| Objective:In this study,female C57BL/6 mice were used to prepare experimental autoimmune encephalomyelitis?EAE?models.The drug delivery method was gavage of the solution of Wuzi Yanzong Pills?WYP?.By observing the clinical symptom of different dose groups of mice,to screen the best medicine dosage.We needed to observe pathological changes of different dose groups of mice as well as the expression of immune cells and inflammatory factors in the spleen and the central nervous system of these groups.The purpose of this study was to explore the efficacy and mechanism of WYP for the prevention and treatment of EAE,and to explore the difference of curative effect between the medium-term of onset and the end-stage of disease.Methods:Female C57BL/6 mice were randomly divided into blank control group,model group and high,medium and low dose group of WYP.One week before the start of modeling,the mice in the blank control group and the model group were given normal saline(50ml·kg-1·d-1).Meanwhile,the mice of the WYP high,middle and low dose groups were given WYP solution,the concentrations were 16g·kg-1·d-1,8g·kg-1·d-1and 4g·kg-1·d-11 respectively.The clinical score was recorded every day from the day of modeling.Half of the mice in each group were killed at day 14 after immunization,and the other half of the mice were killed at day 28 after immunization.The inflammatory cell infiltration and demyelination in spinal cord tissues of mice in each group were observed by pathological sections?eosin hematoxylin staining and luxol fast blue staining?.Flow cytometry was used to detect the splenic macrophage typing and the expression of IL10 and IL12 in the cells.The expression of IL10,IL17,IL12,TNF-?and IFN-?in the supernatant of splenocytes was detected by ELISA.qRT-PCR assay was used to detect the mRNA content of TNF-?and TLR-4 in spleen cells.The protein contents of ROCKII,P-MYPT1,TLR4,NF-?B,MCP-1 and CCR-2 in the brain,spinal cord and spleen were detected by western blot.Results:1.The effect of WYP on the curative effect of EAE model mice.1.1 The effect of WYP on the general signs and neurological function score of EAEmodel mice.?1?WYP could improve nerve function,improve muscular tension,enhance activity and alleviate general signs such as loss of appetite and mental depression.Compared with the blank control group,the mice in the model group had a significouldt decline in appetite,general signs were shown as depression,rough hair,yellow urine,dry shit,as well as severe weakness of the muscle tension of the extremities,activity disorder and even paralysis.Compared with the model group,the mice in WYP high dose group were able to have a good appetite,clear urine,normal stool,soft hair and a slight decrease in limb movement;the mice in the middle and low doses of WYP had poor appetite,yellowish urine,normal stool,rough hair and unfavourable extremities.?2?WYP could delay the onset of disease and reduce the degree of disease.Compared with the blank control group,the clinical score of the model group increased?p<0.05?;the anverage onset time and the anverage highest clinical score and the accumulative clinical score of the model group all increased?p<0.001?.Compared with the model group,the clinical scores of WYP high,middle and low dose groups decreased?p<0.05?,the anverage onset time and the anverage highest clinical scores and the accumulative clinical scores of WYP high,middle and low dose groups all decreased?p<0.05?.The high dose group had the longest onset time and the clinical score was the best.The dose effect relationship was not observed between the high,middle and low dose groups.The highest dosage of WYP was the best dosage.1.2 Effect of WYP on immune inflammatory response in EAE mice undermicroscope.WYP could inhibit inflammatory cells infiltrating spinal cord tissue.Compared with the blank control group,the content of the inflammatory cells of the spinal cord in the model group increased significouldtly?p<0.001?.Compared with the model group,the content of the inflammatory cells infiltrating the spinal cord in the WYP high dose group decreased significantl?p<0.05?.1.3 Effect of WYP on demyelination of spinal cord tissue in EAE mice undermicroscope WYP could reduce demyelination.Compared with the blank control group,the demyelination in the spinal cord of the model group was significantly increased?p<0.001?.Compared with the model group,the demyelination in the spinal cord was significantly reduced?p<0.05?.2.Study on the mechanism of WYP on EAE2.1 Effect of WYP on immune cell function for EAE mice.WYP could promote the expression of anti-inflammatory macrophages?M2?and enhance the expression of anti-inflammatory factors?IL10?.Compared with the blank control group,the expression of M2 type macrophage increased and the expression of anti-inflammatory factor IL10 increased in the model group.Compared with the model group,the expression of M2 type macrophage increased and the expression of anti-inflammatory factor IL10 increased?p<0.05?in the high dose group of WYP.2.2 Effect of WYP on inflammatory response for EAE mice.WYP could down regulate the expression of inflammatory cytokines such as IL17,IL12,IFN-?.Compared with the blank control group,the secretion of inflammatory factors IL17,IL12 and IFN-?in the splenocytes supernatant of the model group increased significantly?p<0.01?;Compared with the model group,the secretion of inflammatory factors IL17,IL12 and IFN-?in the splenocytes supernatant of the WYP high dose group decreased significantly?p<0.01?.WYP could down regulate the mRNA expression of TLR-4 and TNF-?.Compared with the blank control group,the mRNA expression of inflammatory receptor TLR-4 and inflammatory factor TNF-?in the splenocytes of the model group increased?p<0.01?;Compared with the model group,the mRNA expression of inflammatory receptor TLR-4 and inflammatory factor TNF-?in the splenocytes of the WYP high dose group decreased?p<0.01?.2.3 Effects of WYP on Rho/ROCK signaling pathway and NF-?B signaling pathwayin EAE miceWYP inhibited the activation of Rho/ROCK signaling pathway by inhibiting the activity of ROCKII receptor and decreasing the expression level of the substrate of phosphorylation.By inhibiting the activity of TLR4 receptor and inhibiting the expression of nuclear transcription factors,WYP could block the inflammatory reaction process.Compared with the model group,in the Rho/ROCK signaling pathway,the expression of ROCKII and P-MYPT1 protein in brain,spinal cord and spleen cells of WYP high dose group decreased significantly?p<0.05?;in NF-?B inflammatory signal pathway,the expression of TLR4 and NF-?B protein in brain,spinal cord and spleen cells of WYP high dose group decreased significantly?p<0.05?.Compared with the model group,the expression levels of chemokine?MCP-1?and chemokine receptor?CCR-2?in brain,spinal cord and spleen cells of WYP high dose group were significantly reduced?p<0.05?.Conclusion:WYP could improve the nerve function,relieve the clinical symptoms and delay onset time of EAE mice.WYP could regulate Rho/ROCK signaling pathway and NF-?B signaling pathway,reduce nerve tissue damage and inhibit the expression of inflammatory protein.WYP could promote the expression of anti-inflammatory macrophage cells and increase the expression of anti-inflammatory factors as well as reduce the expression of pro-inflammatory factors.Its mechanism may be related to the inhibition of inflammatory response and immunoregulation. |
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