| Multiple sclerosis is a common and unpredictable demyelinating in the central nervous system as the main pathological changes of Inflammatory diseases in the nervous system. MS is autoimmune diseases based on genetic predisposition and environmental factors. Its incidence is higher, a chronic course and high morbidity, tend to suffer from in young adults, physical and cognitive function can damage at the same time. Multiple sclerosis had brings huge economic and social burden with long-term and the serious Influencing the patient’s life. According to incomplete statistics,the prevalence of MS in the world’s population is approximately2.5million. Epidemiological investigation in our country there is no exact statistics,but learn from the literature and case reports, the incidence of Multiple Sclerosis in China is also gradually rising trend.ObjectiveThe experiment proposed by comparative study to observe the effect WenDan decoction on rats with experimental autoimmune encephalomyelitis, improve the clinical symtoms, the course of immune regulation, and other aspects of pathology encephalomyelitis. To explore WenDan decoction in the treatment mechanism and function of Multiple Sclerosis, beneficial exploration for the clinical treatment of MS.Methods1Randomly assigned to40SPF level only six to eight weeks of female Wistar rats were divided into5groups of eight. Group A is the normal control group, and group B is the model group, while the group C is the western medicine treatment group, group D is the traditional Chinese group, group E is the combination treatment group.2To build the EAE model using the immunization for two times.3Filled in the day one:As the normal control group, group A filled the normal saline irrigation during24h intervals for16days. As the model group, group B filled the normal saline irrigation during24h intervals for16days. As the western medicine treatment group, group C filled the Prednisone acetate(1mg/kg) irrigation during24h intervals for16days. As the traditional Chinese group, group D filled the WenDan decoction (11.88g/kg) irrigation during24h intervals for16days. As the combination treatment group, group E filled the WenDan decoction (11.88g/kg) and Prednisone acetate(lmg/kg) irrigation during24h intervals for16days.4In immunization day17executed, executed in rats with excessive anesthesia and collecting specimens, and observe the behavior of rats, histopathological, immunohistochemical for TLR9, IFN-γ and IL-2content by ELISA detection, check the neutrophils of TLR2,4by QPCR.Results1Group A rats do not appear the clinical symptoms. The rats in group B began at the onset in the first two days, the symptoms of central nervous system damaged gradually appear in the first three days,10st days reach the peak of incidence, the incidence rate of8/8, and the mortality rate was1/4. The rats in group C began at the onset in the first two days, the symptoms of central nervous system damaged gradually appear in the first three days,12st days reach the peak of incidence, the incidence rate of8/8, and the mortality rate was1/4. The rats in group D began at the onset in the first two days, the symptoms of central nervous system damaged gradually appear in the first five days,13th days reach the peak of incidence, the incidence rate of8/8, and the mortality rate was1/8. The rats in group E began at the onset in the first two days, the symptoms of central nervous system damaged gradually appear in the first five days, the incidence rate of3/4, and the mortality rate was0/8.2Group A is asymptomatic after treatment, while the highest clinical symptoms of neurological function scores is group B. Compared with group A, the differences of group B, C, D and E was statistically significant (P<0.05); compared with group B,the differences of group C and E was statistically significant (P<0.05);and compared with group C, the differences of group B, D and E was statistically significant (P<0.05); compared with group D and E, the difference was statistically significant (P<0.05).3Compared the incubation period, group A is normal, group B is the shortest and the fastest, while group E is the longest and the slowest. Compared with group B, the differences of group D and E was statistically significant (P<0.05); compared with group C, the differences of group D and E was statistically significant (P<0.05);Compared with group D and E, the difference there was statistically significant (P<0.05); compared with group B and C showed no significant difference (P>0.05).4Compared with the peak time of each group after treatment, group A is normal, and group B is the fastest. While the group E has the longest incubation period, the rats has just be onset when the end of the experiment, it will not be discussed in group E. Compared with group B, the differences of group C and D were statistically significant (P<0.05);Compared with group C and D, the differences were statistically significant (P<0.05).5The normal control group did not appear the clinical symptoms;but the model group and the western medicine treatment group and the traditional Chinese group are all onset. There are six rats get sick in western medicine treatment group. The model group and western medicine treatment group has died two rats in each group; the traditional Chinese group has died one;the normal control group and the combination treatment group were no dead rats.6Weight of Rats in each group was no statistically significance on the day began the experiment (P>0.05). Compared with the weight of the rats on the10st and16st days, group A is the heavest, group B and C are the lightest. Compared with the group A, the differences of group B, C, D and E were statistically significant (P<0.05); compared with group B, the differences of group D and E were statistically significant (P<0.05);compared with group C, the differences of group D and E were statistically significant (P<0.05);there was no statistically significant difference compared with the group B and C.(P>0.05);there was no statistically significant difference compared with the group D and E(P>0.05). Compared with the weight of the rats on the10st and16st days, the differences of each group were statistically significant (P<0.05), and the weight of the16st day were heaver than the10st day. 7HE staining:The slices of brain and spinal cord of group A were normal. The slices of brain and spinal cord of group B was found small blood vessels to dilate in the white matter, while some of the small vessel wall get thickening, with a large number of IFNlammatory cell surrounded the blood vessels, which IFNiltrated of lymphocytes and neutrophils IFNiltrating cells, formed the "sleeve" shape change. The structure of "sleeve" appear in the white matter and gray and white matter junction of spinal cord commonly, IFNlammatory IFNiltration are mainly distributed in the white matter forefoot, gray and white matter junction of spinal cord and also in the median crack around the blood vessels. And the location and extent of IFNlammatory IFNiltration are closely related to the clinical symptoms. The white matter structure is loose, showing vacuolar changes, and the change of the brain parenchyma is mainly distributed in the ventricle and white matter, gray matter are basically not affected. Neurons showed no lesions. Compared with group B, The structure of "sleeve" of group C and D are reduced, the IFNlammatory cells around the vascular are significantly reduce. There can see much more IFNlammatory cell IFNiltration in the white matter. The structure of "sleeve" of the brain and spinal cord white matter in group E are significantly reduced. And it did not find obvious IFNlammatory cell IFNiltration around the vascular. There are scattered IFNlammatory cell IFNiltration in the white matter.8Demyelination stain:The white matter myelin of spinal cord of group A are stained blue uniform, it shows that is not demyelination. The spinal cord white matter of group B appears focal or flake demyelination, axonal swelling or even disappear, blue myelin staining region decreased lighter and the color became shallow, which shows the demyelination. Groups C, D and E demyelinating ease, blue myelin stained area are increased, the most obvious is group E.9Immunohistochemistry:The Positive cells of TLR9have brown granules located in the cytoplasm and cell membrane. Each layers of cerebral cortex cells of the rats in group A, which content the surface layer of cells, such as molecules, the external granular layer and the outer layer of pyramidal cells can almost not find the coloration, indicating that the neutrophils of TLR9is little. Deep in the cerebral, as the granular layer and the pyramidal cell layer, which are colouration in astrocytes,there are also in pyramidal cells, but colors are pale, were weakly positive. TLR9neutrophils of cerebral cortex, which content the molecular layer, external granular layer, the outer layer of pyramidal cells and polymorphonuclear cell layer in group B were increased significantly than the normal group. The number and the positive strength of TLR9positive cells in group C and D were increased than group B. Compared to the group B, the area and color intensity of TRL-9-Positive cell in group E declined significantly.10After the treatment, the highest of the neutrophils of IL-2is group B. Compared with group A and B, the difference was statistically significant (P<0.05). The differences of groupA, C, D and E showed no significant difference (P>0.05).11After the treatment, the highest of the neutrophils of IFN-γis group B. Compared with group A and B, the difference was statistically significant (P<0.05). The differences of group A, C, D and E showed no significant difference (P>0.05).12After the treatment, the lowest of the neutrophils of TLR-2is group A, and the highest is group B. Compared with group A, the differences of group B and C were statistically significant (P<0.05). Compared with group B, The differences of groupA, C, D and E was statistically significant (P<0.05). The differences of group A, D and E showed no significant difference (P>0.05).13After the treatment, the lowest of the neutrophils of TLR-4is group A, and the highest is group B. Compared with group A, the difference of group B and D was statistically significant (P<0.05). Compared with group B, The differences of groupA, C, D and E was statistically significant (P<0.05). The differences of group A, C and E showed no significant difference (P>0.05).Conclusions1Wistar rats were injected through the secondary immune guinea pig spinal cord homogenate EAE rats can be successfully produced.2EAE rats’general condition is poor weight loss, obviously damaged nervous system symptoms.3EAE rats’incubation period is short, fast and reached a peak after the onset of clinical symptoms, IFNlammatory cells IFNiltration and demyelinating in central nervous system, brain tissue IFNlammatory cytokines IL-2, IFN-gamma and TLR2,4,9have increased.4Wendan decoction can reduce morbidity and mortality of EAE rats, prolong its prolong its incubation period, prolonged clinical symptoms reached a peak time, while reducing the peak of clinical symptoms, reducing Encephalomyelitis disease cell IFNiltration and demyelination, indicating WenDan decoction on the incidence of EAE rats have a preventive effect.5WenDan decoction can reduce mRNA neutrophils in rat IL-2and IFN-gamma.6WenDan decoction can reduce mRNA neutrophils in rat TLR2,4,9.7WenDan decoction may inhibit antigen presentation DC Toll-like receptors by inhibiting Thl cells proliferation and Thl cells secrete IFNlammatory. Inhibition of EAE abnormal immune response cells from Multiple links, inhibits the body’s cells cytotoxic T cells, NK, activation of macrophages, monocytes, B cell, regulate the neutrophils of IFNlammatory cells and activation of the medium, to break the amplification of IFNlammation and sustained positive feedback loop, and to suppress the occurrence of reversal of EAE and development.8Combined using WenDan decoction and prednisone acetate to treatment EAE rats can be effected better. |
