| Objective: This experiment studied the Therapeutic effect of teriflunomide(TIF)combined with total glucosides of paeony(TGP)on experimental autoimmune encephalomyelitis(EAE)mouse,a animal model of multiple sclerosis(MS).We observed the behavior of EAE mice and set HE staining,LFB staining and electron microscopy on the lumbar enlargement of spinal cord in order to explore the effect of teriflunomide combined with total glucosides of paeony on the treatment of EAE mice,which may provide a new idea for the combined treatment of multiple sclerosis.Methods:1 C57BL/6 mice(8-10 weeks old,healthy,female,weighing about 18g-20g)were selected for 50 weeks.They were randomly divided into 5 groups: Control group,EAE group,teriflunomide group,TGP group and teriflunomide,total glucosides of paeony in combination group,10 mice in each group.EAE group,teriflunomide group,TGP group and teriflunomide plus TGP group established the mouse model of EAE using MOG35-55 assisted by Mycobacterium tuberculosis,Complete Freund's Adjuvant and Pertussis Toxin.,while the control group were not immuned.The day on which the mice were immunized was recorded as Day 0.At the first days drugs were given,teriflunomide group 15mg/kg TIF,TGP group 500mg/kg TGP,combination group TGP 15mg/kg combined with TIF 500mg/kg,while control group and EAE group mice were received equal volume of 1.5% Carboxymethyl cellulose.From the beginning of the first day of immunization,the mice were weighed and the neurological function was evaluated by the method of double blind method(8:00 and 16:00)by the two observers at the same time every day.The neurological function score was observed by Weaver(15 minutes),and the incidence and course of the mouse were recorded.2 40 female C57BL/6 mice(8 to 10 weeks old,18 g to 20 g body weight),were randomly divided into 5 groups: Control group,EAE group,teriflunomide group,TGP group and teriflunomide plus TGP group,with 8 mouse in each group.The animal model was established with MOG35-55,Mycobacterium tuberculosis,Complete Freund's Adjuvant and Pertussis Toxin,but the control group was granted immunity.The day on which the mice were immunized was recorded as Day 0.The teriflunomide group received the solution of TIF 15mg/kg,while TGP group 500mg/kg,the combination group received 15mg/kg and 500mg/kg.Mice in other groups received equal volume of 1.5% Carboxymethyl cellulose.The weight and clinical of mice symptoms were observed two times every day.The mice in each group were sacrificed on the 26 th day.Five of the mouse in each group were selected at 26 days.The lumbar enlargement of spinal cord of mice were embedded in paraffin.The spinal cords were stained with HE in order to observe the inflammation and Roque Schall fast blue(Luxol Fast Blue)to observe the myelin lost and the axon damage;The other 3 rats in each group was collected for perfusion,which will be obvioused by scanning electron microscopy applications.By means of the above methods,the inflammatory cells and demyelination in the lesion area of mouse in each groups were observed.SPSS 21 statistical software was used for statistical analysis.Measurement data were expressed as mean±standard deviation.The statistical method for comparisons of several groups was one-way ANOVA.P<0.05 was considered that it has statistically significance.Results:1 The incidence of control groups was none.The incidence of other groups: EAE group 100%,TIF group of 90%,TGP group of 80%,the combination group was 30%.Mouse were compared among each groups about the Onset time of disease: mouse in Control group did not occur;the Onset time of TGP+TIF group was later than other groups(P <0.05).However,there was no significant difference between TGP group and TIF group(P >0.05).2 HE staining: the control group had clear structure,no inflammatory cell infiltration.The drug group and EAE group had different degrees of demyelination.EAE group was the most serious one.There are varying degrees' infiltration of inflammatory cells at the junction of the spinal cord,white matter,gray matter,the spinal cord and the central canal near,which included some clear "vascular cuff" phenomenon.Compared with the EAE group,the infiltration of inflammatory cell of mouse in each treatment group were significantly reduced,mainly concentrated around the small blood vessels,and there was significant difference(P<0.05).THE inflammatory cells in in spinal cord tissue of group TGP+TIF were decreased compared with the mouse of TIF or TGP alone group.There was significant difference(P <0.05).However,there was no significant difference between the TGP group and the TIF group in the number of inflammatory cells in the spinal cord of mouse,and there was no significant difference(P >0.05).3 LFB staining: There was no significant demyelination in normal control group.There were different degrees of demyelination in each treated groups and model group,most of which were located in the white matter.EAE group can be observed different size of demyelinating region in the spinal cord,which were mostly in the white matter of spinal Anterior horn and Posterior horn.The blue myelin parts were reduced,the color faded,and even a large area of demyelination appeared,which was vacuolar degeneration like a balloon.It was often accompanied by a large part of the infiltration of inflammatory cells,which indicated that there was a certain relationship between inflammatory infiltration and demyelination;TGP group and TIF group were showed different degrees of demyelination,mostly in the anterior and posterior horns of spinal cord around the white matter,but lighter than EAE group(P<0.05).In the combined treatment group,compared with the contral group,although some of the myelin sheath became thinner,the color became lighter,and there were some small areas of demyelination,there were no large areas of demyelination.Compared with the other two groups,the band of myelin loss was very small,and the difference was statistically significant(P<0.05).However,there was no significant difference between TIF and TGP group(P >0.05).4 Transmission electron microscope scanning: In the normal control group,myelin sheath was complete and arranged in a regular concentric circle in the white matter of the lumbar enlargement of the mouse.There were leukoaraiosis,loose myelin,myelin disintegration,visible fracture loss,at the lumbar enlargement of EAE mouse.The white matter structure was relatively complete at the lumbar enlargement of each treated group and the concentric circle was arranged into the rules.There was no obvious myelin disintegration and loss in the combined treatment group and the demyelination was relatively mildConclusions:1 Teriflunomide combined with total glucosides of paeony could delay the onset time of EAE mice,reduce neurological damage in mice and have the protective effect on EAE,and the effect is better than that of each single drug.2 Teriflunomide combined with total glucosides of paeony in EAE mice can significantly reduce inflammatory cell infiltration and demyelination of spinal cord... |
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