| Abstract:bjective:To explore the effect and possible mechanism of osteopontin(OPN) on experimental allergic encephalomyelitis(EAE),to explore the atorvastatin's effect on OPN and treatment on EAE. By observing the OPN on the EAE about its incidence, pathology, immunohistochemistry, expression of CD44 and OPN, peripheral blood IL-10, IFN-γexpression, Th1/Th2 balance,to explore the effect of OPN on the possible immunological mechanisms of EAE.Method:40 female rats were randomly divided into four groups:normal control group, EAE control group, high-dose atorvastatin treatment group and the low-dose atorvastatin treatment group, n= 10. Myelin basic protein(myelin basic protein, MBP) by crude antigen were injectde(0.2ml/100g) into the EAE control group, atorvastatin high and low dose treatment groups, normal control group was injected with equivalent saline. Since the date of modeling, low-dose atorvastatin treatment group was administered atorvastatin 1mg/kg daily, high-dose atorvastatin treatment group administered 4mg/kg daily, normal control group and the EAE control group administered equivalent saline daily until the end of the experiment. EAE control group, all dose of atorvastatin treatment groups had the symptom for 3 consecutive days without aggravating or quadriplegia,or the rats being dead, would be considered as the peak of EAE disease. Record onset latency, advanced and peak disease score; the rats would be killed at he peak incidence, normal control group were killed 4 weeks later. Then we would keep the brain and spinal cord tissue,and they would be used for HE staining, to observe the pathological changes; Immunohistochemical method and the mean optical density measurement to detect the rat brain and spinal cord white matter tissue OPN and CD44 expression,the capacity of peripheral blood mononuclear cells (peripheral blood mononuclear cell, PBMC) secretion of IFN-γ, IL-10 would be teasted by enzyme-linked immunosorbent assay (ELISA).Result:(1) The incidence of rats:normal control rats did not have the disease. EAE control group, atorvastatin high, low-dose treatment group had varying degrees of disease. EAE control group, the delitescence was 12.5±1.1 days, atorvastatin treatment group,14.2±1.3 days was longer than the EAE control group,this was statistically significant (P＜0.05), high-dose atorvastatin treatment group was 19.4±2.2 days, compared with EAE control group and low-dose group were significantly longer (P＜0.01, P＜0.05); the progression of EAE control group was 6.9±1.4days, low-dose atorvastatin treatment group was 4.7±0.6days,this was shorter than the EAE control group (P＜0.01), high-dose atorvastatin. treatment group was 4.4±0.8 days, was significantly shorter than the EAE control group (P＜0.01),but compared with the low-dose atorvastatin treatment group,this was not statistically significant (P＞0.05); EAE control group, the peak incidence of disease score was 3.6±0.5, low-dose atorvastatin group disease scored 1.8±0.8, compared with EAE,this was significantly decreased (P＜0.01), high-dose atorvastatin group disease scored 1.6±0.8,this was lower than the EAE control group (P＜0.01), but not statistically significant when compared with the low-dose(P＞0.05). (2) Pathological changes in brain and spinal cord:By light microscope, we found the slice of cerebrum, cerebellum, brain stem and spinal cord in the normal group normal.The pathological changes of EAE in the process of maximal disease showed inflammatory cuff around small blood vessels, perivascular inflammatory cell infiltration and demyelination in white matter, especially in spinal cord. The pathological changes of atorvastatin treatment group lessened. (3) OPN and CD44 immunohistochemistry and image analysis:OPN, CD44 was mainly expressed in the brain, brain stem, spinal cord,white matter and gray matter around the junction of the small blood vessels. OPN mainly expressed in the nerve cell nucleus and the part of the cytoplasm also expressed; CD44 mainly e-xpressed in the cytoplasm of nerve cells. Expression of these two parts had heavier inflammation. Normal control group had no OPN and CD44 positive cells. White matter and gray matter could be seen at the junction of a large number of positive cells whith was dark brown in EAE control group; high-dose atorvastatin and low-doses atorvastatin groups had only sparsely stained positive cells, low-dose atorvastatin group was higher than high-dose atorvastatin group. Image analysis showed that with high-dose atorvastatin group and low-dose atorvastatin group, EAE group of CNS white matter of OPN, CD44 expression level was significantly increased (P＜0.01);the expression of OPN and CD44 expression in low-dose and high-dose atorvastatin group was significantly different (P＜0.05). (4) IFN-γand IL-10 of each group:IFN-y of normal control group was 0.21±0.02ng/ml, IFN-γof EAE control group 0.38±0.04ng/ml,whith were significantly higher than the normal control group (P＜0.01), IFN-γof the high-dose and low-dose atorvastatin groups were 0.24±0.01ng/ml,0.27±0.02ng/ml, compared with EAE control group, these were significantly decreased (P＜0.05, P＜0.01), high-dose atorvastatin group was decreased significantly than the low-dose(P＜0.05); IL-10 of normal control group was 2.25±0.28pg/ml, IL-10 of EAE control group was 2.91±0.39pg/ml, compared with the nomal group whith was decreased significantly (P＜0.01), IL-10 of high-dose and low-doses atorvastatin groups were 7.05±1.46pg/ml,5.80±1.10pg/ml, compared with EAE control group whith were significantly increased (P＜0.01), low-dose atorvastatin group compared with high dose atorvastatin group, was obvious (P＜0.05); EAE control group IFN-γ/IL-10 ratio was significantly higher than the normal control group (P＜0.01), high-dose and low-dose atorvastatin groups of IFN-γ/IL-10 ratio compared with EAE control group were significantly decreased (P＜0.01), high-dose and low-dose group had significant difference (P＜0.05). (5) Correlative analysis:the expression of OPN and CD44 in EAE contral group, the low-dose atorvastatin group and the high-dose atorvastatin group were significantly negatively correlated with the delitescence and positive with the progression and miximal disease scores;the ratio of IFN-y/IL-10 were significantly positively correlated with and the maximal disease score and the progression and negetive with delitescence;the ratio of IFN-y/IL-10 were significantly positively correlated with the expression of CD44 and OPN;the expression of OPN was positive with the expression of CD44.Conclusion:1.In this experiment, crude spinal cord of guinea pigs were used to induce EAE model.EAE rats had obvious nerve dysfunction, brain and spinal cord perivascular inflammatory cell had infiltration and demyelination. The EAE model in this study was successful, reliable and stable.2.EAE rats existed immune imbalance. EAE rats had increased Th1 type cytokines IFN-γ, and decreased Th2 type cytokines IL-10.Th1/Th2 cell had imbalance, immune style shifted to the Thl type pattern.3.EAE rats had upregulation of OPN. OPN expression was negatively correlated with delitescence, and positively correlated with progression, disease score.After atorvastatin treatment,OPN expression reduced, whith indicated that OPN had a relationship with the development of EAE.4.The possible reason that OPN was important in the mechanism of EAE was OPN had combined with CD44.The combination of OPN and CD44 might cause Thl/Th2 imbalance.5.Atorvastatin on EAE had a therapeutic effect, whith could improve the clinical symptoms, delay the onset time, improve the central CNS inflammation,decrease the demyelination and axonal injury, and even promote the regeneration of neuraxon. But the mechanism might be achieved through the CD44 pathway of OPN.
|
PDF Full Text Request