| Objective: Multiple sclerosis is a kind of central nervous system (CNS)chronic inflammatory demyelinating diseases. The typical pathologicalfeatures is that demyelination of CNS, glial cell proliferation and varyingdegrees of axonal damage. At present a lot of studies have shown that patientswith multiple sclerosis exist blood-brain barrier damage at the early stage, andspeculate that it may be a critical component of the pathogenesis of multiplesclerosis. However the concrete pathogenesy is not clear and there is no activetreatment. EAE with the similar symptom and pathogenesis as MS is therecognized ideal model of MS, and is the best animal model for studying theimmunopathology and treatment of MS.Matrix metalloproteinase-9degradates matrix proteins which is in thevascular endothelial cells, and leading to an increased microvascula rendothelial permeability,so that T lymphocytes and other inflammatory cellspenetrate through the BBB basement membrane.In the past, we thought Th1couse MS. However with the deep researchof Th17, it is found that IL-23/IL-17inflammatory axis played a moreimportant role in MS and IL-17is a kind of highly inflammatory cytokines.Many studies have found that oxidative stress plays an important role inMS.Probucol as a cholesterol-lowering drug also has a significant effect ofantioxidation, anti-inflammation, clearance of free radical. Current studieshave found that probucol also has a significant effect in a variety of diseases,however there is few study about MS. Whether probucol can reduce the injuryto CNS of MS by it，s antioxidation,anti-inflammation,clearance of free radicaland adjustment of IL-17and MMP-9. There are few researches in this respect.In our study,We administrate probucol to the rats of experimental autoimmune encephalomyelitis(EAE)，to observe the dynamic expression of MMP-9andIL-17,and to see whether probucol has protection of EAE,so as to provide newexperimental evidence.And it can be introduced into clinic.Methods:1The group of experiment and intervention120female wistar rats are divided into four groups (the control group,theprednisone-EAE group,the probucol-EAE group and the EAE group)randomly,each group with30rats.The control group immunized byphysiological saline and CFA. Other rats were immunized by1:10freshguinea pig spinal cord homogenate (GPSCH).The rats of the prednisone-EAEgroup are given prednisone for5mg/kg d since immunized until killed.Therats of the probucol group are given probucol for500mg/kg d sinceimmunized until killed. The other two groups are given right amount ofphysiological saline at the same time.Each group is divided into6groupsaccording to the day of immune,the0day group,the3day,the7day group,the10day group,the14day group and the21day group.2Establish animal modelWe anesthetize the guinea pig, take the whole spinal cord, then weight it.The weight of spinal cord and the volume of brine ice is1:10, is made forguinea pig spinal cord homogenate (GPSCH). It is mixed with the completeFreud`s adjuvant with6mg/ml of bcg vaccine. Each animal is0.5ml for limbsfoot pad and the back subcutaneous injection.3The clinical scoresThe rats are weighed everyday after immunization, and the situations areobserved such as mental state, eating and drinking, urine and shit, and theactivities. The clinical scores are assessed in accordance with the standardfrom Kono.4The observation of indexWe anesthesia the animals, then take the spinal cord to the4%formalin.The lumbar enlargements are made to paraffin blocks. We observe theinflammatory cells infiltration with HE, and count the immune positive MMP-9cells by immunohistochemical (IHC). The sera from rats wereachieved and were detected the levels of IL-17with all enzyme-linkedimmunosorbent assay.Results:1The observation of morbidityThe rats were listlessness, furunsmooth, had no appetite, weight loss afterimmunization gradually. Then tail feeble appear at first, hind limbs and forelimbs, incontinence of urine and defecate. The morbidity time of theprednisone-EAE group and the probucol group are all later than the EAEgroup,also the symptom of the both group are lighter than the EAE group.2The comparison of mean clinical scoresThe mean clinical score of the EAE group in the14th day is higher thanthe prednisone-EAE group and the probucol group, there is statisticallysignificant (P<0.05). The mean clinical score of the prednisone-EAE groupand the probucol group compared in the14th day, there is no statisticallysignificant (P>0.05).3The observation of histopathologyHaematoxylin&Eosin (HE) staining showed an infiltration ofinflammation at different stages of EAE.The histopathologic changes werelighter in the the prednisone-EAE group and the probucol group.4the observation of immunohistochemical (IHC)4.1The EAE group compared with the control groupThe expression of MMP-9in the third day,the7th day,the10th day andthe21day are higher than the control group,there is statistically significant(P<0.05).4.2The expression of MMP-9in different time of EAEThe EAE14day group compared with the group of other time, there isstatistically significant (P<0.05).4.3The EAE group compared with the prednisone-EAE group and theprobucol group.The EAE group compared with the prednisone-EAE group and the probucol group in the day of7,10,14,21, the expression of MMP-9washigher, there is statistically significant (P<0.05).5the levels of IL-175.1The EAE group compared with the control groupThe EAE group compared with the control group in the day of7,10,14,21, the level of IL-17is higher, there is statistically significant (P<0.05).5.2The levels of IL-17in different time of EAEThe EAE14day group compared with the group of other time, there isstatistically significant (P<0.05).5.3The EAE group compared with the prednisone-EAE group and theprobucol groupThe EAE group compared with the prednisone-EAE group and theprobucol group in the day of7,10,14,21, the level of IL-17is higher, there isstatistically significant (P<0.05).Conclusions:1The serum levels of IL-17and the expression of MMP-9correlated with theverity and activity of EAE.2Probucol can delay morbidity time, alleviate clinical symptoms.3Probucol can relieve inflammatory cells infiltration of the spinal cord inEAE rats.4Probucol can reduce the expression of MMP-9in the spinal cord of the EAErats.5Probucol has good safiy and tolerance compared with prednisone, althoughit is worse in delaying the disease. These results suggest that probucol maybe used in the diseases like MS in future. |
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