| 1.Background and objectiveMultiple sclerosis(MS)is a chronic,self-reactive inflammatory disease of the central nervous system(CNS),which can eventually lead to demyelination and neuronal degeneration.Experimental autoimmune encephalomyelitis(EAE)is an autoimmune disease characterized by diffuse infiltration and demyelination of a large number of mononuclear cells around the small blood vessels in the CNS.It is a relatively recognized animal model for the study of MS.The pathogenic factors and the driving mechanism of MS are various and complex.In recent years,researches on immune microenvironment of CNS showed that microglia play an important role in the pathogenesis and progression of MS[1].Therefore,we focused on the regulation of microglia and changed the properties of immune microenvironment in the CNS,which had core and common value for the treatment of many CNS diseases,including MS.At present,the clinical treatment for MS is in the state of "being able to slow down or not to cure,treating the symptoms or not to cure the root".Under the strong contrast of "high incidence and difficult treatment",the disease mechanism understanding and treatment means of MS have become the common focus and application in modern immunology,neurobiology and new drug research development,which has clear scientific innovation significance and practical application values.Dihydroartemisinin(DHA)is a sesquiterpenoid compound.Its chemical formula is C15H24O5,molecular weight is 284.35,the physical and chemical properties are white crystals[2].With the development of research on DHA,the pharmacodynamic and pharmacological advantages of DHA are not only limited to the treatment of malaria,but also show great application prospects in anti-inflammatory and immune regulation Previous studies have found that DHA is effective in the treatment of immune diseases.especially for autoimmune diseases.It is preliminarily confirmed that DHA can inhibit inflammatory bowel disease(IBD)by regulating the balance of Th17/Treg cells.However,there are some gaps in the research of key scientific issues:(1)the common mechanisms of DHA in the regulation of immune inflammation and the disease spectrum are unclear,especially its application in the regulation of autoimmune inflammation in the CNS is lack of systematic research;(2)the balanced regulation of T cell by DHA is unclear and its regulation of immune inflammatory microenvironment is lack of systematic interpretation release and experimental description[2];the potential molecular mechanism of immunomodulatory activity of DHA is unclear and the specific molecular target and related signal pathway are lack of detailed analysis and disclosure.Based on the above analysis,the comprehensive and systematic study of DHA from three aspects of efficacy evaluation,immune function disclosure,and molecular mechanism exploration is to improve the level of DHA drug understanding and promote the rational and effective application of DHA Key research content.As we all know,the balanced regulation of T cells is a complex and huge system,since the integration of "APCs-T cells" unit plays an important role in the balanced regulation of immune function of T cells,which is the core pathological basis for the initiation and sustained activation of autoimmune inflammatory damage.However,based on above theory,the research of DHA is still blank.Therefore,in this paper,combined with the pharmacodynamic characteristics of DHA and the pathological characteristics of MS,a systematic EAE pharmacodynamic evaluation system was established to carry out a comprehensive DHA pharmacodynamic evaluation.Based on the infiltration and activation of microglia in the antigen-presenting cells of the CNS,the research strategy of immune activity based on the interaction of microglia-T cells was established to expand the knowledge of inflammatory regulatory activity of DHA The functional changes of microglia in CNS inflammation were taken as the starting point to excavate and study the mechanism,improve the molecular pharmacological mechanism of "point surface aggregation",clarify the molecular mechanism and activity characteristics,and explore the role of DHA in the remodeling of EAE pathological environment.2.Methods and contents1.The pharmacodynamic effect of DHA in EAE model mice(1)We established the EAE animal model with oligodendrocyte glycoprotein recombinant(MOG35-55).DHA 2mg/kg/day,10mg/kg/day,20mg/kg/day were used as low,medium,high dose and methylprednisolone lmg/kg/day as positive drugs.The mice were administrated by gavage on the second day after modeling.The normal control group and the model group were given equal doses of solvent.The state changes of mice were observed regularly every day.RRMS animal model:after 23 days of continuous administration,the behavioral changes of mice were detected by gait analysis system.After behavioral test,the brain and spinal cord were taken out for histological test(LFB and transmission electron microscope test).Serum was separated and cerebrospinal fluid was taken out from the other part of mice.SPMS animal model:after 53 days of continuous administration,behavioral changes of mice were detected by gait analysis system.After the behavioral experiment,Oligo 2 expression in brain and spinal cord tissues was detected by immunohistochemistry.(2)After behavioral detection of RRMS mice,the brain and spinal cord tissues were taken out for histological detection.After 4%paraformaldehyde was fixed,paraffin embedded,sectioned and stained with H&E to detect the degree of infiltration of inflammatory cells.The MNCs were isolated and the mRNA expressions of TNF-?,IL-1?,IL-6,IL-10 and TGF-? in MNCs were detected by qRT-PCR2.Studying on the anti-inflammatory immune activity of DHA in EAE model mice(1)Single cells were isolated from the CNS of mice,with the number of cells to a concentration of 1 × 106/ml;10x labeling of cDNA fragments,building a database,sequencing,and obtaining sequencing data.According to the top 10 principal components of PCA results with the largest explanatory variance,t-SNE(t-distributed stochastic neighbor embedding)was used to visualize the single cell cluster,and then the cell subsets and differential genes related to EAE diseases were obtained(2)RAW264.7 cells and BV2 cells were cultured in vitro,PM was isolated in vitro.The cells were treated with DHA 1 and 4 ? m for 24 hours.First,RT-PCR(reverse transcription polymerase chain reaction)was used to detect the expression of co-inhibitory signal PDL1 in RAW264.7 and PM cells;indirect immunofluorescence was used to detect the protein expression level of PDL1 on RAW264.7,BV2 and PM cells;jurkat and BV2 cells were used to establish a co culture model in vitro,and the differentiation level of Treg cells(Foxp3 expression)was detected in jurkat cells by DHA in the co culture system by flow cytometry[2]The expression of CCL5 was detected by qRT-PCR and concentration of and CCL5 in serum and cerebrospinal fluid from EAE model mice by ELISA.In chemotaxis experiment,the concentration of CCL5 in the supernatant of BV2 culture was detected by ELISA,and the chemotaxis ability of CCL5 to microglia was detected by transwell(4)In the phagocytosis assay,PC 12 cells were cultured in DMEM medium for 48h without FBS,annexin V-FITC for 48h,indirect immunofluorescence for PC 12 cells The phagocytosis of apoptosis PC 12 cells by BV2 cells was observed by fluorescence microscopy.3 Focusing on the molecular mechanism of "microglial function regulation" of DHA inhibiting inflammation in CNS.(1)In order to verify the dependence of DHA on AXL in molecular mechanism level,we used SGI7079(0.2 ?m),the inhibitor of AXL,to block the activation of AXL.First,we used BV2 cells to phagocytize apoptotic PC 12 cells to detect the change of phagocytic capacity of BV2 cells;the chemotaxis of CCL5 on mouse microglia BV2 was measured by transwell assay.Finally,the co-culture model of macrophage-t cells was established by jurkat and mouse microglia BV2.The differentiation level of Treg cells in jurkat cells(Foxp3 expression)was detected by indirect immunofluorescence(2)Western blot was used to detect the expression of AXL,phospho-AXL,SOCS3 and phospo-STAT1 in BV2 cells treated with DHA and LPS.On the basis of the above study,the expression of AXL,phospho-AXL,SOCS3 and phospo-STAT1 were also detected after sgi7079 was added.[2]After treated with DHA 1 and 4 ?M for 24 hours,the expression of PDL1,Foxp3 and CYSE/F480 in BV2 cells were detected by indirect immunofluorescence,the content of CCL5 in the supernatant of BV2 cells by ELISA and the chemotaxis of BV2 cells by transwell.After BV2 cells were treated with IFN-? and fludarabine,the expression of CYSE+/F480+was detected by indirect immunofluorescence method,and the protein expression of phospo-STAT1,SOCS3,AXL and phospho-AXL were detected by Western blot.3.Results3.1 The pharmacodynamic effect of DHA on EAE model miceModel validation:under the induction of MOG35-55,the clinical score increased and mean weight reduced gradually.Histological and ultrastructural studies of the spinal cord showed that demyelination was accompanied.The above results show that the EAE model is successful and meets the experimental requirements for drug evaluation.1.1 DHA has an effect of behavioral function improvement and neuroprotection in EAE mice.The efficacy validation of RRMS animal model:RRMS is mainly at the early stage of the onset of the disease,which is dominated by CNS inflammation and accompanied by demyelination.Therefore,inflammation damage and neuroprotection are detected.Compared with the EAE model group,DHA administration groups and met group can improve the skill activity disorder of EAE model mice.At the histological level,it can protect the myelin sheath.On the level of neuroprotection,the effect of middle dose DHA was better than that of met group.The efficacy validation of SPMS animal model:SPMS is the late stage of the disease,mainly due to the inactivation of oligodendrocytes,leading to the loss of myelin sheath,and oligo 2 is the active marker of oligodendrocytes,so the activity of oligodendrocytes can be reflected through expression of oligo 2.Compared with EAE model mice,DHA administration groups and MET group can improve the functional activity of EAE model mice.At the histological level,the positive expression rate of oligo 2 was dose-dependently increased in each DHA dose group and met group1.2 DHA can inhibit inflammation of central nervous system in RRMS miceH&E results showed that DHA and MET significantly reduced the infiltration level of inflammatory cell in brain and spinal cord;DHA could down-regulate the mRNA expression of TNF-?,IL-1?,IL-6 in each administration group and MET group,on the contrary,the transcription of IL-10,TGF-? was significantly activated;meanwhile,DHA could dose-dependently balance the activity of inflammatory response and in the high-dose group was better than the MET group.2.The study of DHA on the anti-inflammatory and immune activities in EAE model mice2.1 Based on the analysis of mononuclear cell subpopulation in central nervous system of EAE mice regulated by DHA by 10x genomics TechnologyCell subsets analysis:14 cell subpopulations were obtained,and the two types of cell subpopulations most related to the process of EAE disease model were macrophages and microglia.Differential Gene analysis:a total of 305 base heterogenes regulated by DHA were screened out from 14 cell subsets of EAE model mice,while 25 genes with significant difference(foldchange>2)were enriched in macrophages and microglia.Finally,through literature mining and molecular verification,the candidate genes AXL and CCL5,which have the highest concentration and are closely related to the disease process of EAE,are selected for further pharmacological molecular mechanism research.At the same time,based on the previous research tips and the latest progress of immunology research,the "microglia-T cell integrated regulatory unit" is focused,from the double signals presented by antigens Start with the system and carry out pharmacology research from another angle.2.2 The study of the drug activity of DHA on the presentation of autoimmune antigens.DHA significantly increased the expression of PDL1 on the cell surface of RAW264.7 and peritoneal macrophages in the molecular level;when come to functional level,DHA can also promote the differentiation of jurkat cells into Treg cells,and up regulate the expression of Treg surface marker Foxp3.2.3 The drug activity of DHA in autoimmune chemotaxisIn vivo experiment,qRT-PCR results showed that DHA dose-dependently inhibited the expression of CCL5 at transcription level in MNCs,which was better than MET group.The ELISA results showed that DHA could reduce the CCL5 concentration in serum and cerebrospinal fluid of EAE model mice.In vitro,the results of ELISA showed that LPS could increase the content of CCL5 in the culture supernatant of BV2 cells,while DHA could reduce the content of CCL5;in the test results of transwell,DHA could weaken the chemotaxis of BV2 cells.2.4 The activity of DHA in the clearance of autoinflammatory cytokinesIn vivo experiments,qRT PCR results showed that DHA and MET could up regulate the expression of AXL in MNCs.The results of Western blot showed that DHA could significantly up regulate the expression of AXL in BV2 cells.At the same time,the results of fluorescence microscopy and flow cytometry showed that DHA could successfully and effectively promote the phagocytosis of microglia to apoptotic cells and the clearance of apoptotic cell fragments.3.Focusing on the molecular mechanism of "microglial function regulation" of DHA in inhibiting inflammation of central nervous system3.1 the mechanism of DHA targeting AXL based on microglial function system.Functionally,we found that after BV2 cells were treated with SGI7079,AXL inhibitor,indirect immunofluorescence showed that the ability of DHA to present autoimmune antigens could be eliminated by SGI7079.Through fluorescence microscopy and indirect immunofluorescence detection,we found that the scavenging ability of DHA to autoimmune inflammatory cytokines was also inhibited by SGI7079;transwell results showed that the ability of DHA to autoimmune inflammatory chemotaxis was also blocked by SGI7079.3.2 The molecular pathway of DHA based on Axl regulating autoimmune inflammationIn the study of the molecular pathway level of regulation of autoimmune inflammation by DHA based on AXL,western blot results showed that DHA could up regulate the expression of Total-AXL,SOCS3,and phosphor-STAT1 respectively,the difference is statistically significant.When BV2 cells was added to SGI7079,the expression of other phospho-AXL,SOCS3 and phosphor-STAT1 proteins were inhibited.3.3 Preliminary study on the pathological state specificity of DHA in the regulation of "pro-inflammatory" balanceThe results of indirect immunofluorescence showed that DHA alone had no significant difference in the presentation of autoimmune antigens,the chemotaxis of autoimmune inflammation and the clearance of autoimmune inflammatory factors compared with the normal group;The indirect immunofluorescence results showed that DHA could increase the number of CYSE+/F480+subsets and enhance the phagocytic ability of BV2 cells after IFN-? treatment,while the phagocytic ability of BV2 cells disappeared after fludarabine treatment.Western blot results showed that DHA could up regulate the expression of phosphor-AXL,SOCS3 and phospso-STAT1 protein under the effect of IFN-?.When fludarabine was added,DHA had no significant difference in the expression of these proteins.4.conclusions1.DHA could improve the clinical score and behavioral disorder in EAE mice,has the function in neuroprotection and regulation of inflammation at the histological level,and then alleviate the central nervous system inflammation of EAE mice;2.Macrophages and microglia in the central nervous system are the main effector cells of DHA in EAE model.Meanwhile,the changes of AXL,CCL5 and other related inflammatory molecules in microglia constitute the main molecular basis for DHA to exert its efficacy;3.DHA plays a systematic and diverse regulatory role in antigen presentation,inflammatory chemotaxis and antigen clearance of autoimmune inflammation in the central nervous system,and finally realizes the balance remodeling and myelin sheath protection of autoimmune inflammation.They are summarized as follows:(1)DHA can regulate the process of antigen processing and presentation,reduce the high response of T cells to their own antigens.It can promote the surface expression of PDL1,reverse the overactive state of autoimmune inflammation in the central nervous system,and achieve the remodeling of T-cell inflammatory suppressor balance(Th 17/Treg);(2)DHA could reduce inflammatory chemotaxis,recruitment level and infiltration of inflammatory cells in the central nervous system.DHA could also significantly inhibit the expression and secretion of inflammatory chemokine CCL5,reduce the infiltration of inflammatory cells in the site of inflammatory injury,reduce the motor chemotaxis of inflammatory cells,and then delay the progress of inflammatory injury;[2]DHA could enhance the effective clearance of microglia to injured myelin sheath and promote the active dissipation of inflammatory process.DHA could significantly and specifically up regulate the expression of phagocytic receptor AXL,enhance the clearance ability of apoptotic cell fragments,reduce the exposure of autoimmune inflammatory factors,and weaken the induced level of inflammatory response;4.The regulatory activity of DHA on autoimmune inflammatory response focused on the effective regulation of AXL as a molecular target and depends on the high-level activation of IFNAR-STAT1-SOCS3 signaling pathway.These molecular mechanism studies also provide preliminary experimental hints for the pathological selectivity and efficacy specificity of DHA in the treatment of autoimmune inflammation... |
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