Spatiotemporal Expression Of Poly(rC)-Binding Protein PCBP2 Modulates Schwann Cell Proliferation After Sciatic Nerve Injury

ObjectiveTo investigate the expression and role of PCBP2 in rat sciatic nerve after crush, clarify the relationship of PCBP2 expression and schwann cell proliferation and provide theoretical basis for the role of PCBP2 in peripheral nerve injury and repair and contributed to the management of peripheral nerve injury.Methods1. Adult male Sprague Dawley rats weighing 220–275 g were used in this study. The Sprague-dawley(SD) rats were subjected to sciatic nerve crush. All animals(n = 54) were randomly divided into groups consisting of one normal group and eight SNC groups at several time points after surgery(6, 12 h, 1, 3, 5 days, 1, 2 and 4 weeks; n = 6 for each time point), and after extracting the protein, the expression of PCBP2 protein in sciatic nerve was analyzed by Western blot. The tendency of PCBP2 expression after peripheral nerve injury was analysised in general.2. The expression of PCBP2 in sciatic nerve was detected by immunohistochemistry, and double immunofluorescence showed the distribution and location of PCBP2 expression in sciatic nerve.3. To determine whether the expression of FHL3 and p21 was changed when knocking down PCBP2, we generated PCBP2 stable knockdown Schwann cells. We used the flow cytometry and cck-8 assays to test the effect of knocking down PCBP2 on Schwann cell proliferation.Results1. Western blot analysis showed PCBP2 protein levels began to rise 3 d after injury, peaking in the 7 d, returned to the normal level at 4 w.2. Immunohistochemical results revealed that the positive rate of PCBP2 in nerve tissues was significantly enhanced after sciatic nerve injury. Double Immunofluorescence indicated that PCBP2 was mainly located in Schwann cells signed by S100 and it had no location with axons signed by NF200. Besides, in proliferating Schwann cells which could be marked by PCNA in the nerve tissue segment, we could observed colocation between PCNA and PCBP2 in injured sciatic nerve.3. In the model of proliferation of Schwann cell cultured in vitro, we observed the expression of PCBP2 became gradually increased which was consistent with the expression of PCNA. While the expression of FHL3 and p21 was attenuated. The flow cytometry and cck-8 analysis revealed that down-regulated PCBP2 could inhibit Schwann cell cycle.4. The protein level of FHL3 and p21 was increased when PCBP2 was knocked down in Schwann cells, and down-regulated FHL3 decreased the expression of p21. Immunoflurescent staining confirmed the co-localization of PCBP2, FHL3 and p21 in Schwann cells.Conclusions1. The expression of PCBP2 in rat sciatic nerve was upregulated 3-5d following crush by Western blot analysis. It gradually reduced to normal level at 4 week. PCBP2 was widely expressed in Schwann cells of the distal sciatic nerve segment but was barely localized in the axons of either normal or crushed sciatic nerve. Meanwhile, the upregulation of PCBP2 expressions in the sciatic nerve was concomitant with Schwann cell proliferation.2. PCBP2 might exert its function in SCs proliferation through the FHL3-p21 signaling pathway.