were randomlyassigned to adjuvant control group, EAE control group, TGP is low,medium and high dose treatment group,10in each group. Guineapigspinal cord homogenate (GPSCH) with complete Freund Zuo Qi (CFA)and pertussis vaccine slurry (PV) after subcutaneous injection of EAEcontrol group and the high, medium and low-dose TGP treatment groupproduced EAE model, using the CAF plus PV subcutaneous injectionadjuvant control group. Since the date of the modeling, low, middle andhigh dose TGP treatment group of TGP75mg/kg· d, respectively, TGP150mg/kg· d of TGP300mg/kg· d dose gavage once. EAE control groupand adjuvant control group conventional breeding. Every morning from time to time on the adjuvant control group, EAE control group and thelow, medium and high doses of TGP symptoms of rats were observed toscore, non-aggravated symptoms in rats for3consecutive days, all limbsparalysis or death as EAE onset of the peak of disease. Record the rats tothe incubation time, the progress of time and the peak of neurologicaldysfunction score. The rats were killed at the peak incidence of disease.Keep the rat orbital venous blood, brain spinal cord tissue. Slice cerebraltissue, HE stain, to observe the pathological changesunder the lightmicroscope. Enzyme-linked immunosorbent assay (ELISA) was used toassay the leves of INF-γ, IL-4in blood and brain tissue cytokines IL-2,TNF-α content.We used flow cytometry to detect T lymphocyte subsetsof blood of the rats in each group at the peak incidence.we usedSPSS17.0software to analysis each group’ s data.Results:clinical symptons of eachgroup: adjuvant group: no rats the incidence,all rat of EAE in the controlgroup and low, middle and high dose TGP treatment group are incidence.(2) the incubation period of EAE control rats was9.7±2.4days, low,medium, high-dose TGP froup rat’s incubation period were11.4±2.1days,13.5±1.8days,16.3±3.0days, low, memdiu, high incubationperiod of the different dose-TGP group compared with EAE group wassignificantly prolonged (P <0.05)，difference was statistically significant;high-dose TGP group ’ s incubation period compared with lower dosegroup was significantly prolonged (P <0.05)，difference was statistically significant; high doses TGP group The incubation period than the middledose group was significantly prolonged (P <0.05)， difference wasstatisticallysignificant.(3) advanced stage of EAE group rats was7.3±1.6days, low, medium and high doses TGP group incidence of advanced5.3±1.7days,4.1±1.5days,3.2±0.8days; low, medium, highdose TGP group advanced shorter than the EAE group (P <0.05)，difference was statistically significant; the progress of higher doses TGPgroup is shorter than the progress of lower-dose TGP group (P <0.05)，difference was statistically significant;(4) Neurological dysfunctionscore of EAE rats at peak incidence was3.0±0.8, low, medium andhigh doses of TGP rats peak incidence of neurological dysfunction scoreswere2.3±0.5,1.5±0.3,1.1±0.2.The nerve dysfunction scoreof Low, medium, high doses TGP group rats at the peak incidencecompared with the EAE control group was significantly lower (P <0.05),the difference was statistically significant; the nerve dysfunction score ofhigher dose TGP group rats at the peak incidence was lower than lowerdoses TGP group rats (P <0.05), the difference was statisticallysignificant. The brain tissue of each group: no abnormal change in theadjuvant control group of rat,brain tissue of EAE control group and lowin the high dose of TGP treatment group rat brain tissue the EAE typicalpathological change, small perivascular inflammatory cell infiltration, showing the sleeve-like changes, perivascular white matter demyelinationsignificantly. Compared to EAE control group,the degree ofdemyelination and inflammatory cell’ infiltration of all TGP treat groupwere alleviated; TGP higher-dose treatment group was lighter than theTGP lower does group at the aspect of demyelination and inflammatorycell’ infiltration(P <0.05), difference was statistically significant.3.Thelevel of IFN-γ in EAE group rats’ blood at the peak incidence is higherthan CFA group (P <0.01), but it’s IL-4levels compared withadjuvantcontrol group was lower (P <0.01), the difference wasstatisticallysignificant; the leave of IFN-γ in blood of each doseTGPtreated rats islower than the EAE group (P <0.01), it’s IL4levelcompared with theEAE group was increased (P <0.01),difference was statisticallysignificance; the levels of IFN-γ in blood of the high and middle doseTGP group rats were lower than the EAE group(P <0.01),but it’s IL-4levels in the higer than the EAEgroup’(P <0.01), the difference wasstatistically significant; the higher does TGP group,the lower the levelsIFN-γ(P <0.05), the higher does TGP group,the higher levels of IL-4(P<0.05), difference was statistically significant; the ratio of IFN-γ/IL-4in the blood of EAE rats was higher than the CAF group(P <0.05),difference was statistically significant; the ratio of IFN-γ/IL-4in theblood of each does treat group rat is lower than the EAE group(P <0.05),difference was statistically significant.The higher does TGP group,the lower ratio of IFN-γ/IL-4ratio (P <0.05) difference wasstatisticallysignificance.4. The content of IL-2, TNF-α in rat brain tissue of eachgroup: the content of IL-2, TNF-α in brain tissue of EAE rats at thepeak incidence were higher than the adjuvant control group (P <0.05),the difference was statistically significance;the content of IL-2, TNF-αinbrain tissue of each TGP grpup were lower than the EAE gropu(P <0.05),the difference was statistically significance;the higher does of TGP,thelower content of IL-2, TNF-αin the brain tissuse(P <0.05), the differencewas statistically significance.5The ratio of T lymphocyte cell subsetsin peripheral blood of rats in each group at the peak incidence: The ratioof CD4+lymphocyte and CD8+lymphocyte in peripheral blood of EAEgroup were lower than the CAF group (P <0.01), the ratio of CD4+/CD8+were higer than CAF group (P <0.01),the difference wasstatistically significant; The ratio of CD4+and CD8+in peripheral bloodof each TGP group is higer than EAE(P <0.01),the ratio of CD4+/CD8+were lower than EAE(P <0.01),the difference was statisticallysignificant;there is no singinficant difference of the ratio of CD4+,CD8+, CD4+/CD8+in different does TGP groups(P>0.05).6correlation analysis: the ratio of IFN-γ/IL-4at the peak of theperipheral blood， the content of IL-2,tTNF-alpha in brain tissue,theratio of CD4+/CD8+was negatively correlated to incubation time;butthey are positively correlated to the incidence of progression and neurological dysfunction score (P <0.05or P <0.01). Conclusion:1.We use GPSCH as inmunogen to induce EAE mode, EAE ratsneurological disorder is obvious、brain tissue perivascularly infiltrated ininflammatory cell, demyelinati on of white matter, so we think thismethod is stable and reliable.2.EAE rat exists immune imbalance; invivo of EAE rat， Th1-type cytokines INF-γ increased， Th2-typecytokines IL-4decreased, the Th1/Th2imbalance, brain tissueinflammatory cytokines (IL-2and TNF-α)increased， production ofimmune pattern offset to the Th1type,at the peak of EAE,the CD4+/CD8+imbalance。3.TGP extended EAE rats incubation period，shorten the progress, reduce the EAE rats symptoms and the degree ofinflammatory cells’ damage to cerebral tissue.4.TGP can increase thesecretion of IL-4, inhibit the secretion of INF-γ, reduce the content ofIL-2, TNF-alpha in EAE rat brain,regulate the Th1/Th2imbalance.promote Th cell differentiation to Th2cell,reduce the ratio of CD4+/CD8+,djust the ratio of T lymphocyte subsets,play a treatment role inthe EAE rats. |
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