| Objective:To study pathogenesis and relapsing mechanism of experimental autoimmune encephalomyelitis (EAE) and efficacy and therapeutic mechanism of Yishendaluo decoction, which may provide theoretical evidence to remedy multiple sclerosis(MS), based on changes of microglias, extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway, Thl and Th2 cells, and oligodendrocytes.Materials and methods:(1) Grouping: in experiment 1, 40 female SJL mice (8- to 12-wk-old) were randomly divided into normal group, model group, Chinese medicine group and hormone group, 10 mice per each group. In experiment 2, 60 female SJL mice (8-to 12-wk-old) were randomly divided into normal group, model group, Chinese medicine group, hormone group and PD98059 group, 12 mice per each group.(2) Establishment of EAE: each mouse in the model, Chinese medicine,hormone and PD98059 group, was injected subcutaneously with 200μl emulsion containing 150μg of PLP139-151 in CFA supplemented with 4 mg/ml M. tuberculosis H37RA at two different sites of epigastrium on the first day. 6 × 105 Bordetella pertussis in 100μ1 of saline was injected twice by caudal vein, first at the time of the PLP immunization and at the second time 48 hours later. The mice in the normal group with no dispose.(3) Intervention: 7 days after immunization, mice in the normal and model group were given double distilled water (0. 1m1/10g), Chinese medicine group were Yishendaluo decoction (0. 1ml/10g, 2g crude herb/ml) and hormone group were prednisone (0. lml/10g,0.39mg/ml) by intragastric administration every day for 54 days in experiment 1, and for 15 days in experiment 2. The mice in the PD98059 group were intraperitoneally injected with PD98059 (0. 5mg/kg) once a week.(4)Execution: the mice were executed on 60 days after immunization in experiment 1 and on 21 days after immunization in experiment 2.(5) Evaluation index: the ethology, weight and neurological function score were observed daily. Hematoxylin-eosin (H-E) and Luxol fast blue (LFB) staining were used to detect the infiltration of inflammatory cells and loss of myelin.Western blot was used to detect the level of IL-2, IL-4, PLP, Oligl, O1ig2,Iba-1, Ras, p-c-Raf, c-Raf, p-MEK1/2, MEK1/2, p-ERK1/2 and ERK1/2. The immunohistochemistry was used to observe the morphology of microglial cells.Results:Part one: effects and functional mechanisms of Yishendaluo decoction on the mice with EAE in the relapsing phase.(1)Effects of Yishendaluo decoction on the time of onset, incidence,relapsing time and rate of the mice with EAE: the mean time of onset of the mice in the model group was 12. 4±0.2 days, the incidence was 80%, the mean relapsing time was 48. 2±10. 3 days and the relapsing rate was 62. 5%; the mean time of onset of the mice in the Chinese medicine group was 12. 7±0. 3 days, the incidence was 77. 80%, the mean relapsing time was 56 days and the relapsing rate was 14. 3%; the mean time of onset of the mice in the hormone group was 11. 8±0. 3 days, the incidence was 60%, the mean relapsing time was 56 days and the relapsing rate was 33.3%. The time of onset, incidence, relapsing time and rate of the mice with EAE in the Chinese medicine and hormone group did not reduce or delay, compared with the model group (P>0. 05).(2)Effects of Yishendaluo decoction on neurological score of mice with EAE:the highest score of the mice with EAE in the model, Chinese medicine and hormone group were all appeared on the 14th day, and there were no difference among the three groups (P>0. 05). On the 21 days after immunization, the score in the hormone group was lower than the model group (P<0. 05). On the 60 days after immunization, the score in the Chinese medicine group was lower than the model group (P<0. 05).(3)Effects of Yishendaluo decoction on the pathological changes of the mice with EAE: H-E staining results indicated that the cerebral and spinal tissue of the mice in the normal group showed uniform dyeing and integrated cell structure. There were many circumscribed inflammatory sites with massive inflammatory cells, and the inflammatory cells around the vein to form sleeve sample in tissue of the mice in the model group. The inflammatory sites were reduced in the mice with EAE in the Chinese medicine and hormone group.(4)Effects of Yishendaluo decoction on the demyelination of the mice with EAE: the luxol fast blue staining results indicated that the myelin tissue in the normal group was pyknotic and orderly. There was significant demyelination in the mice with EAE in the model group, even vacuolar sample. The demyelination in the mice with EAE in the Chinese medicine and hormone group was alleviated.(5)Effects of Yishendaluo decoction on the expression of IL-2 and IL-4:western blot results showed that the level of IL-2 in the model group was higher than the normal group (P<0. 05), and the level of IL-2 in the Chinese medicine and hormone group was lower than the model group (P<0. 05). The level of IL-4 in the model and hormone group was little higher than the normal group(P>0. 05), and the level of IL-4 in the Chinese medicine was higher than the model and hormone group (P<0. 05).(6)Effects of Yishendaluo decoction on the remyelination in the mice with EAE: Western results indicated that the level of PLP in the model group was lower than the normal group (P<0.05),and the level of PLP in the Chinese medicine and hormone group increased in comparison of the model group (P<0. 05).The expression of Oligl in the Chinese group was increased significantly compare with the model and hormone group (P<0. 05), and the expression of O1ig2 in the Chinese group was higher than the model group (P<0. 05).Part two: effects and functional mechanisms of Yishendaluo decoction on the mice with EAE in the acute phase.(1)Effects of Yishendaluo decoction on the time of onset and incidence of the mice with EAE: the mean time of onset of the mice in the model group was 11. 4±0. 2 days, the incidence was 75%; the mean time of onset of the mice in the Chinese medicine group was 12. 8±0. 6 days, the incidence was 75%; the mean time of onset of the mice in the hormone group was 12. 7±0. 5 days, the incidence was 91. 7%; the mean time of onset of the mice in the PD98059 group was 11.9±0.4 days, the incidence was 75%. There was no difference among the mice with EAE in the Chinese medicine, hormone and PD98059 group on the incidence(P>0. 05). The onset time of EAE mice in the Chinese medicine and hormone group was delayed compared with the model group (P<0. 05).(2)Effects of Yishendaluo decoction on neurological score of mice with EAE:there were no difference among the model, Chinese medicine, hormone and PD98059 group on the highest score of the mice with EAE (P>0. 05). On the 21 days after immunization, the score in the Chinese medicine, hormone, and PD98059 group was lower than the model group (P<0.05).(3)Effects of Yishendaluo decoction on the activation of microglia: the immunohistochemistry results exhibited that the number of microglia in the mice of normal group was less than the model group and with slender nucleus and fine Dendron, low Iba-1 expression. The number of microglia, the cell volume, and the expression of Iba-1 in the model group were increased, compared with the normal group. The number of microglia, the cell volume, and the expression of Iba-1 in the Chinese medicine, hormone and PD98059 group were decreased, compared with the model group. Western blot results indicated that the expression of Iba-1 in the model group was increased compared with the normal group (P<0. 05), and in the Chinese medicine, hormone and PD98059 group was decrease vs the model group (P<0. 05).(4) The expression of p-c-Raf. p-MEK1/2 and p-ERK1/2 in the mice of model group were higher than normal group (P<0.05). The level of p-c-Raf in the mice of Chinese medicine, hormone and PD98059 group were higher than normal group(P<0. 05). The level of p-MEK1/2 in the mice of Chinese medicine, hormone and PD98059 group were lower than model group (P<0. 05, P<0. 05, P<0. 01), and PD98059 group was lower than normal group (P<0. 05). The expression of p-ERK1/2 in the mice of Chinese medicine, hormone and PD98059 group were lower than model group (P<0. 05). There were no difference among the normal, model, Chinese medicine, hormone and PD98059 group on the expression of Ras, c-Raf, MEK1/2 and ERK1/2.Conclusion:(1)The disorder of Thl and Th2 cells is involved in the relapse of EAE.Yishendaluo decoction may reduce the neurological function score, and alleviate inflammatory infiltration and demyelination of the mice with EAE by mean of decreasing the level of IL-2, increasing the level of IL-4 and regulating the dysfunction of Thl and Th2 cells.(2) Oligodendrocytes participate in remyelination that occurs in the mice with EAE. Yishendaluo decoction increases the level of Oligl and O1ig2 to enhance remyelination, in turn to alleviate neurological defictis.(3)The activation of microglial cells and ERK1/2 cell signal pathway are related to onset of EAE. Yishendaluo decoction may delay onset and reduce neurological deficits of EAE mice by inhibiting the activation of microglial cells and ERK1/2 pathway which inducing inflammatory responses. |
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