The Efficacy And Mechanism Study Of DHA In Treating Secondary Progressive Multiple Sclerosis By Promoting Remyelination

Background and objectiveMultiple Sclerosis(MS)is a demyelinating disease of the central nervous system caused by autoimmune inflammation,which is an important cause of acquired non-traumatic disability in young people.In the past twenty years,with the development of immunotherapy,inhibit inflammation,reduce the recurrence of the disease,but not enough to prevent axon and neuronal damage and loss caused by the accumulation of permanent disability,especially in the progress of the disease stage,it also led to a research strategy to focus on how to prevent neural degenerative diseases,in order to avoid the progressive MS disability.Therefore,for MS with demyelination and myelin regeneration disorders,especially secondary progressive multiple sclerosis(SPMS),it is the most direct and effective way to improve patients' quality of life to break through the myelin repair barrier and achieve the improvement of patients'function.The repair of myelin sheath mainly depends on Oligodendrocyte precursor cells(OPCs)to proliferate,migrate and differentiate into new Oligodendrocyte(OLs)immediately after myelin sheath injury,thus initiating the process of myelin regeneration.In this process,promoting the effective differentiation of OPCs into mature oligodendrocytes is the main way to promote myelin regeneration.M2-type Microglia(MG)can promote the inflammation in the lesion site by secreting IL-4,IL-13,TGF-? and other factors,and maintain the microenvironment for the survival and differentiation of OPCs.MG also supports the proliferation and differentiation of OPCs by secreting Insulin-like growth factors-1(IGF-1),Brain-derived neurotropic factor(BDNF)and other neurotrophic factors.The animal models commonly used for MS are Experimental Autoimmune Encephalomyelitis(EAE)and Cuprizone-induced demyelinating model(CPZ model).The chronic EAE animal models is induced by MOG35-55.It is the recognized model of MS pharmacodynamics due to its similar pathological features and progression pattern with SPMS.However,due to the recurrent disease conditions in chronic EAE models,the degree of demyelination and myelination regeneration varies greatly among individuals.In the study of myelination regeneration mechanism,demyelination model induced by Cuprizone(CPZ)is usually used.The time and mechanism of demyelination and myelin regeneration are clear in this model,which is a classic pharmacological model to study the mechanism of myelin regeneration and its drugs.After 6 weeks of modeling,the CPZ model mice showed behavioral impairments in SPMS patients,such as motor sensory cognition.After 6 weeks of modeling,CPZ model mice showed behavioral disorders similar to those of SPMS patients,such as motor sensory cognition and other impairments.Therefore,CPZ model is a good research carrier for improving function based on myelin regeneration,and also meets the research objectives of this study.In the previous experiments,we found that Dihydroartemisinin(DHA)can play a good role in the intervention of a variety of animal models of autoimmune diseases(including animal models of MS,EAE),which has a good prospect of drug development and application.Preliminary results showed that the administration of DHA during the construction of an acute EAE model had a clear inhibitory effect on the progression of the disease and the level of inflammation in the lesions.However,the preliminary experimental studies only evaluated the effectiveness of DHA from the perspective of overall disease intervention.In particular,in the process of lesion repair after myelin sheath injury,whether DHA can play the role of drugs to promote the repair of damaged myelin sheath and thus achieve the improvement of overall function has not been concluded,which is also the main focus of this study.On the basis of pure anti-inflammatory,our study focused on the process of lesion repair to realize the effective expansion of the understanding of the efficacy of DHA,and further complete the efficacy evaluation and pharmacological research of DHA.In addition,the specific pharmacological process of DHA in promoting myelin regeneration and its potential signaling pathways were identified,so as to provide laboratory evidence support for the further study of its molecular mechanism and scientific and rational application in the future.Methods and content1.The pharmacodynamic effect of DHA on SPMS model mice(1)Chronic EAE model induced by MOG35-55 was established as the efficacy study model of SPMS.Body weight and status were monitored from the first day of autoimmunity induction.After 14 days of immunization induction,all model mice developed disease.Different doses of DHA(2 mg,10 mg,20 mg/kg/d,respectively,in low,medium and high doses)and MS first-line treatment drug methylprednisolone(MET,1 mg/kg/d)were treated for 36 consecutive days.The normal control group and the model group were given equal doses of solvent.The state changes of mice were observed regularly every day.We take the body weight and KONO'S clinical score as the criteria for efficacy evaluation.(2)CPZ-induced demyelination model was established as a research model for functional recovery of SPMS.Grouping and administration dose are the same as above,the normal group was fed with normal diet throughout the whole process,and the model group and the administration group were fed with 0.2%CPZ diet for 6 weeks to create demyelination model.2.Pharmacological mechanism of DHA promoting myelin regeneration in SPMS model mice(1)The lumbar pulp of mice in the chronic EAE model and brain tissue of mice in the CPZ model were stained with Luxol Fast Blue(LFB)to analyze the positive myelin area in the lumbar pulp and corpus callosum.The myelin sheath microstructure was observed by transmission electron microscopy(TEM),and the myelin axon density was statistically analyzed.The G-ratio value was used as the index of myelin sheath regeneration.(2)Chronic EAE and CPZ model mice were intraperitoneally injected with BrdU 4 days before sampling to mark the new cells,and brain tissues of the model mice were stained with BrdU and Oligo 2 fluorescence double label.The number of new cells(Brdu+cells),oligodendrocyte lines(Olig2+cells)and new oligodendrocyte lines(Brdu+/Olig2+cells)in the corpus callosum were analyzed.(3)The lumbar pulp of chronic EAE model mice and the brain tissues of CPZ model mice were stained with MBP immunofluorescence,and the MBP positive area was used as an indicator of myelin regeneration degree.3.Based on "microglia-oligodendrocytes",the mechanism of DHA promoting myelin regeneration was explored.(1)M2 polarization(Arg1+cells)ratio of macrophages/microglia in the lumbar spinal cord and corpus callosum was detected by immunofluorescence method after tissue fixation in the lumbar spinal cord and CPZ model mouse brains.In vitro,the effect of DHA on the M2 polarization of microglia was further verified with the BV-2 microglia cell line.LPS(20 ng/mL)was used to model the microglia,and each dose of DHA(1 ?M,2 ?M,4 ?M)and MET was used to intervene.Flow cytometry was used to detect the proportion of microglia M2 polarization cells(CD206+cells)in the case of single dosing and model building.(2)Extraction,isolation,purification and identification of mouse primary OPCs:The cortex of C57BL/6J Suckling mice within 24 hours was taken.After removing the meninges,cut them into pieces for tissue adherent culture.After 9-15 days of proliferation culture,OPCs were isolated and purified by differential centrifugation method.After 3 days of proliferation culture,the proportion of OPCs(A2B5+cells)was detected by immunofluorescence method.(3)Proliferation and apoptosis:LPS(20 ng/mL)was used to prepare the model.OPCS was treated with DHA(1 ?M,2 ?M,4 ?M)and MET for 72 h.EdU Apollo567 In Vitro Kit and TUNEL Kit were used to detect the proliferation and anti-apoptosis effects of DHA on OPCs,respectively.(4)The proportion of oligoendrocytes(MBP+cells)was detected by immunofluorescence method in the adoptive culture system of "microglia-OPCs".The direct effect of DHA on OPCs differentiation was excluded in the case of direct dosing.(5)The relative levels of IGF-1 and BDNF mRNA in BV-2 cells were determined by RT-qPCR,and the level of IL-4 in BV-2 supernatant was determined by ELISA.(6)The activity of OPCs PPAR? was detected in adoptive culture system,and the nuclear transfer of PPAR? in oligoendrocytes was detected in CPZ animal model using PPAR? and Oligo 2 fluorescence dual-label techniques.Results1.Efficacy evaluation of DHA on SPMS model mice(1)DHA can relieve the clinical symptoms of chronic EAE model miceFrom the 8th day after immunization,the chronic EAE model mice developed the disease successively,until the 14th day,all the mice developed the disease,and reached the peak at the 24th day,indicating that the modeling was successful.After 36 days of continuous intervention,each dose of DHA could significantly reduce KONO's score and alleviate the weight loss of model mice.MET reduced the KONO's score of the model mice,but the difference in body weight was not statistically significant compared with the model group.(2)DHA can promote the improvement of learning and memory,motor balance and mood disorders in demyelinated miceIn the functional recovery experiment,with the extension of the training cycle,the escape latency of each group showed a decreasing trend during the location navigation phase 5 days before the water maze test.After the fifth day of training,DHA dose groups could significantly reduce the escape latency of model mice,and the swimming distance and the percentage of swimming time in the target quadrant of DHA medium and high dose groups were significantly higher than that of the model group on the 5th day.On the 6th day of the test,during the space exploration experiment,DHA increased the number of platform crossing and swimming distance in the target quadrant of the model mice,and the swimming distance and swimming time in the target quadrant of the escape latency period 5 days before MET.There were no statistical differences between the number of platform crossing and swimming distance in the target quadrant of the model group and the model group on the 6th day.In the rotating rod experiment,all doses of DHA intervention could prolong the rod stay time of model mice,while there was no statistical difference between the rod stay time of MET group and the model group.In the elevated cross maze experiment,the high dose of DHA could significantly increase the percentage of the times of entering the open arm of the model mice,and the percentage of the time of entering the open arm had an increasing trend,but there was no statistically significant difference between the MET group and the model group in The Times and percentage of the time of entering the open arm.2.Pharmacological mechanism of DHA promoting myelin regeneration in SPMS model mice(1)DHA can maintain the integrity of myelin sheath in the lesion of SPMS miceLumbar pulp and corpus callosum were typical LFB staining sites of chronic EAE model mice and CPZ model mice,respectively.It was found that DHA could effectively increase the positive area of LFB in the lumbar pulp of chronic EAE model mice and the corpus callosum of CPZ model mice,and promote the degree of myelination in the lesion of model mice.Further adopt the method of transmission electron microscopy observation the change of microstructure,mice at the myelin myelin structural integrity in normal mice and model groups outside the small axon myelin to intermittent depigmentation,with cavity sample structure,axon density decrease,DHA after the intervention,the structure of myelin sheath were improved,and obviously increase the density of the myelinated axons.The structure of axons at the lesion was analyzed,and G-ratio represented the ratio of inner and outer diameter of axons,and the lower the ratio was,the higher the degree of myelin regeneration was.The experimental results showed that DHA could effectively reduce the G-ratio value,suggesting that DHA could promote myelin regeneration.(2)DHA promoted the regeneration of oligodendrocyte cells in the focal area of SPMS model miceBrdU can be used as a marker of new cells in vivo.The BrdU+/Oligo 2+fluorescence dual labeling method was used to investigate whether the function of DHA in maintaining myelin integrity was based on the role of oliodendrocyte cell line in cell regeneration.The results showed that DHA could increase the proportion of new cells(BrdU+cells)and new oligoendrocytes(BrdU+/Oligo 2+cells)in the lesions of both models,while there was no statistical significance in the MET group compared with the model group.(3)DHA promoted myelin regeneration in SPMS model miceMBP is a marker of mature oligoendrocytes.Immunofluorescence was used to stain the lesion tissues of the two models,and it was found that the MBP positive area in the lesion sites of the two models was significantly reduced,while DHA could effectively increase the MBP positive area.Compared with the model group,there was no statistical difference in the MBP positive area in the MET group.3.Based on "microglia-oligodendrocytes",the mechanism of DHA promoting myelin sheath regeneration was explored(1)DHA promoted the proliferation of oligodendroprogenitor cells and inhibited their apoptosisFirst of all,tissue adherent culture was conducted in the cortex of C57BL/6J Suckling mice.On day 3-6,cells were seen crawling out of the tissues.After 6-15 days of adding OPCS proliferation medium,OPCS-like cells proliferated massively and adhered to the adherent cell layer.After separation and purification by the method of shaking table differential separation at the end of 15 days.After separation and purification by the method of shaking table differential separation at the 15th day,the OPCs were proliferated for 3 days,and the proportion of typical unipolar and bipolar OPCs(A2B5+cells)was up to 83.63±11.86%,which could be used for further experiments.After direct dosing,it was found that DHA could significantly promote the proliferation of OPCs.In the anti-apoptotic experiment,it was found that both DHA and MET could effectively inhibit the apoptosis of OPCs,but the anti-apoptotic ability of all doses of DHA were significantly higher than that of MET group.(2)Based on the adoptive culture system of "microglia-OPCs",DHA could promote the differentiation of OPCsThe proportion of Argl+macrophages/microglia in the lesion site of corpus callosum in the lumbar spinal cord of chronic EAE model and CPZ model mice was significantly increased in all DHA dose groups.MET group increased the ratio of M2 polarization of macrophages/microglia(Arg1+cells)in the lumbar spinal cord in the chronic EAE model,but had no significant effect on the lesion site of corpus callosum in CPZ model mice.In vitro experiments,DHA and MET could significantly promote the M2 polarization of microglia,both alone and in inflammatory environment.In addition,the ability to induce M2 polarization in the inflammatory environment of each dose of DHA group and MET were improved to a certain extent compared with that of the single intervention group.Compared with the single intervention group,the ability to induce M2 polarization of high-dose DHA in the inflammatory environment was significantly increased,even better than that of MET.The proportion of mature oligoendrocytes(MBP+cells)differentiated into OPCs by DHA was not significantly changed,suggesting that the role of DHA in promoting myelin regeneration and lesion repair may be dependent on the involvement of other cells.Combined with previous single cell sequencing results,we further evaluated its drug activity in"microglia-OPCS" unit.The results showed that the number of mature oligoendrocytes(MBP+cells)in the model of adoptive transfer of microglia cells in the condition medium could be increased in each dose of DHA,and the number of oligoendrocytes in the high dose group was 2.1 times that in the model group.These results indicated that the efficacy of DHA was dependent on the "microglia-OPCS" unit.(3)In the adoptive culture system of "microglia-OPCs",the mechanism of DHA promoting OPCs differentiation was studiedIn BV-2 microglia cells,DHA could effectively increase the mRNA levels of IGF-1 and BDNF,and provide nutritional support for the proliferation and differentiation of OPCs.The content of IL-4 in the supernatant of BV-2 microglia cells in each group was detected,and it was found that the content of IL-4 was significantly increased in each DHA group.The activity of PPAR? in OPCs of DHA medium dose group was significantly increased after co-culture of microglia in conditioned medium after DHA intervention.In the brain tissue of the CPZ model,the Oligo 2+/PPAR? immunofluorescence analysis showed that all doses of DHA could increase the proportion of PPAR? nuclear transfer cells in Oligo 2+cells,but there was no significant change in the activity of PPAR? in the MET groupConclusion and discussionThrough this study,we draw the following conclusions:1.In the overall efficacy study,DHA improved cognitive,motor,and affective dysfunction in SPMS mice(chronic EAE model and CPZ-induced demyelination model).2.At the histological level,DHA can effectively maintain the integrity of myelin sheath in the two different lesion sites(lumbar spinal cord in chronic EAE model mice and corpus callosum in CPZ-induced demyelinating model mice),improve the microstructure of myelin sheath in the lesion site,and increase the density of myelinated axons in the lesion site.3.The G-ratio value of axonal structure in the lesion site suggested that DHA could effectively promote myelin regeneration in the lesion site of SPMS mice.Combined with the co-expression analysis of BrdU and oligodendrocyte cell line markers,it was found that DHA could promote the regeneration of oligodendrocyte cell line in SPMS model mice.4.At the cell level,we extracted,isolated and purified mouse primary OPCs in vitro,and from the number and differentiation of OPCs,a key factor in myelin regeneration,we found that DHA could promote the proliferation,resist apoptosis and promote the differentiation of primary OPCs.5.In terms of mechanism,using "microglia-OPCs" as an in vitro model,it was found that DHA could promote the M2 polarization of microglia cells,secrete neurotrophic factors IGF-1 and BDNF,and promote the proliferation and differentiation of OPCs,and the potential mechanism of IL-4/PPAR? pathway was suggested.This study expand the efficacy of DHA in SPMS,provide experimental evidence support for drug research and development of SPMS,and have suggestive significance for the treatment of neurodegenerative diseases with myelin regeneration disorder as a common pathological feature.