| Objective: To observe the protective effects of paclitaxel(PTX) on experimental autoimmune encephalomyelitis(EAE) and to look for the possible immunological mechanisms. Methods :Fifty female Wistar rats were randomly divided into five groups according to the randomly digital table:normal control group, EAE control group, low dose PTX group, median dose PTX group and high dose PTX group. The complexes of the guinea pig spinal cord homogenate(GPSCH)and the complete Fr Reud’s adjuvant(CFA) as immunizing antigen(2mg/kg) were used in the induction of EAE group and the PTX groups Wistar rat model of EAE. PTX was injected into the abdominal cavity in different doses starting from the day when rats were injected by antigen to the 10 th day. The dose respectively were 1mg/kg,2mg/kg,4mg/kg.Executing the rats when the scores of symptoms were highest or one month later for the rats in the normal control group. If the score of symptom was zero after immunization, making the rats executed one month later too. The scores of symptoms were recorded in this period. The brain,spleen tissue,orbital venous blood were retained. Part of brain were kept for the pathology observation with the aid of hematoxylin-eosin staining and the severity of the pathological changes were scored according to the Okuda standard. Other brain tissue were used for measuring the expressions of CD28 and CTLA-4 by flow cytometry;Using the method of enzyme-linked immunosorbent(ELISA) to measure the peripheral blood mononuclear cell(PBMC) secretion of interferon gamma(INF-γ),interleukin-4(IL-4); the levels of transforming growth factor beta(TGF- β) in the blood were also measured by the radiative immunoassay;Using flow cytometry to measure the ratio of Th1 cell and Th2 cell in the spleen tissue;Finally the levels of Stimulating factor of B cell(BAFF) was measured by ELISA.All the results were analyzed with SPSS17.0.Results:(1)clinical symptoms: ①No rats in the normal control group were ill. All the rats were ill in the EAE control group,the seven of ten were ill in the low dose PTX group,five of ten were ill in the middle dose,fore of ten were ill in the high dose. The clinical manifestation of EAE rats were decreased tone of tail,ataxia,quadriplegia,articulo mortis,death,But the PTX groups had anesis than EAE control group,the higher the dose,the more relax the symptoms Compared with EAE control group,② all the PTX groups showed significantly prolonged latent period(P<0.05)and the latent period of the high dose PTX group was longest.the difference between the high dose PTX group and the low dose PTX group was statistically significant(P<0.05).the difference between the median dose PTX group and the low dose PTX group was statistically significant(P<0.05).the difference between the high dose PTX group and the median dose PTX group was not statistically significant(P>0.05).③Compared with EAE control group, the progressive stage of the disease among all the PTX groups were short, and when the dose of the PTX became higher, the progressive stage of the disease became much shorter(P<0.05).the differences among all the PTX groups showed statistically significant(P<0.05).④There were decreases in the scores of symptoms among all the PTX groups when it came to the EAE control group, and when the dose of the PTX became higher,The scores of symptoms became much lower, the differences among all the PTX groups showed statistically significant(P<0.05).(2)Pathological changes observed through hematoxylin-eosin staining:There was no central nervous system(CNS) inflammatory cell infiltration in the normal control group while there were infiltrations around small blood vessels which sometimes typically formed a cuff kind of change both in EAE control group and all the PTX groups. According to the Okuda standard, the pathological changes were lessened in all the PTX groups compared with EAE control group(P<0.05). And when the dose of PTX groups was higher, the score of the pathological changes became smaller. There were fewest inflammatory cell infiltrations in the high dose PTX group and the differences among all the doses PTX groups were significant(P<0.05).(3)The changed levels of CD28 and CTLA-4 of EAE control group and PTX groups in the brain:①The levels of CD28 in EAE control group and all the PTX groups were higher than normal control group, the differences were statistically significant(P<0.01). The levels of CD28 in all the PTX groups were lower than EAE control group, the differences were statistically significant(P<0.01).And when the dose of PTX groups was higher,the level of CD28 was much lower, the differences among all the doses PTX groups were significant(P<0.01).②The levels of CTLA-4 in EAE control group and all the PTX groups were lower than normal control group, the difference between normal control group and EAE group were statistically significant(P<0.01). The differences between normal control group and middle dose PTX group,low dose PTX group were same.And when the dose of PTX groups was higher,the level of CTLA-4 was much Higher, the difference between high dose PTX group and low dose PTX group were statistically significant(P<0.01). The differences between middle dose PTX group and low,high dose PTX group were not statistically significant(P>0.05).(4)The PBMC secretion of INF-γ,IL-4 and the ratio of INF-γ/ IL-4 of EAE control group and PTX groups :①The levels of INF-γ in EAE control group and all the PTX groups were higher than normal control group(P<0.01), the level of INF-γ in high dose PTX group were lower than EAE group(P<0.01),while the differences among all the doses PTX groups were not significant(P>0.05).②The levels of IL-4 in EAE control group and all the PTX groups were lower than normal control group(P<0.01), the levels of IL-4 in all PTX groups were higher than EAE control group(P<0.01).when the dose of PTX groups was higher,the level of IL-4 was much higher.The differences between high dose PTX and middle,low dose PTX was statistically significant(P<0.01 or P<0.05),while the difference between middle dose PTX and low dose PTX was not statistically significant(P>0.05).③The ratio of INF-γ/ IL-4 in EAE control group and all the PTX groups were higher than normal control group(P<0.01), the ratio of INF-γ/IL-4 in all PTX groups were lower than EAE control group(P<0.01).when the dose of PTX groups was higher,the ratio of INF-γ/IL-4 was much lower.the differences among all the doses PTX groups were significant(P<0.01).(5)The TGF-β expression in serum of EAE control group and all PTX groups: Compared with the normal control group, there were significantly low-regulated expressions of TGF-β both in EAE control group and all the PTX groups(P<0.05), the expression of TGF-β in all PTX groups were higher than EAE control group(P<0.05),when the dose of PTX groups was higher,the expressions of TGF-β was higher,the differences among all the doses PTX groups were statistically significant(P<0.05).(6)Th1 cell,Th2 cell,Th1/Th2,BAFF in spleen tissue of EAE control group and all PTX groups:①The levels of Th1 cell and ratio of Th1/Th2 were higher,the levels of Th2 cell was lower in EAE control group and all the PTX groups than normal control group,the differences were statistically significant(P<0.01 or P<0.05).the levels of Th1 cell and ratio of Th1/Th2 were lower, the levels of Th2 cell was higher in all PTX groups than EAE control group.The difference of Th1 cell in spleen tissue between high dose PTX group and EAE group were statistically significant(P<0.01 or P<0.05),it was same to Th2 cell.The ratio of Th1/Th2 in all PTX groups were lower than EAE group,the differences were statistically significant(P<0.05).When the dose of PTX was higher,the levels of Th1 cell and ratio of Th1/Th2 were lower,the levels of Th2 cell was higher.The difference of Th1 cell in spleen tissue between high dose PTX group and low dose PTX group were statistically significant(P<0.05).the difference Th2 cell between low dose PTX group and middle dose,high dose were statistically significant(P<0.01).The differences of Th1/Th2 among the different dose PTX groups were statistically significant(P<0.01 or P<0.05).②The levels of BAFF in EAE control group and all the PTX groups were higher than normal control group(P<0.01),the levels of BAFF in all PTX groups were lower than EAE control group(P<0.01 or P<0.05).When the dose of PTX groups was higher,the levels of BAFF were lower.The differences among all the doses PTX groups were statistically significant(P<0.01).(7) Correlative analysis:①Both in the EAE control group and all the PTX groups, the latent period of this disease were positively correlated with CTLA-4,TGF- β,Th2 cell(P<0.01),while negatively correlated with the expressions of CD28,Th1 cell,BAFF,the ratio of Th1/Th2 and INF-γ/IL-4(P<0.01 or P<0.05).②In all the groups except the normal control group, the progressive stage of this disease were positively correlated with the expressions of CD28,Th1 cell,BAFF,the ratio of Th1/Th2 and INF-γ/IL-4(P<0.01 or P<0.05), while negatively correlated with the ratio of CTLA-4,TGF-β,Th2 cell(P<0.01).③Both in the EAE control group and all the PTX groups, the scores of symptom in the peak time of this disease were positively correlated with expressions of CD28,Th1 cell,BAFF,the ratio of Th1/Th2 and INF-γ/IL-4(P<0.01 or P<0.05).while negatively correlated with CTLA-4,TGF- β,Th2 cell(P<0.01 or P<0.05).Conclusion:1.The ratio of Th1/Th2 and INF-γ/IL-4,the content of CD28,the level of BAFF were up-regulated, while the content of CTLA-4 were just reduced in the development of EAE, which were all correlated with the EAE rats,s state of an illness,the pathology change of myelencephalon.The results indicated the change of Th1/Th2 and its representative cytokines INF-γ/IL-4,the abnormity level of BAFF had relationships with the development of EAE. 2.PTX could mitigate the clinical symptoms of rats,Prolong latent period, shorten progressive stage,reduce the scores of symptoms and lessen pathological chages,the greater of dose,the greater of the effects.which suggested PTX have prevention and cure effects on rats with EAE and the effects were related to the dose. 3.The mechanism related to the protective effects of PTX on EAE might include rectifying excursion of Th1/Th2,reducing the ratio of INF-γ/IL-4, reducing the content of CD28 and BAFF,increasing content of CTLA-4. |
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