The Chondroitin Sulfate Proteoglycans In Glial Scar Area Regulate Migration Of Meningeal Fibroblasts Via P38 MAPK Signal Pathway

BackgroundAfter brain injury,the adult mammalian brain constructs a dense wall around the injury site,called the glial scar,preventing the inflammation to spread and protects the intact neural tissue.However,the dense "fence" has become a physical and chemical barrier to the regeneration of axons in the late stage,which is the reasons why the axon cannot regenerate through the injured area leading into poor recovery of nerve function.How to reduce the formation of glial scar in the late stage of injury and benefit axon regeneration is an urgent problem to be solved.It was reported that glial scar is forming by reactive astrocytes,meningeal fibroblasts and extra cellular matrix,such as chondroitin sulfate proteoglycan(CSPGs).There is no meningeal fibroblasts in normal brain tissue,and they only migrate to the injured area after brain injury,then form glial scar with reactive astrocytes.However,it is rarely known that the mechanism of meningeal fibroblast migration to the lesion CSPGs,as the main extra cellular matrixin,were suggested that inhibited axon regeneration and neural stem cell migration.Whether CSPGs is involved in the migration of meningeal fibroblasts to the injured area or not have rarely been reported.P38 MAPK signaling pathway plays important regulatory roles in cellular proliferation and migration.Moreover,increasing expression of phosph-p38 was also observed during the migration of fibroma cells.So we examine the effects of chondroitin sulfate on meningeal fibroblasts migration and the related downstream pathways like p38 MAPK.The purpose and potential mechanism of the migration of meningeal fibroblasts to the injured area may promote the exploration of glial scar formation and its pathophysiological significance.ObjectiveTo investigate the purpose and mechanism of the migration of meningeal fibroblasts to injury area after brain injury.MethodAstrocytes and meningeal fibroblasts were isolated from newborn rat brain for primary culture and identified by fluorescence staining.Astrocytes(A OGD)were treated with oxygen glucose deprivation(oxygen-glucose deprivation,OGD)in vitro and then co-cultured with(A OGD+F).The expression of GFAP was detected by fluorescence staining and qPCR,while CSPGs mRNA,protein was detected by qPCR,western blot respectively.Meningeal fibroblasts were co-cultured with the following groups:A OGD group,A OGD+ChABC group and A without OGD group in transwell chamber to observe migration.The supernatants from 3 groups were co-cultured with meningeal fibroblasts respectively for 0 min,15min,30min,1h,2h,3h,then the expression of p38 and phosphorylated p38 was detected by western blot.What's more,p38 MAPK pathway inhibitor SB203580 was added in A OGD group to ensure the migration of meningeal fibroblasts induced by activation of p38 MAPK signaling pathway.Downstream protein cofilin of p38 MAPK signaling pathway expression was detected also.Results1.Astrocytes transformed into reactive astrocytes after OGD,which showed high expression of GFAP,coarse cell axis mutation and increased expression of CSPGs.Reactive astrocytes were co-cultured with meningeal fibroblasts that reduced its the expression of GFAP and CSPGs.2.The migrate cells of the meningeal fibroblasts in the A OGD group was significantly higher than that in the A OGD+ChABC group or A without OGD group(P<0.05).The expression of phospho-p38 was significantly higher in the A OGD group(P<0.05),while the other two groups did not change significantly,and the expression of p38 have no difference among 3 group.3.The migrate cells of meningeal fibroblasts was significantly reduce after adding SB203580 than that in A OGD group(P<0.05).Moreover,the expression of cofilin increased significantly in A OGD culture medium treatment group,but decreased significantly after ChABC digestion of CSPGs or SB203580(p38 pathway inhibitor)treatment.Conclusion1.After OGD treatment in vitro,astrocytes can be induced into reactive astrocytes and overexpression of CSPGs.What's more,meningeal fibroblasts reduced the activation of astrocytes.2.Reactive astrocytes promote the migration of meningeal fibroblasts through CSPGs,while the regulation of the migration of meningeal fibroblasts might accomplished by activating p38 MAPK signaling pathway.3.The increase of meningeal fibroblasts migration is related to the activation of cytoskeleton protein cofilin.