Astrocytes Are Dedifferentiated And Induced By Neuregulin-1 To Participate In Remyelination And Repair Of Spinal Cord Injury And Its Underlying Mechanism

Background Spinal cord injury(SCI)is a disabling disease.Axonal demyelination is one of the main pathological features of SCI.Promoting remyelination is a new breakthrough for the repair of SCI.Oligodendrocytes are the main cells of myelin formation and remyelination in the spinal cord.Promoting oligodendrocyte differentiation is of great significance for myelin regeneration.SCI is often accompanied by inflammation.Astrocytes in the spinal cord can re-acquire stem cell potential and dedifferentiate under inflammation,thereby becoming a promising source of cells for the research of regenerative medicine.Transforming astrocytes with stem cell potential into oligodendrocytes is an innovative strategy for SCI repair.However,the mechanism of astrocyte dedifferentiation is still unclear.There is still a big gap in the exploration of methods to transform astrocytes into oligodendrocytes and participate in remyelination after SCI.Nanog is an essential transcription factor to maintain the characteristics of stem cells,and the role of Nanog in astrocyte dedifferentiation process remains unclear.The in-depth understanding of astrocyte dedifferentiation provides a large number of potential cell resources and lays a theory foundation for the repair of SCI.Remyelination repair after SCI also requires effective differentiation of oligodendrocytes.Neuregulin-1(Nrg1)plays an essential role in the differentiation of oligodendrocytes.Therefore,it's a potential therapeutic strategy for demyelination in SCI that using Nrg1 to drive dedifferentiated astrocytes to differentiate into oligodendrocytes.ObjectiveTo explore the role of Nanog in the dedifferentiation of astrocytes and its possible mechanism.To explore the possibility of Nrg1-induced transformation of dedifferentiated astrocytes into oligodendrocytes to participate in SCI remyelination repair and its possible mechanism.MethodsIn this study,in order to simulate the inflammatory response in vivo and explore the possible mechanism,tumor necrosis factor-a(TNF-a)was used to stimulate primary rat spinal cord astrocytes to dedifferentiate into reactive state.At the same time,Nrg1 was used to induce dedifferentiated astrocytes in vivo and in vitro to increase oligodendrocyte differentiation and remyelination after SCI.The expression of Nanog was detected by q RT-PCR and Western blotting,and the expressions of immature markers CD44,Musashi-1 and oligodendrocyte markers PDGFR-? and O4 were detected by q RT-PCR and immunofluorescence.Western blotting was used to detect the expression of key protein molecules in NF-kB and PI3K-Akt-m TOR signaling pathway,including Akt,p-Akt,m TOR,p-m TOR,IKK,p-IKK,NF-kB p65,p-NF-kB p65,IkB?,p-IkB? and PI3 K.In order to further explore the role of Nanog in astrocyte dedifferentiation,the small interference RNA(si RNA)of Nanog was used to down-regulate the expression of Nanog.In order to perform functional recovery experiments,the pathway inhibitor BAY11-7082 was used to inhibit the NF-kB signaling pathway,and Erb B inhibitor was used to inhibit the effect of Nrg1.The regeneration and protection of myelin were detected by LFB myelin staining and myelin basic protein(MBP)content.The expression of myelin marker protein MBP,axon marker protein NF-H and astrogliosis marker protein glial fibrillary acidic protein(GFAP)was detected by western blotting and immunohistochemistry.BBB scoring system was used to evaluate the recovery of motor function in rats.ResultsThe results o showed that TNF-a could promote the re-expression of immature markers CD44 and Musashi-1 in astrocytes,suggesting that TNF-a induced astrocytes to dedifferentiate into stem cell state.Moreover,since TNF-a is a classic inflammatory factor that activates the NF-kB signaling pathway,the results of western blotting showed that TNF-a stimulation could increase the activation of NF-kB signal pathway in rat spinal cord astrocytes and significantly up-regulate the expression of m RNA and protein of Nanog.Knocking down Nanog by Si RNA could reverse the expression of CD44 and Musashi-1 in astrocytes induced by TNF-a,suggesting that Nanog was involved in the dedifferentiation of astrocytes.In addition,Nanog knockdown did not affect NF-kB signaling pathway.Both NF-kB signal pathway and Nanog are involved in the dedifferentiation of astrocytes induced by inflammation.In order to fully understand the relationship between them,BAY11-7082,a pathway inhibitor,was used to inhibit NF-kB signal pathway.The results showed that BAY11-7082 blocked the NF-kB signal pathway and inhibited the expression of immature markers,which fully demonstrated that TNF-a induced astrocyte dedifferentiation through the NF-kB signal pathway.BAY 11-7082 also inhibited the expression of Nanog,indicating that Nanog was regulated by the NF-kB signal pathway.In addition,Nrg1 could induce primary dedifferentiated astrocytes to express oligodendrocyte markers PDGFR-? and O4 at both translation and transcriptional levels.Intrathecal delivery of Nrg1 to rats with SCI in vivo could also promote the expression of oligodendrocyte lineage marker proteins in astrocytes,indicating that Nrg1 could directly transform astrocytes into oligodendrocyte lineage cells.In addition,PI3K-AKT-m TOR signal pathway and many transcription factors are involved in the regulation of oligodendrocyte differentiation.In order to further explore the mechanism of Nrg1 action,the results of western blotting showed that Nrg1 could significantly activate PI3K-AKT-m TOR signal pathway and up-regulate the expression of key transcription factors promoting oligodendrocyte differentiation including Olig1,Nkx2.2,Olig2,SOX10 and Myrf.The above effects of Nrg1 were significantly reversed by the co-administration of Nrg1 and Erb B inhibitor,suggesting that Nrg1 transduced signals into cells through Erb B receptors and functioned through PI3K-AKT-m TOR signaling pathways and the key transcription factors.Nrg1 promotes the transformation of astrocytes to oligodendrocyte lineage cells,which provides the possibility for the repair of SCI.LFB myelin staining combined with detection of MBP content showed that Nrg1 could significantly inhibit the pathological changes of demyelination after SCI.The increase of myelin sheath is often accompanied by the increase and protection of axons.The observation of the expression of NF-H also showed that Nrg1 also played a role in protecting axons after SCI.The change of astrocyte differentiation fate induced by Nrg1 may affect astrogliosis characterized by astrocyte proliferation in SCI.The results of GFAP detection by the same method showed that Nrg1 could significantly reduce astrogliosis after SCI.All the functional effects of Nrg1 suggest that it may be beneficial to the locomotor recovery of SCI rats.Hence,we observed the locomotor recovery of rats for 42 days.The results showed that Nrg1 could improve the BBB score of SCI rats.In addition,the co-administration of Nrg1 and Erb B inhibitor could inhibit the therapeutic effects of Nrg1,indicating the specificity of Nrg1.ConclusionThese results indicate that activation of the NF-?B signaling pathway through TNF-a leads to astrocytes dedifferentiation via Nanog.In addition,Nrg1 can convert dedifferentiated astrocytes to oligodendrocyte lineage cells through PI3K-AKT-m TOR signal pathway and key transcription factors,inhibit astrogliosis,promote remyelination,protect axons and eventually improve locomotor function after SCI.In conclusion,these results increase the understanding of the mechanism of astrocyte dedifferentiation and enrich the mechanism in repairing SCI by Nrg1,providing a theoretical basis for the development of new therapeutic strategies for SCI.