| Objective: Research the prevention and cure function ofLaquinimod(LQ) on experimental autoimmune encephalomyelitis(EAE).Investigate the prevention and controul function with immunologicalmechanisms of LQ effect on EAE. Methods:75female Wistar rats wererandomly divided into five groups: normal control group, EAE group andlow, meddle, high doses of LQ treatment group(n=15).Of three daysbefore the start of the buiding, namely the LQ low, meddle, and high dosegroup intervention, respectively be LQ0.03mg/kg·d,0.06mg/kg·d,0.12mg/kg·d to fill the stomach, normal control group and EAE groupwith normal saline2ml/d lavage. After using the crude myelin basicprotein(MBP) plus complete Freund’s adjuvant(CFA) make EAE modelby subcutaneous injection, the normal control group subcutaneousinjection the same amount of complete Freund’s adjuvant. The LQ eachdose group continue to treatment for a week after the EAE building.Wistar rats should have nerve dysfunction scores and records for everyday, the nerve dysfunction score for three consecutive days withoutaggravating, quadriplegia, or close to death as the onset of rush hour. Atthe same time record Wistar rats onset latency and progress period. In therush to be put to death animals,not disease and normal control group after 4weeks in the buiding to be put to death. To return of rat brain spinaltissue, spleen tissue and orbital venous blood. The rat brain and spinalcord tissue after HE staining for observation. Using the method ofenzyme-linked immunosorbent (ELISA) determination of peripheralblood mononuclear cell (PBMC) secretion of interleukin-4(IL-4),interferon gamma (INF-γ), interleukin-17(IL-17)levels, cells through thedetermination of the ratio of spleen tissue Bregs, By ELISA in spleen cellculture supernatant on interleukin-10(IL-10), transforming growthfactor-β(TGF-β) content. Results:(1)pathogenesis of Wistar rats:Thenormal control group rats did not come on,the rate of the control group ofEAE is100%; low, meddle, high doses of LQ treatment group were80%,66.67%,60%. The incubation period of control group of EAE was10.27±1.67days, The incubation period of low, meddle, high doses of LQtreatment group to some extent, were15.67±1.95,17.27±1.49,20.73±1.58days, compared with EAE control group, difference hasstatistical significance(P<0.01), and has a dose-response relationship.The development period of control group of EAE was6.47±1.19days,The development period of low, meddle, high doses of LQ treatmentgroup were5.40±1.12,4.20±1.15,3.00±0.93days, shortened than theEAE control group（P<0.01or P<0.05）, a dose-response relationship. Thepeak incidence of disease score of control group of EAE was3.73±1.39;LQ low, middle and high dose group of incidence peak nerve dysfunction score were lower than those of EAE group, were2.40±1.72,1.60±1.60,1.07±1.22, the difference between high, middle dose of LQ group andEAE geoup is statistically significant(P<0.01), the difference betweenlow dose of LQ group and EAE group is not significantdifference(P>0.05) although the incidence peak nerve dysfunction scorewas lower than those of EAE group. High dose group and low dose groupcompared with the differences statistically significant(P<0.05); Thedifferences between high dose of LQ group and middle dose of LQ groupis not statistically significant(P>0.05), the same to the meddle dose groupand low dose group.(2) EAE control group, high, meddle, low dose ofLQ treatment group Wistar rats brain and spinal cord pathologicalchanges: no normal control group rats had abnormal; the brain and spinalcord demyelinating changes of EAE control group, high, meddle, lowdose of LQ treatment group rats had appeared different degree of whitematter, gathered a large number of inflammatory cells in the brain andspinal cord parenchyma around the blood vessels, typical form “sleeve”change, accompanied by vascular engorgement. Gather cells is givenpriority to with lymphocytes, with a small amount of mononuclear cells.LQ each dose groups of white matter demyelination and inflammatorycells invasion degree reduce EAE control group, and reduce the effect ofhigh dose group is more obvious.(3) The peak incidence of EAE controlgroup, high, meddle, low dose of LQ treatment group rats PBMC secretion of IL-4, INF-γ, IL-17ability and the ratio of IL-4/INF-γ: thePBMC secretion of IL-4ability, IL-4/INF-γvalue on EAE control group isdecreased compared with normal control group(P<0.01), secrete INF-γ,IL-17ability increased compared with normal control group(P<0.01);high, meddle, low dose of LQ treatment group PBMC secretion of IL-4ability, IL-4/INF-γvalue increased compared with EAE controlgroup(P<0.01), secrete INF-γ, IL-17ability decreased compared withEAE control group(P<0.01or P<0.05);high and meddle dose of LQtreatment group PBMC secretion of IL-4ability, IL-4/INF-γvalueincreased compared with low dose of LQ treatment group(P<0.01),secrete INF-γ, IL-17ability decreased compared with low dose of LQtreatment group(P<0.01or P<0.05); the PBMC secretion of IL-4ability,IL-4/INF-γvalue of LQ high-dose treatment group is higher than LQmeddle dose treatment groups(P<0.01), secrete INF-γ, IL-17abilityreduce in the LQ meddle-dose group(P<0.01or P<0.05).(4) EAE controlgroup, high, meddle, low dose of LQ treatment group rats in proportion tothe peak incidence of spleen tissues Bregs and the content of IL-10andTGF-β in culture supernatant of spleen cells: EAE control group peakincidence of IL-10+Bregs and TGF-β+Bregs ratio in the spleen and spleencells culture supernatant of IL-10, TGF-β content significantly lowerthan normal control group (P<0.01); LQ high, meddle, low dosetreatment groups of IL-10+Bregs and TGF-β+Bregs ratio in the spleen and spleen cells culture supernatant of IL-10, TGF-β content increased thanthe EAE group(P<0.01or P<0.05); the high and middle dose of LQgroups of IL-10+Bregs and TGF-β+Bregs ratio in the spleen and spleencells culture supernatant of IL-10, TGF-β content increased in the EAEcontrol group(P<0.01); LQ high-dose treatment group of of IL-10+Bregsand TGF-β+Bregs ratio in the spleen and spleen cells culture supernatantof IL-10, TGF-β content increased in the LQ meddle-dose group(P<0.01).(5) correlation analysis: EAE control group, low, meddle, high lose LQtreatment groups pathogenesis incubation period with the peak of IL-17was negatively related(P<0.01), and IL-4, IL-4/INF-γvalue, spleen tissuesIL-10+Bregs, TGF-β+Bregs proportion and supernatant of IL-10,TGF-βlevels were positively correlated(P<0.01); EAE control group, low,meddle, high lose LQ treatment groups the incidence of progress and theincidence of IL-17was positively correlated(P<0.01), with IL-4,IL-4/INF-γvalue, IL-10, TGF-β, IL-10+Bregs, TGF-β+Bregs proportionwere negatively related(P<0.01); EAE control group, low, meddle, highlose LQ treatment groups morbidity peak nerve dysfunction score andmorbidity peak of IL-17positively correlated(P<0.01), and IL-4,IL-4/INF-γvalue, IL-10, TGF-β, IL-10+Bregs, TGF-β+Bregs proportionwere negatively related(P<0.01). Conclusion:1. The guinea pig crudeMBP can be induced to establish EAE model on Wistar rats, EAE ratswith clinical symptoms, brain tissue perivascular inflammatory cell infiltration, white matter demyelination, shows that this method can besuccessfully established EAE model.2. EAE rats the peak of PBMCsecrete inflammatory cytokines(IL-17, INF-γ) increased, andimmunosuppressive cytokines(IL-4) to reduce. This case the Th1/Th2imbalance. Th0differentiation to Th1drift.3. Bregs has a negativeregulatory role of EAE rats and its mechanism may be: Bregs can throughthe secretion of IL-10and TGF-β inhibitory cytokines or throughinteraction with other immune cells to exert negative regulatory role.4.LQ can reduce EAE rats incidence, prolong the incubation period, shortenthe development period,reduce EAE rats nerve dysfunction andinflammatory cells invasion, LQ plays a protective role.5. LQ byinhibiting EAE rat PBMCs of inflammatory cytokines and promote theimmune inhibitory cytokines(IL-4), which are able to correct the Th1/Th2imbalance, prompting Th0differentiation to Th2drift.6. LQ can increaseEAE rats spleen Bregs ratio, thus through Bregs and its inhibitorycytokine secretion(IL-10, TGF-β) and play a role of negation regulation,so as to achieve the protection of EAE. |
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