| Objective: To explore the neuroprotective effects of melatonin(MT)on the pathogenesis of experimental autoimmune cerebrospinal meningitis(EAE)mice and its potential anti-oxidative stress mechanism,and to search for new therapeutic drugs for MS.Method: There were 48 female C57BL/6 mice(weighted 18-20 g,aged 8-10 weeks)into the study,which were assigned randomly into three groups: the normal group,the EAE group,the MT group.Then each group randomly divided into two subgroups(A,B).In accordance with the above group information,mice were got corresponding number.Using the MOG35-55 peptide,CFA,H37 Ra,PTX,the mice of EAE group and MT group were injected those substances to set up animal model of EAE.These materials are the key drugs to build the animal model.The normal group mice were only injected with CFA without MOG35-55 to reduce the experimental error.Two investigators assessed the clinical signs of mice 1 times a day(10:00 am)by Benson5 score standard.Since the date of modeling,the MT group were treated with melatonin 10 mg / kg(10mg/ml)by intraperitoneal injection until the peak stage of disease,then the mice were sacrificed to collect materials.The normal group and EAE group were only used normal saline 1ml / kg by similar way.The criterion for judging the peak period of the disease was that the symptom of mice did not aggravated for three consecutive daysor quadriplegia or death.Because of sympotom-free,the normal group and the remaining mice were sacrificed at 28 days after modeling.The distinction between A,B subgroups was the different in materials.A subgroup mice were fixed with 4% formalin to get the spinal cord for checking;while the brain tissue of B subgroup mice were quickly removed on the ice tray,and were made brain tissue homogenate,which stored in the-80 ℃.After all the materials were finished,the expression of Nrf2 and HO-1 in spinal cord were observed by immunohistochemistry in each A subgroup mice,and we observed the spinal cord perivascular inflammatory infiltration and vascular "cuff" phenomenon by HE staining.B subgroup mice were determined brain SOD,CAT activities and TBARS,ROS levels.Results:(1)The morbidities of disease in different groups:There was no ill mice in normal group,but various incidence occurred in the EAE group and MT group.The morbidity of EAE group and MT group respectively was 81.2%,56.3%.(2)The change of neurological deficits score,incubation period and progress periodin each group:at the pick stage of disease the mean neurological deficits score of the EAE group and MT group respectively was 2.8±1.0,1.4±0.7.The mean neurological deficits score of EAE control group and MT group were higher than the normal group,and MT group was apparently lower than EAE group.(P<0.05).The incubation period of EAE group and MT group respectively was 12.77±1.92,16.14±2.12.The latency of MT group prolonged clearly than EAE group,and the difference was statistically significant(P<0.05).The progress period of EAE group and MT group respectively was 6.92±1.80,4.00±0.87.The advanced period of MT group shortened clearly than EAE group,and the difference was statistically significant(P<0.05).(3)The changes of the pathomorphology in different group:by light microscope,we found that the spinal cord of normal group kept normal.The pathological changes of EAE group in the process of maximal disease showed that a mass of inflammatory cell infiltrating in the spinal cord,especially around the small venous.The typical change was inflammatory cells gathering around small blood vessels,and we described this funny phenomenon as “vascular cuff”.In the anterior median fissure around of lumbar enlargement this phenomenon was more obvious,which is easy to find.Comparing with EAE group,The inflammatory cell infiltration of MT group were relieved."vascular cuff" phenomenon is a rare.(4)The brain SOD and CAT activities:at the peak stage of disease the mean SOD activities of the normal group,EAE group and MT group respectively was 164.72±6.68,107.09±26.41,132.13±17.72U/mgprot.The mean CAT activities respectively was 10.69±0.70,5.69±1.67,7.50±2.24umol/mgprot.For those two markers,the EAE group and MT group both lower than normal group,and MT group apparently higher than EAE group,both differences have statistically significant(P<0.05).(5)The brain TBARS and ROS concentration in different group:at the peak stage the mean TBARS content of normal group,MT group and EAE group respectively was 19.50±3.47,38.77±12.32,28.86±7.52 ug/g.The ROS levels respectively was 47.00±4.53,92.63±25.03,69.00±17.25ηnmol/mgprot.For those two indicators,EAE group and MT group both higher than that of normal group,but MT group markedly lower than EAE group.All differences have a statistically significant difference(P<0.05).(6)The express of Nrf2 and HO-1 in spinal cord:at peak stage of the disease the mean number of Nrf2 positive cells respectively were 15.13±3.27,27.63±3.58,54.88±10.01.And the HO-1 positive cells in each group mice respectively were 11.50±2.82,30.38±9.72,48.88±13.97.Both EAE group and MT group showed high expression of Nrf2 and HO-1 than that of normal group.MT group was markedly higher than EAE group,which has a statistically significant difference(P<0.05).(7)Correlative analysis:at the peak stage of disease,the activities of SOD,CAT and the express of Nrf2,HO-1 in EAE group and MT group were significantly negatively correlated with the neurological dysfunction score and the progressive stage(P<0.01),and were significantly positively correlated with incubation period.The levels of TBARS and ROS were significantly positively correlated with the neurological dysfunction score and the progressive stage(P<0.05),and were significantly negatively correlated with incubation period.Conclusion:(1)In this study,the EAE models were established by subcutaneous injection of MOG35-55 peptide fragment.We found that EAE mice had a remarkable dysfunction,spinal cords had lots of lymphocytes infiltration,and the typical changes were the “vascular cuff “ phenomenon around small blood vessels.These showed that this method was successful,and had a good repeatability.(2)There are oxidative stress damage in the pathogenesis of EAE.The levels of TBARS,ROS increased;the activities of antioxidant enzymes SOD,CAT decreased.Those all indicated that oxidative stress damage may be an important factor leading to the onset of EAE.(3)MT can reduce the clinical deficits score,prolong the latency period,shorten the progress period,and lessen the infiltration of lymphocytes in the spinal cord tissue,which all indicated the protective effect ofMT on EAE.(4)MT played an important role on antioxidative stress,we suspected that this function can be achieved via activating the Nrf2/ARE signaling pathway probably,then up-regularting the activities of antioxidant enzyme,inhibiting the degree of lipid peroxidation. |
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