Study On The Role Of Spp1 In The Wallerian Degeneration And Its Molecular Mechanism After Rat Sciatic Nerve Injury

ObjectivesWallerian degeneration(WD)in injured peripheral nerves is associated with a large number of regulatory genes,secreted phosphosprotein 1(Sppl)is one of these critical genes.Some studies have suggested that Sppl has been significantly up-regulated in Schwann cells(SCs)during WD and plays an important role in the axonal regeneration after injury.However,it is not clear how the up-regulated Sppl in Schwann cells effects the regeneration in WD and which molecular mechanism involved.The purpose of this study is to investigate how Sppl promotes nerve regeneration and related signal pathways after peripheral nerve injury.The results can not only enrich the theoretical basis of regeneration after peripheral nerve injury,but also may provide new ideas for the effective repair and reconstruction of injured nerve in clinic.Materials and Methods1.Using the sciatic nerve transection injury model of adult male SD rats,the expression and localization of Sppl were determined in the distal injured nerve tissue by tissue immunofluorescence(IHC)technology,and the dynamic expression of Sppl at different time points after nerve tissue injury was observed;2.Primary SCs were cultured from the sciatic nerves of newborn SD rats,and the expression and localization of Sppl in SCs were further insured by cell immunofluorescence(ICC)analysis;3.Primary cultured SCs were transfected with sppl-siRNA or pcDNA3.1(+)-sppl to silence the expression of Sppl or overexpression of Sppl.The proliferation level of SCs was detected by EdU assay,Transwell assay and Annexin V assay were used to detect the migration and the apoptosis of SCs respectively,so as to determine the effect of changes in Sppl expression on the biological function of SCs;4.We continued to use the primary cultured Schwann cells to silence express and overexpress Sppl by transfecting SCs,and then perform real-time fluorescent quantitative PCR(Real-time PCR)on the related factors(PKCa,Bax,Bcl2 and Nf2)affecting the biological activity of SCs and use protein imprinting(Western Blot)analytical technique to farther clarify the expression changes of genes and proteins related to proliferation,migration and apoptosis of SCs;5.In vivo experiments:An animal model of sciatic nerve defects in adult SD rats was implanted with a nerve conduit carrying Sppl-siRNA transfection reagent to bridge the repair of nerve defects.7 days and 14 days after surgery,we detected c-Fos,PKCa and ERK with Real-time PCR and Western Blot for the purpose of gene and protein detection to further explore the relationship between Sppl and p-ERK/ERK signaling pathway-related proteins,in order to clarify the molecular regulation mechanism of Spp 1 during regeneration after peripheral nerve injury.Results1.In the sciatic nerve,Sppl was co-located with the specific marker S100 of SCs,and its expression gradually increased from 6h to 14d after the nerve injury.Sppl was co-located with S100 in SCs,indicating its stable expression in SCs.2.Altered expression of Sppl in SCs resulted in altered mRNA and protein expression levels for cytokines,c-Fos,PKC?,and phospho-ERK/ERK and affected SC apoptosis in vitro.3.Silencing of Sppl expression in SCs using siRNA technology reduced proliferation and promoted migration of SCs in vitro.Overexpression of Sppl promoted proliferation and reduced migration in SCs in vitro.4.Real-time PCR results showed that the mRNA expression levels of Bax,Bc129 Nf2 and PKCa decreased after siRNA interference with Sppl expression in SCs,while the mRNA expression level of the above factors increased after Sppl overexpression.The mRNA expression level of NT3 increased after Sppl interference and decreased after overexpression.5.Differential expression of Sppl after sciatic nerve injury in vivo altered the expression of cytokines,c-Fos,PKC?,and the p-ERK/ERK pathway.Conclusions1.Sppl is differentially expressed in the course of Wallerian degeneration after sciatic nerve injury in rats.2.Sppl promotes SCs proliferation,inhibits the migration and apoptosis of SCs.3.Sppl may prevent the apoptosis of SCs by maintaining the relative stability of Bcl2 and Bax.4.Sppl is a key regulatory factor that affects nerve degeneration and regeneration through c-Fos,PKCa and p-ERK/ERK pathways after rat sciatic nerve injury.