Study Of SPP1 On Proliferation And Apoptosis Of Schwann Cells In Wallerian Degeneration After Peripheral Nerve Injury And Its Molecular Mechanism

Objective:Schwann cells play an important role in Wallerian degeneration after peripheral nerve injury and participate in the whole process of Wallerian degeneration.During this regeneration process,proliferation and apoptosis will occur in Schwann cells at different stages,and at the same time undergo a series of changes in gene expression,which affect the fate of peripheral nerve regeneration.However,how do these genes control the proliferation and survival of Schwann cells remains unclear.Our previous study showed that the expression of SPP1 and PKCa were up-regulated in SCs after the rat sciatic nerve injury,and the elevated SPP1 promoted the proliferation and inhibited the apoptosis of Schwann cells,and the elevated PKCa also promoted the proliferation of Schwann cells.It has been reported that SPP1 could promote the expression of PKCα,but it is not clear whether SPP1 affects the proliferation and apoptosis of Schwann cells through PKCα,and how SPP1 affects the intracellular kinase PKCα,as a secreted glycoprotein.Previous studies have shown that SPP1 bind to the membrane surface receptors CD44 and αvβ3 of numerous cell types and affect the intracellular signal pathways,including PKCα and its downstream signal molecules.However,it is not clear whether receptor CD44 and αvβ3 are involved in the regulation of Schwann cell proliferation and apoptosis by SPP1 after peripheral nerve injury,and whether participated the signal transmission from SPP1 to PKCα.PKCa could promote cell proliferation and inhibit apoptosis through ERK and Bcl-2,respectively.But whether SPP1 promoting Schwann cells proliferation and inhibiting Schwann cell apoptosis through PKC a is also related to the above signal pathway needs to be further discussed.Therefore,the main purpose of this study was to explore whether SPP1 play its role through PKCα,to determine whether the receptors CD44 and αvβ3 are involved in the regulation of PKCα by SPP1,and to reveal the signal pathways of SPP1/PKCαregulating Schwann cells proliferation and apoptosis respectively,and to provide a reliable molecular target for clinical intervention in peripheral nerve repair.Methods:1.Collect the distal tissue of median nerve after clinical traumatic injury;2.RT-qPCR,Western blot and immunofluorescence staining techniques were used to detect the expression and localization of SPP1 and PKCa after human peripheral nerve injury.3.Primary Schwann cells were cultured.siRNA targeting SPP1 combined with PMA,an activated of PKCα,or plasmid overexpression of SPP1 combined with the inhibition of PKCα by Go6976 were used to explore the relationship between SPP1 and PKCα,as well as their effects on proliferation and apoptosis of Schwann cells.The proliferation and apoptosis of Schwann cells was detected by EdU and flow cytometry,respectively.The expression of p-ERK/ERK,Bcl-2/Bax and cleaved Caspase-3/Caspase-3 was detected by Western blot.4.The rat model of sciatic nerve injury model was established.The expression of cell surface protein CD44 and αvβ3 after sciatic nerve injury were detected by RT-qPCR and Western blot,and its localization in rat sciatic nerve and primary Schwann cells was detected by immunofluorescence staining.5.In primary Schwann cells,CD44 and β3 were silenced by siRNA.EdU and flow cytometry were used to detect their effects on proliferation and apoptosis of Schwann cells,and Western blot was used to detect the expression of p-ERK/ERK,Bcl-2/Bax and cleaved Caspase-3/Caspase-3.6.Exogenous SPP1(rOPN)was introduced to mimic the secreted SPP1.rOPN combined with si-CD44 or si-β3 were used to explore the signal pathway of the effect of SPP1 on proliferation and apoptosis of Schwann cells.Results:1.In the distal specimens of clinical median nerve injury,both SPP1 and PKCαwere located in Schwann cells,and the expression was up-regulated after the nerve injury.2.Like SPP1,PKCα could inhibited Schwann cell apoptosis and promoted Schwann cell proliferation,while both SPP1 and PKCα could up-regulated the phosphorylation of ERK,the relative expression of Bcl-2/Bax,and inhibit the cleaved Caspase-3.and SPP1 could regulated these cytokines by activating PKCα,thus promoted Schwann cells proliferation and inhibited Schwann cells apoptosis.3.Both CD44 and αvβ3 were localized in Schwann cells in rat sciatic nerve,and their expressions were up-regulated after nerve injury.CD44 was mainly expressed on the cell membrane of the primary Schwann cells,while αvβ3 was expressed on the focal adhesion on the surface of the cell membrane.4.Both CD44 and αvβ3 could inhibited apoptosis and promoted proliferation in primary Schwann cells.After silencing CD44 or αvβ3,it was found that the expression of p-ERK/ERK and Bcl-2/Bax was down-regulated and the expression of cleaved Caspase-3/Caspase-3 was up-regulated in Schwann cells.5.Similar to the overexpression of endogenous SPP1,the addition of exogenous SPP1(rOPN)could up-regulated the expression of PKCa in Schwann cells,up-regulated p-ERK/ERK,Bcl-2/Bax,and down-regulated cleaved Caspase-3/Caspase-3.6.After silencing CD44 or β3 by siRNAs,the expression of PKCα,phosphorylation of ERK and the relative expression of Bcl-2/Bax were down-regulated,and Caspase-3 was cleaved.Under the background that CD44 orβ3 was silenced,rOPN could not upregulated the expression of PKCα,p-ERK/ERK,and Bcl-2/Bax in Schwann cells,and inhibited cleaved Caspase-3.The effect of SPP1 on the promotion of PKCa and downstream signal pathway was blocked by si-CD44 or si-β3,indicating that CD44 and β3 are involved in the promotion of proliferation and inhibition of apoptosis of Schwann cells by SPP1-PKCα.Conclusions:Through the study of the molecular mechanism that SPP1 may be involved in promoting Schwann cell proliferation and inhibiting Schwann cell apoptosis in the process of Wallerian degeneration after peripheral nerve injury,we draw the following conclusions:1.The expression of SPP1 and PKCα increased significantly after clinical peripheral nerve injury.2.SPP1 activates PKCa and downstream signal pathways to promote Schwann cell proliferation and inhibit apoptosis through CD44 and αvβ3 on the cell membrance.3.SPP1 promotes the proliferation of Schwann cells through PKCα-ERK.4.SPP1 mediates the anti-apoptotic effect of Schwann cells by regulating PKCα-Bcl2/Bax-cleaved Caspase-3/Caspase-3.