| 1.Research background and objectivesDegenerative disease represented by Multiple Sclerosis(MS)is a demyelinating disease in the Central Nervous System(CNS),which is characterized by myelin and axonal damage caused by focal inflammatory infiltration.The successful development of immunotherapy has changed the current immunosuppressive monotherapy regimen for multiple sclerosis,which is effective in reducing disease activity and associated clinical relapse during relapsing remissive disease.However,research advances to alleviate disability due to axonal and neuronal damage and loss have not been achieved,especially in the progressive stages of the disease,and remain challenging in terms of treatment.One strategy to combat neurodegeneration is to promote neuroprotection by enhancing myelin regeneration to restore nerve conduction and metabolic support for axons.One of the effective strategies to achieve this goal is to promote endogenous remyelination,thereby restoring nerve conduction and preventing neurodegeneration.Microglia are macrophage-like cells of the central nervous system,which can manifest as pro-inflammatory M1 type and anti-inflammatory M2 type in different disease states.M1 microglia have the role of tissue damage.In the active stage of MS,a large number of M1 microglia damage the CNS tissue by releasing inflammatory factors.On the one hand,M2 microglia can secrete inflammatory dissipating factors to slow down the damage caused by M1 microglia,and on the other hand,it can release many neurotrophic factors(such as IGF-1,BDNF,Chi313,TGF-β),which promote the repair of nerve tissue and the generation of oligodendrocytes.Therefore,enhancing myelin regeneration by modulating microglia-mediated microenvironment and thereby enhancing the endogenous oligodendrocyte generation process has emerged as one of the most promising measures to delay,prevent,or reverse the progression of MS.At present,a large number of clinical and experimental studies have achieved positive results in the promotion of myelin regeneration by neural stem/progenitor cells.Normal cell renewal or functional tissue repair after injury is the result of a finely regulated balance between stem cell quiescence and activation,self-renewal,proliferation,cell fate,and differentiation,in which the microenvironment plays a crucial role.We believe that neural stem/progenitor cell therapy will make a beneficial contribution to the treatment of demyelinating patients in the near future.Taken together,given the special properties of microglia,their close association with neural stem cells and their progeny,and the myriad molecules they can secrete that influence the differentiation fate of stem cells,it is conceivable that microglia are an important immune cell regulating adult neurogenesis.In previous studies,we investigated the efficacy of DHA in the treatment of Experimental Autoimmune Encephalomyelitis(EAE)and cuprizone(CPZ)-induced demyelination models.Experiments have demonstrated the effectiveness of DHA in promoting myelin regeneration and suggested that microglia may be an important effector cell for DHA to exert its action,but whether DHA is dependent on microglia to exert the drug effect of myelin regeneration is still unknown.In addition,previous studies have found that DHA can promote the generation of oligodendrocyte lines in the corpus callosum of EAE and CPZ induced mice,suggesting that DHA may promote the generation of oligodendrocytes at the source.Given that the promotion of neural stem cell differentiation is the driving force for the generation of oligodendrocyte lines,we hypothesized that DHA-dependent microglial promotion of neural stem cell differentiation plays a role in promoting remyelination,thereby promoting white matter repair in demyelinating neurodegenerative diseases.In this study,we established the "microglia-neural stem cell" unit to establish EAE model mice with microglia-ablated DHA,and investigated whether DHA exerts therapeutic effect on EAE by promoting myelin regeneration through the "microglia-neural stem cell" unit after administration.In addition,the mechanism by which DHA promotes remyelination through the microglia-neural stem cell unit was explored.Firstly,the bridge molecule of DHA promoting microglia to neural stem cells was explored,and then how DHA promotes microglia to express this bridge molecule and its molecular pathway were further explored,and the dependence of DHA on this pathway was verified by experiments.2.Research methods and content2.1 Pharmacological study of DHA on the establishment of immune microenvironment conducive to myelin regeneration by reshaping the inflammatory balance of microglia(1)Lipopolysaccharide(LPS)was used to stimulate microglia to mimic the immune microenvironment of microglia-mediated inflammation imbalance.qRT-PCR and ELISA were used to detect the mRNA and secreted protein expression of pro-inflammatory cytokines IL-1β and TNF-α and anti-inflammatory factor IL-10 mediated by microglia.The proportion of pro-inflammatory microglia labeled with CD 16/32 was detected by flow cytometry.Immunofluorescence was used to double-stain the M1 pro-inflammatory iNOS and M2 anti-inflammatory Argl in microglia to evaluate the role of DHA in balancing microglia-mediated inflammatory microenvironment.(2)Network pharmacology was used to predict the targets and pathways of DHA in the treatment of neuroinflammatory diseases,and to explore the pathways of DHA in balancing inflammation.(3)The expressions of key genes Axl,p-Axl(Y702),PI3K,p-PI3K(Tyr317),Akt,p-Akt(Ser473),mTOR and p-mTOR(S2448)in the key pathways predicted by network pharmacology were verified by Western Blot.2.2Evaluation of the efficacy of DHA in promoting myelin regeneration through microglia in EAE model(1)MOG35-55 was used to induce EAE model.Mice were fed with maintenance diet containing 0.029%PLX3397(a selective CSF1R/c-kit inhibitor that can cross the blood-brain barrier orally)on the 8th day after immunization.Microglia were ablated in EAE model mice.DHA was administered 10 days after feeding the PLX3397 diet.The body weight and state of the mice were detected from the first day of induction,and the body weight and clinical KONO’s score were used as indicators of drug efficacy.(2)Immunohistochemical staining of CD11b,a molecular marker of microglia,was used to verify the ablation of microglia.(3)The brain tissue of mice was stained by fast blue staining to observe whether DHA could maintain the integrity of myelin sheath and promote myelin regeneration through microglia.2.3Evaluation of the activity of DHA in promoting neural stem cell differentiation via microglia(1)The microglia-neural stem cells adoptive culture system was established.After the microglia were treated with DHA for 24 hours,the supernatant containing DHA was discarded,and the supernatant was added to fresh medium for another 24 hours.(2)qRT-PCR and Western Blot were used to detect the expression of Nestin,Glial Fibrillary Acidic Portein and glial fibrillary acidic portein.GFAP),Neuronal nuclear antigen(NeuN),Myelin Proteolipid Protein(Myelin Proteolipid Protein),and oligodendrocyte line(myelin proteolipid protein).PLP),Myelin Basic Protein(MBP),a molecular marker of mature oligodendrocytes,mRNA and protein were detected to observe the effect of DHA treated microglia supernatant on neural stem cell differentiation.Immunohistochemical staining of oligodendrocyte progenitor cell molecular marker NG2 and mature oligodendrocyte molecular marker MBP in the Subependymal Ventricular Zone(SVZ)of EAE mice was performed.To verify the differentiation of neural stem cells into oligodendrocytes in vivo.(3)The expression of MiR-34a and Chi313 mRNA was detected by RT-qRCR method,and Chi313 secreted protein was detected by ELISA method.To observe whether miR-34a and Chi313 are the active genes of DHA in myelin regeneration.2.4 Exploration of the mechanism of DHA promoting myelin regeneration through the "microglia-neural stem" unit(1)Microglial cells were co-cultured with AS1517499 to inhibit STAT6 activation.After DHA administration,the expression of Chi313 mRNA and protein in microglia was detected by qRT-PCR and ELISA.Western Blot and qRT-PCR were used to detect the activation of EGFR and the protein and mRNA expressions of SOX10,PLP,and MBP.(2)Axl shRNA was used to block the Axl transcription in microglia.After DHA administration,the expression of Chi313 mRNA and protein in microglia was detected by qRT-PCR and ELISA.Western Blot and qRT-PCR were used to detect the activation of EGFR and the protein and mRNA expressions of SOX 10,PLP,and MBP.3.Study results3.1 Pharmacological study of DHA on the establishment of immune microenvironment conducive to myelin regeneration by reshaping the inflammatory balance of microglia(1)The imbalance of microenvironment mediated by LPS stimulated microglia was characterized by the overexpression of a large number of pro-inflammatory factors and the insufficient expression of inflammatory dissipation factors.The proportion of M1 microglia labeled by CD 16/32 and iNOS was significantly higher than that of M2 microglia labeled by Argl.The results showed that DHA could reduce the increase of proinflammatory molecules IL-1β and TNF-α mediated by microglias after LPS stimulation,and increase the expression of inflammatory resolution factor IL-10.Flow cytometry results showed that DHA could reduce the proportion of CD 16/32 labeled M1 microglia.Immunofluorescence results showed that the fluorescence intensity of Argl labeled microglia was increased and the fluorescence intensity of iNOS was decreased after DHA treatment.It can be seen that DHA can balance the LPS-mediated inflammatory microenvironment of microglia and promote the transformation of microglia from M1 to M2 under LPS stimulation.(2)A total of 27 drug-disease intersection genes were obtained by mapping disease targets with drug targets;The DAVID database was used for GO gene function enrichment analysis.A total of 275 GO terms were screened out,including 285 terms related to biological process,51 terms related to cellular component and 39 terms related to molecular function.According to the network pharmacology results,in addition to Axl,PI3K is also one of the key targets regulated by DHA,indicating that Axl/PI3K/Akt/mTOR pathway may be one of the important pathways for DHA to balance inflammation.(3)PI3K/Akt/m-TOR is one of the important pathways regulated by Axl,and its activation plays an important role in promoting the M2 phenotype of microglia and reducing the release of pro-inflammatory molecules.The regulation of Axl/PI3K/Akt/mTOR pathway in microglia after DHA co-culture was verified experimentally.Western Blot results showed that DHA could increase the expression of Axl,p-Axl(Y702),p-PI3K(Tyr317),p-Akt(Ser473),and p-mTOR(S2448)proteins in microglia under inflammatory conditions.The total protein expression of PI3K,Akt and mTOR had no significant regulatory effect.These results indicated that DHA could activate the Axl/PI3K/Akt/m-TOR pathway in microglial cells stimulated by LPS.Conclusion:DHA can balance the inflammatory microenvironment of microglia induced by LPS and establish an environmental basis conducive to myelin regeneration,and Axl/PI3K/Akt/mTOR is an important pathway for DHA to regulate the inflammatory balance.3.2 Evaluation of the efficacy of DHA in promoting myelin regeneration through microglia in EAE model(1)The results of immunohistochemistry showed that the number of positive microglia labeled with CD11b in the brain of EAE mice fed with 0.029%PLX3397 diet was reduced,indicating that the experimental method successfully ablated microglia in EAE mice.After intragastric administration of DHA,KONO’s score in EAE+DHA group was significantly lower than that in EAE group(P<0.05,P<0.01);There was no significant difference between EAE+PLX3397+DHA group and EAE+PLX3397 group fed with diet containing PLX3397(P<0.05),suggesting that DHA was dependent on microglia to alleviate the degree of disability in the EAE model.In terms of body weight,the EAE+DHA group had significantly higher body weight than the EAE group(P<0.05,P<0.01);There was no significant difference between EAE+PLX3397+DHA group and EAE+PLX3397 group fed with diet containing PLX3397(P<0.05),suggesting that DHA promoted body weight gain in EAE model mice in a microglial dependent manner.(2)Compared with EAE group,the EAE+DHA group showed significantly increased myelination(P<0.05,P<0.01);There was no significant difference between EAE+PLX3397+DHA group and EAE+PLX3397 group fed with diet containing PLX3397(P<0.05),the results showed that DHA promoted myelin integrity and remyelination in the brain dependent on microglia.Conclusion:DHA plays a role in treating EAE,alleviating the degree of disability in EAE,and promoting myelin integrity in EAE through microglia.3.3 Evaluation of the activity of DHA dependent microglia to promote neural stem cell differentiation(1)The results showed that DHA treatment of microglia supernatant significantly increased the mRNA transcription and protein expression of oligodendrocyte markers PLP and MBP,but had no effect on the mRNA expression of astrocyte marker GFAP and neuronal cell marker NeuN in neural stem cells C17.2.As the expression of Nestin,a molecular marker of neural stem cells,was decreased,it was observed that the supernatant of microglia treated with DHA promoted the directional differentiation of neural stem cells C17.2 into oligodendrocytes.(2)After demonstrating that DHA can promote the differentiation of neural stem cells into oligodendrocytes through microglia in vitro,we found that DHA can promote the differentiation of neural stem cells into oligodendrocytes in the contralateral Subependymal Ventricular Zone(SVZ)in vivo.SVZ)oligodendrocyte progenitor cells(labeled NG2)were analyzed by IHC with mature oligodendrocyte cells(labeled MBP).The results showed that the increased numbers of mature oligodendrocyte progenitor cells and oligodendrocyte progenitor cells in the SVZ induced by DHA were abolished after ablation of microglia.The results showed that DHA promoted the differentiation of neural stem cells into oligodendrocytes in EAE mice in a microglia-dependent manner.(3)DHA inhibited the expression of miR-34a and promoted the expression of Chi313 mRNA and Chi313 protein in microglia.Conclusion:DHA promotes the differentiation of neural stem cells(NSCs)into oligodendrocytes via microglia,and the active genes are miR-34a and Chi313.3.4 Exploration of the mechanism of DHA promoting myelin regeneration through "microglia-neural stem cell" unit(1)It has been reported that Chi313 promotes the differentiation of neural stem cells into oligodendrocytes through CLp-EGFR(Y1068)-Pyk2-MEK-ERK pathway,which promotes SOX10 transcription.Having established that DHA could promote oligodendroglial generation through the microglia-neural stem cell unit and promote Chi313 secretion by microglia,we experimentally verified EGFR receptor activation and SOX10 expression in neural stem cells.The results showed that the expression of p-EGFR(Y1068)and SOX10 in neural stem cells was enhanced by the supernatant of microglia cultured with DHA.These results indicated that DHA could activate the surface receptor EGFR and promote the expression of SOX10 in neural stem cells through microglia.(2)STAT6 is a direct transcription factor of Chi313.In our previous study,we found that DHA could promote Chi313 mRNA transcription in microglia,so here we tested whether DHA could activate JAK2/STAT6.DHA enhanced the protein expression of p-JAK2(Y1007)and p-STAT6(Y641),but had no effect on the total protein expression of JAK2 and STAT6.The results showed that DHA could activate the JAK2/STAT6 pathway in microglia.(3)After confirming that DHA could activate STAT6 and promote Chi313 secretion,we next performed reverse experiments to test whether DHA promoted oligodendrocyte differentiation by activating STAT6 to promote Chi313 expression.The results showed that depletion of DHA promoted Chi313m RNA and protein expression in microglia co-cultured with STAT6 activation inhibitor AS1517499,while p-EGFR(Y1068)protein was not significantly changed after the supernatant was adopted from neural stem cells.SOX10,PLP and MBP mRNA and total protein did not change significantly.These results indicated that DHA could secrete Chi313 and promote the differentiation of neural stem cells into oligodendrocytes,which was dependent on STAT6 activation.(4)(4)As the focus gene of our previous study,Axl is one of the key target genes of DHA in EAE treatment and is closely related to myelin regeneration.Since JAK2 is activated by Axl,we hypothesized that DHA could activate JAK2/STAT6/Chi313 via Axl to promote the differentiation of neural stem cells into oligodendrocytes.The results showed that the effect of DHA on promoting the expression of Chi313m RNA and protein in microglia was lost after microglial cells were transfected with lentivirus carrying Axl shRNA.At the same time,p-EGFR(Y1068)protein,SOX10,PLP,MBP mRNA and total protein were not significantly changed after the supernatant was adopted from neural stem cells.These results indicate that DHA is AxL-dependent to secrete Chi313 and promote the differentiation of neural stem cells into oligodendrocytes.Conclusion:DHA promotes the differentiation of neural stem cells into oligodendrocytes by activating the Axl/JAK2/STAT6 pathway to promote the release of Chi313,thereby activating the EGFR receptor and promoting the expression of SOX 10 in neural stem cells.4.Conclusions(1)DHA could balance microglia-mediated inflammation and establish a microenvironment conducive to myelin regeneration.(2)DHA can promote myelin regeneration in EAE model mice depending on microglia.(3)DHA promoted the differentiation of neural stem cells in a microglia-dependent manner;miR-34a and Chi313 are the active genes of DHA on microglia.(4)DHA promotes the release of Chi313 from microglia by activating the Axl/JAK2/STAT6 pathway in microglia,which activates the EGFR receptor and promotes the expression of SOX 10 in neural stem cells,leading to the directional differentiation of neural stem cells into oligodendrocytes.5.MeaningThis study revealed for the first time that DHA promoted myelin regeneration in EAE model dependent on microglia.It is verified that one of the main links of promoting myelin regeneration is promoting the differentiation of endogenous neural stem cells.The intrinsic molecular mechanism of its action is revealed.This study provides new drug guidance for therapeutic strategies to promote endogenous remyelination,and provides reference for future mechanistic studies of DHA. |
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