| Objective:Multiple Sclerosis(MS)is an inflammatory neurodegenerative disease mediated by the central nervous system autoimmune response.Traditional Chinese medicine will be classified as "Flaccid Syndrome",the symptoms of the disease for more sensory abnormalities,limb weakness and visual impairment.MS tends to occur in young women.Because of MS complicated clinical manifestations,high disability rate,and the lack of specific auxiliary diagnostic indicators,there are difficulties in diagnosis and treatment.In this paper,the theory of Etiology and pathogenesis of MS is studied both theoretically and experimentally.In the aspect of theoretical research,the mechanism of Multiple Sclerosis is discussed from the perspective of "Brain Marrow"theory,and the law of drug use in treatment group is explored through literature.The basic pathogenesis characteristics of kidney deficiency and essence deficiency,phlegm-dampness-heat-stasis,turbid-toxin and blocking brain collaterals are expounded and produced "benefit kidney,detoxicate,Tongluo" the rule of law train of thought.Then the target of network pharmacology and experimental research were carried out.In experimental studies,astrocyte plays an important role in the repair of damaged neurons and axon regeneration in multiple sclerosis.How to reduce the multiple sclerosis nerve damage and glial scar formation,promote the repair and so on are the key issues in the field of nerve regeneration.Astrocytes are an important link in the release of inflammatory factors from the central nervous system,the formation of glial scars,and the secretion of Neurotrophin.Based on this,this study induced astrocyte activation in an inflammatory environment to investigate whether the method of invigorating kidney,detoxifying and dredging collaterals plays a role on the signal transduction pathway of Adenosine a 1 membrane receptor(AJAR)and protein kinase a/cAMP response element protein(PKA/cAMP/CREB)by β-arrestin1(Arrb1).This study provides a theoretical basis for the study of the mechanism of reactive astrogliosis and inflammatory microenvironment in the treatment of multiple sclerosis by tonifying kidney,detoxicating and dredging collaterals.Method:1.Taking "Multiple Sclerosis"as the theme,CNKI was counted to treat multiple sclerosis in the past 10 years,and then through the auxiliary platform of Traditional Chinese Medicine Inheritance(TCMISS V2.5)to drug frequency,sex and taste channel,potential drug combination data mining.2.The treatment of multiple sclerosis with Yishendaluo decoction was analyzed by the method of Internet pharmacology.Using TCMSP database and GeneCards,OMIM,PharmGkb,TTD and Drugbank database,the active components,targets and multiple sclerosis targets of Yishendaluo decoction were obtained.Using cytoscape 3.8.0,STRING,CytoNCA,R software,the active components-target Gene Network,PPI network,GO,KEGG enrichment analysis results were obtained and verified by molecular docking.3.Firstly,the primary astrocyte was extracted from suckling mice,and the Glial Fibrillary Acidic Protein(GFAP)was identified by Immunofluorescence staining.The effect of Lipopolysaccharide(LPS)on the growth activity of astrocyte cells was detected by CCK-8 method.The expression and secretion of astrocyte IL-1β and TNF-α were determined by Elisa.The expression of GFAP and GDNF was detected by Western blotting,and the inflammatory model induced by LPS was established.4.The lentivirus-silenced Arrb1 gene(RNAi-Arrb1)was cloned and the expression of Arrb1 and histone H4 was detected by Western blotting.5.The expression of GFAP,PKA cAMP Creb,GDNF,Arrb1 and histone H4 was detected by immunofluorescence and Western blotting after LPS stimulation of primary cultured astrocyte cells in vitro.RESULT:1.The literature on the treatment of Multiple Sclerosis with traditional Chinese medicine compound prescription has the largest proportion of drugs for invigorating Qi,and the results of Meridian Tropism of nature and taste have the largest proportion of drugs for warming nature,sweet taste,regulating the liver,spleen,kidney and stomach,pay attention to supplementing Qi and nourishing blood,nourishing liver and kidney,strengthening spleen and resolving phlegm,warming Meridians and dredging collaterals.2.There are 115 active components in Yishendaluo decoction,225 predicted targets,8098 multiple sclerosis targets and 199 overlapping targets.The biological process mainly focuses on inflammatory reaction and oxidative stress reaction,which is closely related to IL-17 signal pathway and TNF pathway.The results of molecular docking show that there is a strong binding activity between the core active components and the core target.3.Morphological observation of the primary astrocyte:The Primary Cultured astrocyte showed aggregative growth,the edges were not easy to distinguish,and were polygonal.Then gradually began to spread,the cell body increased,part of the cell processes extension,spindle-shaped,pseudopodia increased,the cells are more transparent,glial cells basically full bottle bottom,3 generations of astrocyte can be used for the next experiment.Immunofluorescence assay:The astrocyte protein GFAP shows a positive cell count of more than 95%,which can be used in the following experiments.1 mg/mL LPS for 24 h induced astrocyte activation.Compared with the blank group,LPS showed an increase in astrocyte activity,but there was no significant difference(P<0.05).LPS could increase the expression of IL-1β and TNF-α(P<0.01),increase the expression of GFAP Protein(P<0.05),and decrease the expression of GDNF(P<0.05).The High Dose Group of Yishendaluo decoction could significantly decrease the expression of IL-1β and TNF α induced by LPS(P<0.01),decrease the expression of GFAP Protein(P<0.05)and increase the expression of GDNF Protein(P<0.01).4.Arrb1 is distributed in both cytoplasm and nucleus of astrocyte.In the inflammatory environment,the Arrb1 protein expression increased with the increase of Arrb1 distribution in the nucleus(P<0.05).Yishendaluo decoction may regulate histone H4 expression by reducing Arrb1(P<0.05).5.The shRNA-Arrb1 lentivirus has been shown to reduce the astrocytes Arrb1 expression(P<0.05).The shRNA-Arrb1 lentivirus silencing astrocytes Arrb1 reduced histone H4 expression(P<0.05).Yishendaluo decoction had no significant effect on the expression of H4 of shRNA-Arrb1 lentivirus.6.In inflammatory environment,the levels of cAMP,PKA,p-CREB protein and A1AR were decreased P<0.05),and the expression of astrocyte was decreased(P<0.05).The expression of cAMP,PKA,Creb,p-CREB and A1AR increased in the high dose group of Yishendaluo decoction.Conclusion:1.The traditional Chinese medicine compound prescription in treating multiple sclerosis pays attention to the method of supplementing Qi and nourishing blood,nourishing liver and kidney,strengthening spleen and resolving phlegm,warming Meridians and dredging collaterals.2.Through the analysis of Network Pharmacology,it is concluded that Yishendaluo Yin exerts anti-inflammatory and regulating immune responses through Pi3k-akt,MAPK,IL-17,TNF-α,camp and other key targets such as MAPK,Akt,TNF.3.In Vitro,Yishendaluo decoction could improve the activation of astrocyte,decrease the release of IL-1 B and TNF-A,decrease the expression of GFAP,increase the expression of GDNF and improve the glial response.4.Yishendaluo Yin may affect the expression of histone H4 and histone Epigenetics by reducing Arrb1 in the activated astrocyte.5.Yishendaluo decoction can promote the expression of astrocyte/cAMP/CREB signal transduction pathway. |
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