| BackgroundIntervertebral disc(IVD)degeneration(IDD)is the rimary cause of low back pain(LBP)with no currently long-lasting and effective treatment.Recently,activation and recruitment of stem/progenitor cells has provided a novel and promising therapeutic strategy for endogenous repair of the degenerative IVD.Many studies have demonstrated tha various chemotactic factors highly released in damaged tissues play an important role in tissue self-repairing.Stromal cell-derived factor-la(SDF-la)regulates stem cell proliferation.migration and differentiation through interaction with CXC chemokine receptor 4(CXCR4).Although SDF-la has also been reported to be highly upregulated in the degenerative IVD,its biological effects on the endogenous stem/progenitor cells residing in IVDs and the possible underlying mechanisms are still unclear.ObjectivesTo verify the upregulation of SDF-la in the degenerative IVD and the endogenous expression of CXCR4 in nucleus pulposus-derived stem cells(NPSCs).To investigate the effects of SDF-1/CXCR4 axis on the capacities of proliferation,chondrogenic differentiation,and migration of NPSCs.To determine the role of autophagy in regulating SDF-1/CXCR4 axis-induced NPSCs migration.To understand the downstream pathway of SDF-1/CXCR4 axis by which regulating autophagic activity and migration of NPSCs.MethodsIsolated NPSCs from the nucleus pulposus tissues of adult Sprague-Dawley(SD)rats using the method of differential adhesion were identified by colony formation,multilineage differentiation,and the expression of specific surface markers.Real-time RT-PCR,enzyme-linked immunosorbent assay(ELISA),and immunohistochemistry were performed to compare the expression and secretion of SDF-1α between the normal and degenerative IVDs.The effects of SDF-1α on the NPSCs proliferation and apoptosis were analyzed by CCK-8 and EdU proliferation assays,cell cycle distribution,and Annexin V/PI staining.After induction of chondrogenic differentiation of NPSCs,we examined the effect of SDF-1/CXCR4 axis on the expression of chondrogenic markers.In addition to wound healing and Transwell migration assays in vitro,the effect of SDF-1/CXCR4 axis on the migration ability of NPSCs was evaluated by observing the migration profile of NPSCs in the ex vivo organ culture model of bovine tail IVD and in the rat coccygeal degenerative IVD model in vivo,respectively.Moreover,the endogenous expression of CXCR4 in NPSCs and the role of SDF-1/CXCR4 axis on the migration ability of NPSCs were analyzed by real-time RT-PCR,immunoblotting,and immunofluorescence.The effect of SDF-1/CXCR4 axis on autophagic activity of NPSCs were evaluated by detecting autophagosome formation and the expression of autophagy-related proteins.After treatment with rapamycin and 3-methyladenine(3-MA),the effects of mammalian target of rapamycin(mTOR)pathway and autophagy on the migration ability of NPSCs were evaluated by in vitro migration assays.In addition,the phosphorylation level of mTOR/p70 S6K signaling pathway and its associations with F-actin cytoskeletal remodeling and autophagic activity were examined to further clarify the possible mechanism of SDF-1/CXCR4 axis-induced NPSCs migration.ResultsAfter the identification of stem cell characteristics,it was confirmed that the isolated NPSCs had the abilities of colony formation and multilineage differentiation as well as higher expression of mesenchymal stem cells(MSCs)specific surface markers.SDF-1αwas significantly upregulated in the degenerative IVD settings.In addition to improvement of late proliferation and chondrogenic differentiation,SDF-la induced the migration capacity of NPSCs both in vitro and in vivo.SDF-la not only increased CXCR4 expression but also stimulated translocation of CXCR4 from cytoplasm to membrane,accompanied by cytoskeletal rearrangement.Furthermore,blocking CXCR4 with AMD3100 effectively suppressed SDF-la-induced chondrogenic differentiation and migration capacities of NPSCsWith the increased SDF-1α,the autophagic activity of NPSCs was gradually inhibited AMD3100 significantly inhibited SDF-1α-mediated activation of mTOR pathway and inhibition of autophagic expression in NPSCs.Rapamycin inhibited SDF-1α-induced NPSCs migration,but 3-MA enhanced SDF-la-induced NPSCs migration.In addition.SDF-1/CXCR4-activated downstream mTOR/p70 S6K signaling pathway was involved in F-actin cytoskeletal rearrangement and the inhibition of autophagic activity in NPSCs.ConclusionsChemokine SDF-1α is highly expressed and secreted in the degenerative IVD settings and its receptor CXCR4 is endogenously expressed in NPSCs the endogenous stem cells in IVDs.SDF-1α has the potential to promote proliferation,chondrogenic differentiation,and migration of NPSCs through SDF-1/CXCR4 axis,contributing to in situ repair and regeneration of IVD.The migration capacity of NPSCs induced by SDF-la might be related to the activation of the downstream mTOR/p70 S6K pathway and the inhibition of autophagy.In the early stage of IVD degeneration,SDF-la could activate and recruit the endogenous stem/progenitor cells in the IVD,which has the potential to promote endogenous repair of the degenerative IVD,providing a new therapeutic target for in situ tissue engineering to repair the IVD in the future. |
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