Study The Mechanism Of Yishen Daluo Decoction On Experimental Autoimmune Encephalomyelitis Mice By β-arrestin1

Objective:Based on the method of Yishen Jiedu Tongluo,taking Yishen Daluo Decoction as the research objection and β-arrestin1 as the research target,to explore the mechanism of Yishen Daluo Decoction on experimental autoimmune encephalomyelitis(EAE)from the theory of Zang Xiang,immune imbalance and histone acetylation.The aim of this study is to explore the mechanism of Yishen Daluo Decoction in the treatment of EAE.Methods:1.By means of literature integration to study the theoretical basis of Yishen Daluo Decoction in treating Multiple sclerosis(MS).2.Adeno-associated virus(AAV)was used as a vector to package β-arrestin1 si RNA,which was injected into mice to inhibit the expression of β-arrestin1 in vivo.3.Female C57BL/6J mice were divided into blank group,model group,AAV-βgroup,traditional Chinese medicine(TCM)group,AAV-β+TCM group and Western medicine(PA)group.EAE models were made except the blank group.Body weight and clinical score were evaluated dayly.Seven days after modeling,mice in blank group,model group and AAV-β group were perfused with 0.9% sodium chloride solution,mice in TCM group and AAV-β+TCM group were perfused with Yishen Daluo decoction,and mice in PA group were perfused with prednisone acetate,the samples were collected after 14 days of continuous gavage.4.HE staining and LFB myelin sheath staining were used to observe the pathomorphological changes of central nervous system(CNS).5.The contents of IL-2,IL-23,IL-27,IL-35,IFN-γ in serum and the contents of ATP in erythrocyte were measured by ELISA.6.The ratio of Th17/Treg cells in spleen was measured by Flow cytometry method.7.The expressions of β-arrestin1,A1 AR,P2Y1,GDNF,GFAP,P53,H3K9 ac,H4K5ac and HDAC1 in CNS were detected by Western Blot.8.The expressions of β-arrestin1,A1 AR,P2Y1 in CNS were detected by immunohistochemistry.Results:1.MS lies in the dysfunction of zang-fu,mainly in the deficiency of kidney essence and deficiency of liver and spleen.It affects the heart and lung for a long time,and all the zang-fu organs participate in the development and pathogenesis of MS.2.The changes of neurological function scores:no neurological deficit was found in the blank group,but irritability was found in the other groups on the 4th day,and the onset began on the 10 th day and reached the peak on the 18 th day.Compared with model group,AAV-βgroup,TCM group,AAV-β+TCM Group and PA group were significantly decreased.3.Results of HE staining:most of the neurons in the model group had severe cellular edema,cellular body shrinkage and deep staining,and inflammatory cell aggregation.Compared with model group,the pathological changes were milder in TCM Group,PA group,AAV-β group and AAV-β+TCM group,but no abnormality was found in blank group.Results of LFB staining:the demyelination in model group was more severe than that in the other groups,the degree of demyelination in AAV-β group was more serious than that in TCM group,AAV-β+TCM Group and PA group.4.Compared with blank group:the expression of IL-2,IL-23,IL-27,IFN-γ in model group was higher,and the expression of IL-35 was lower compared with model group,the levels of IL-2,IFN-γ,IL-23,IL-27 were decreased and IL-35 was increased in TCM group.ATP content in erythrocytes:compared with blank group,the model and AAV-β group decreased.Compared with model group,the ATP in TCM,AAV-β+TCM,PA group increased.5.The ratio of Th17/Treg cells:compared with blank group,the ratio of Th17/Treg cells in model group increased,while the Treg cells decreased.Compared with model group,the levels of Th17 in AAV-β,TCM,AAV-β+TCM and PA group were lower,while the levels of Treg in PA group and AAV-β+TCM group were higher.6.Compared with blank group,the levels of β-arrestin1,P2Y1,GFAP,P53,HDAC1,H3K9 ac,H4K5ac,in model group increased,while A1 AR and GDNF decreased.Compared with model group,the levels of β-arrestin1,P2Y1,GFAP,P53,HDAC1,H3K9 a,H4K5ac in AAV-β group,TCM group,AAV-β+TCM Group,PA group decreased.A1 AR and GDNF were increased.The above datas were statistically significant mostly(P<0.05,P<0.01).Conclusion:1.MS has dysfunction of five zang-organs,mainly deficiency of kidney essence,deficiency of liver and spleen,and deficiency of heart and lung.All the zang-fu organs participate in the development and pathogenesis of MS.2.By inhibiting β-arrestin1 in EAE mice,Yishen Daluo Decoction could alleviate the inflammatory infiltration and demyelination of CNS,regulate the ratio of Th17/Treg,inhibit the expression of inflammatory factors and promote the secretion of anti-inflammatory factors,therefore the neurological deficits of EAE mice were ameliorated.3.Yishen Daluo Decoction activates A1 AR receptor,inhibits P2Y1 receptor,increases ATP level,promotes the secretion of Neurotrophin GDNF,promotes nerve regeneration and improves energy metabolism in EAE mice.4.Yishen Daluo Decoction can improve the clinical symptoms of EAE by inhibiting the expression of GFAP,P53,HDAC1,H3K9 AC,H4K5AC in CNS,regulating histone acetylation and inhibiting the activation of astrocytes.