Radial Extracorporeal Shock Wave Therapy Enhances the Proliferation and Differentiation of Neural Stem Cells by Notch, PI3K/AKT, and Wnt/β-catenin Signaling

Abstract

Neural stem cell (NSC) proliferation and differentiation play a pivotal role in the repair of brain function in central nervous system (CNS) diseases. Radial extracorporeal shock wave therapy (rESWT) is a non-invasive and innovative treatment for many conditions, yet little is known about the effects of this treatment on NSCs. Mouse NSCs (NE-4C) were exposed to rESWT with 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5 bar (500 impulses, and 2 Hz) in vitro. Cell viability test results indicated that rESWT, at a dose of 2.5 bar, 500 impulses, and 2 Hz, increased NE-4C viability within 72 h, and that the PI3K/AKT pathway was involved in its mechanisms. Exposure to rESWT also affected proliferation and differentiation of NE-4C after 8 weeks, which may be associated with Wnt/β-catenin and Notch pathways. This assessment is corroborated by the ability of inhibitors of Wnt/β-catenin [Dickkopf-1 (Dkk-1)] and the Notch pathway (DAPT) to weaken proliferation and differentiation of NSCs. In summary, a proper dose of rESWT enhanced NSCs augment via the PI3K/AKT pathway initially. Also, Wnt/β-catenin and the Notch pathway play important roles in regulation of the long-term efficacy of rESWT. This study reveals a novel approach to culture NSCs in vitro and support neurogenesis.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1

The effect of different doses of rESWT applied on the cultures. The multiplication of NE-4C cells was analyzed using an MTT cell proliferation assay (Promega, Japan). rESWT at a dose of 2.5 bar, 500 impulses, 2 Hz significantly stimulated the number of NE-4C cells at 72 h compared with the control group. Bar: 20 μm. (A) Different doses of rESWT ranging from 1.0 to 3.5 bar were used in cultures. The proliferation of NE-4C cells was tested at 12, 24, 36, 48, 60, and 72 h after rESWT. Below the 2.5 bar, 500 impulses, 2 Hz dose, rESWT increased proliferation in a quantity-dependent manner. Over the 2.5 bar, 500 impulses, 2 Hz dose, rESWT had an opposite effect (B).

Figure 2

NSE expression. NSE expression was assessed by immunofluorescence, Western blot analysis, and qRT-PCR. Fluorescence signals were analyzed by an Axiovert 200 inverted microscope. Nuclei were stained with DAPI. Bar: 20 μm (A–C). The Western blot shows a visible difference in three groups (D). Data are reported as the mean ± SD of significant difference from the control group at both week 8 (*P < 0.05) and week 12 (**P < 0.01) and from the Dkk-1 inhibitor group (***P < 0.001) (E). NSE mRNA expression was improved at 8 and 12 weeks compared with the control group (**P < 0.01) and Dkk-1 inhibitor group (***P < 0.001) (F).

Figure 3

β-tubulin III expression. β-Tubulin III expression was improved at 8 and 12 weeks compared with the control group. The Dkk-1 inhibitor suppressed β-tubulin III expression with regard to both the protein (A–E) and mRNA (F) levels as determined by immunofluorescence staining (A–C), Western blot (D,E), and qRT-PCR (F) analyses. β-Tubulin III fluorescence is shown in green and nuclei staining is shown in blue. Bar: 20 μm (**p < 0.01, ***P < 0.001).

Figure 4

Western blot and Real-time PCR findings of PI3K and AKT. The Western blot results demonstrate the enhanced expression of PI3K and AKT at 24, 48, and 72 h after rESWT in two independent experiments (***P A,B,D,E). PI3K and AKT mRNA expression, as measured with qRT-PCR, are also increased at 24, 48, and 72 h using rESWT (***P < 0.001) (in C,F).

Figure 5

Wnt3a expression. Wnt3a expression was determined at 1, 4, 8, and 12 weeks. Immunofluorescence microscopic findings of Wnt3a (green color) show that the expression increased compared with the control group and decreased in the Dkk-1 inhibitor group (A–C). Western blot findings demonstrate that the prominent improvement in expression was revealed only at 8 and 12 weeks relative to the control group (D,E). The same results are seen for the mRNA expression (F). (*P < 0.05, **P < 0.01, ***P < 0.001). The data are presented as the mean ± SD.

Figure 6

β-catenin expression. β-catenin (green color) immunofluorescence findings (A–C), and Western blot findings (D,E) demonstrate the augmented expression at weeks 8 and 12 compared with the control group, and reduced expression induced by the Dkk-1 inhibitor (***P < 0.001). β-catenin mRNA expression was also improved relative to control group at 8 weeks (**P < 0.01) and 12 weeks (***P < 0.001) and reduced in the Dkk-1 inhibitor group at 8 weeks (*P < 0.05) and 12 weeks (***P < 0.001) (F). Data are reported as the mean ± SD.

Figure 7

LRP-6, Axin, Frizzled, and GSK3β expression. qRT-PCR was used to test the mRNA expression of LRP-6, Axin, Frizzled, and GSK3β. Results revealed distinct differences between three groups on weeks 8 and 12. LRP-6, Axin, and Frizzled mRNA expression increased after rESWT and was suppressed by the Dkk-1 inhibitor (A–C). GSK3β expression was opposite to that seen for LRP-6, Axin, and Frizzled mRNA (D). (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 8

Notch1 signaling, nestin, and NSE expression after rESWT. Notch1, NICD, and Jagged1 expression was enhanced after rESWT on weeks 8 and 12 (**P E,G,I). mRNA expression for Notch1, Jagged1, and Hes1 was augmented compared with the control group and DAPT group (**P < 0.01, ***P < 0.001) (F,H,J). Notch1 signaling was upregulated and expression of NSE, nestin, and mRNA was increased and was inhibited by DAPT on weeks 8 and 12 (***P < 0.001, **P < 0.01) (A–D).