| Objective: To survey the preventive treatment effect of doxycycline(DOX) on experimental allergic encephalomyelitis. And attempt to find out the possible amynologic mechanism. Method: We randomly subgrouped fifty female Wistar rats to normal group, EAE group, low-dose DOX group,median-dose DOX group and the high-dose group, n=10.Establish EAE model by hypodermic injecting the immune antigen into rats’ vola, which is composed of crude MBP and CFA. The EAE control group only inject the same amount of saline. From the beginning three days before modeling EAE, the low, midian and high does DOX group were intraperitoneally injected with DOX 5mg/kg/d,DOX 10mg/kg/d,DOX 20mg/kg/d for 10 days. The normal control group and EAE control group gave the same amount saline the same way. Such situation below could be considered as the peak of EAE disease: the symptom did not aggravate or quadriplegia for 3 consecutive days, or the rats were dead.Recorded the latentperiod, the progression and the peak incidence of neurological dysfunction score. Killed the rats at the peak time, the normal group and the rat’s score of symptom being zero were abserved for 4 weeks and then killed them. Keeped the brain spinal cord tissue for HE staining to observe the pathological changes. The blood of rats to detect the capacity of peripheral blood mononuclear cells secreting IL-4,IL-17,IL-23,IFN-γ,TGF-β1,the brain tissue content of MMP-2,MMP-9 were to be detected by ELISA. Tested the Th17 cell and the Treg cell rate of spleen by flow cytometry. Test the CSF to serum quotient(QA) to assessment the BBB function. Results:(1)The incidence: Rats in normal group didn’t be attacked. The incidence in EAE control group was 90%.The incidence in the high-dose,median-dose and low-dose DOX treatment group was 40%,60%,80%.The latentperiod of EAE control group and the high-dose,median-dose and low-dose DOX treatment group was 11.3±1.5 days,23.7±1.9 days,20.6±1.2 days,17.7±1.0 days,the latentperiod of each DOX group was longer than the EAE control group(P <0.01), and the high-dose DOX group’s was the longest, the difference between each group had statistic significance. The progression of EAE control group and the high-dose,median-dose and low-dose DOX treatment group was 10.1±1.0 days,4.4±0.6 days,6.9±0.4 days,7.7±0.7 days, the progression of each DOX group was shorter than the EAE control group(P<0.01), and the high-dose DOX group’s was the shortest, the difference between each group had statistic significance(P<0.01). The neurological dysfunction score in peak incidence of EAE control group and the high-dose,median-dose and low-dose DOX treatment group was4.3±0.7 points,1.1±0.4 points,1.9±0.3 points,3.0±0.3 points,the score of each DOX group was lower than the EAE control group(P <0.01), and the high-dose DOX group’s was the lowest, the difference between each group had statistic significance(P<0.01).(2) The brain and spinal cord pathological changes in EAE control group and each DOX group : Through microscope, the normal group had no changes. In the EAE control group, we found lots of inflammatory cell infiltration around small blood vessels, which were mainly lymphocytes and a small amount of mononuclear,typically forming a cuff-like or vessel-like change. The changes in each DOX group all were lighter than EAE control group. The high-dose DOX group was the lightest.(3)The effect the DOX having on the levels of IFN-γ,IL-4 and their ratio in the rats peak incidence: In EAE control group and each DOX group,the levels of IFN-γ and the ratio of IFN-γ/IL-4 was higher than normal group, the levels of IL-4 was lower than normal group(P<0.01), except that there is no significant difference of the levels of IFN-γ between the high-dose DOX group and normal group(P>0.05).In each DOX group, the levels of IFN-γ and the ratio of IFN-γ/IL-4 were lower than EAE control group, the levels of IL-4 were higher than EAE control group(P<0.01),with the amount developing,each DOX group’s levels of IFN-γ and the ratio of IFN-γ/IL-4 became lower and levels of IL-4 became higher.The difference between each group had statistic significance(P<0.01),except that there is no significant difference of the levels of IL4 between the high-dose DOX group and normal group(P>0.05).(4)The effect the DOX having on the levels of IL-17,IL-23,TGF-β1 in the rats peak incidence: In EAE control group and each DOX group,the levels of IL-17 and IL-23 were higher than normal group, the levels of TGF-β1 were lower than normal group, except that there is no significant difference of the levels of IL-17,IL-23 between the high-dose DOX group and normal group(P>0.05).In each DOX group, the levels of IL-17,IL-23 were lower than EAE control group, the levels of TGF-β1 were higher than EAE control group(P<0.01).With the amount developing,each DOX group’s levels of IL17 and IL-23 became lower, and levels of TGF-β1 became higher. The difference between each group had statistic significance(P<0.01),except that there is no significant difference of the levels of TGF-β1 between the high-dose DOX group and the median dose group(P>0.05).(5)The effect the DOX having on the Th17 cell and the Treg cell rate of spleen in the rats peak incidence: In EAE control group,median-dose and low-dose DOX group, the Th17 cell rate of spleen was higher than normal group, the Treg cell rate of spleen was lower than normal group(P<0.01),except that there is no significant difference between high-dose DOX group and normal group(P>0.05).In each DOX group, the Th17 cell rate of spleen was lower than EAE control group, the Treg cell rate of spleen was higher than EAE control group(P<0.01).With the amount developing,in each DOX group, the Th17 cell rate of spleen became lower, and the Treg cell rate of spleen became higher. The difference between each group had statistic significance(P<0.01).(6)The effect the DOX having on the MMP-2,MMP-9 of the brain tissue and QA in the rats’ peak incidence: In EAE control group and each DOX group, the level of MMP-2,MMP-9 and QA were higher than normal group(P<0.01 or P<0.05),except that there is no significant difference of MMP-2 between high-dose DOX group and normal group(P>0.05).In each DOX group, the level of MMP-2,MMP-9 and QA were lower than EAE control group(P<0.01),With the amount developing,the level of MMP-2,MMP-9 and QA were lower in each DOX group. The difference between each group had statistic significance(P<0.01).(7)The correlation analysis: the incidence latentperiod of EAE control group and each DOX treatment group was negatively related to IL-17,MMP-2,MMP-9,QA,the ratio of IFN-γ/IL-4,the ratio of Th17,and positively related to TGF-β1,the ratio of Treg. The incidence progression of EAE control group and each DOX treatment group was positively related to IL-17,MMP-2,MMP-9,QA,the ratio of IFN-γ/IL-4,the ratio of Th17,and positively related to TGF-β1,the ratio of Treg. The peak incidence of neurological dysfunction score in EAE control group and each DOX treatment group was positively related to IL-17, MMP-2,MMP-9,QA,the ratio of IFN-γ/IL-4,the ratio of Th17,and positively related to TGF-β1,the ratio of Treg. Conclusion: 1.In the peak incidence of EAE, the pro-inflammatory cytokines of rat’s peripheral blood increased, the inhibition-inflammatory cytokines decreased. The ratio of Th17 in spleen increased, and the ratio of Treg decreased. The Th1/Th2 balance and Th17/Treg balance in rats were broken.2.In the peak incidence of EAE, the level of MMP-2,MMP-9 and QA in the brain tissue increased, the changes above suggested that the blood brain barrier was broken.3.DOX could reduce the incidence of EAE rats, prolong the latentperiod, shorten the progression and reduce the peak incidence of neurological dysfunction score, lighten the inflammatory cell infiltration of cerebral spinal tissue. The changes above suggested that DOX had preventive effect on the onset of EAE.4.DOX could prevent the onset of EAE, by inhibiting the pro-inflammatory cytokines production, promoting the inhibition-inflammatory cytokines, reducing the ratio of Th17,increasing the ratio of Treg in spleen, reducing the level of MMP-2,MMP-9 and QA in the brain tissue, correcting the Th1/Th2 balance and Th17/Treg balance, protecting the blood brain barrier. |
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