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

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.

the differentiation of many adult stem cells 15 . In the central nervous system (CNS), Wnt/β-catenin signaling is also crucial for instructing cell fate choices and regulating neuronal differentiation process 16,17 . Notch signaling is a novel pathway that influences many aspects of NSCs 18 ; for examples, Notch signaling is important in the proliferation and fate of NSCs by maintaining the self-renewable state of NSCs, both in vivo and in vitro 19 , and plays an essential role in differentiation to neurons 20 . Notch1, a member of Notch pathway receptor, and its cognate ligand Jagged1 are expressed in the neurogenic niches 21 , especially in the adult hippocampus 22 , which take part in the development of the CNS 20 .
The use of extracorporeal shock wave therapy (ESWT) began in the urological field for lithotripsy and gradually extended to many musculoskeletal diseases and regenerative medicine disorders 23 . Presently, two types of ESWT are used in clinical medical therapy; they are, focused ESWT (fESWT) and radial ESWT (rESWT) 24 . fESWT significantly augments migration and proliferation in fibroblasts and keratinocytes, which improves the process of healing 25 , and fESWT has also been shown to have potential as a non-invasive and innovative technology for the treatment of nerve damage, such as sciatic injury 26 and cerebral ischemia 27 . Although corresponding studies about rESWT are few, several have addressed potential mechanisms. Recently, our studies showed that an appropriate rESWT dose delivered to rat brain helped restore neurological function and increased the number and differentiation of NSCs in vivo 28 . In addition, an effect of rESWT on cells has been demonstrated 29 . and different cell types are differentially influenced by the effects of rESWT 30 . In this study, we used NE-4C mouse NSCs to assess the effect by rESWT on proliferation and differentiation and to further explore the possible signaling pathway of PI3K/AKT, Wnt/β-catenin, and Notch signaling in vitro.

Results
rESWT increased proliferation and differentiation of NE-4C stem cells. An rESWT dose of 2.5 bar, 500 impulses, and 2 Hz significantly increased the NE-4C stem cells 72 h after rESWT compared with the control group (Fig. 1A). The rESWT quantity dose-dependently stimulated proliferation below 2.5 bar, 500 impulses, and 2 Hz. Interestingly, when rESWT exceeded the aforementioned dose, an opposite effect was observed (Fig. 1B). An rESWT dosage of 2.5 bar, 500 impulses, and 2 Hz was used in this study. Neuron-specific enolase (NSE) and β-tubulin III expression levels were measured by immunofluorescence, Western blot, and quantitative reversephase polymerase chain reaction (qRT-PCR). All cultures revealed positive expression for NSE and β-tubulin III, the marker of neuronal cells, on weeks 8 and 12 after the onset of rESWT (Figs 2 and 3), but no difference was discovered before week 8. The fluorescent marker of the control group was significantly less than that of the experimental group ( rESWT increased the protein expression of PI3K and mRNA expression of PI3K and AKT. The expression of PI3K and AKT was measured by Western blot and qRT-PCR. Compared with the control group, rESWT distinctly (P < 0.001) promoted PI3K and AKT expression at 24, 48, and 72 h (Fig. 4A,B,D,E). The mRNA expression levels of PI3K and AKT were also increased (P < 0.001) after rESWT (Fig. 4C,F), which is consistent with the time of stimulating the cell proliferation. The Wnt/β-catenin signaling pathway was induced by rESWT. The Wnt3a and β-catenin response was induced on week 8 and maintained steadily to week 12. rESWT unregulated the Wnt3a and β-catenin protein expression and the mRNA expression detected by immunofluorescence, Western blot, and qRT-PCR . 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). (Figs 5 and 6). The mRNA expression of other key points of the Wnt/β-catenin pathway were also altered; for examples, mRNA expression of low-density lipoprotein receptor-related protein 6 (LRP-6), Axin, and Frizzled increased, whereas that of the glycogen synthase kinase 3β (GSK3β) decreased (Fig. 7). Dkk-1, the inhibitor of Wnt/β-catenin pathway, apparently suppressed the expression of Wnt3a and β-catenin, and the other key point of Wnt/β-catenin pathway, as well as NSE and β-tubulin III expression after rESWT (Figs 2, 3, 5, 6, and 7).   . 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. . 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).

Discussion
The results of the current study demonstrate that rESWT promoted the proliferation and differentiation of mouse NSCs in vitro. As the second generation of fESWT, with waves dispersing eccentrically from the applicator tip 31 , rESWT is a breakthrough in clinical medicine because of its better security than fESWT, its simplicity of use, and especially for its excellent therapeutic results 32 ; for example, rESWT simplifies application without anesthesia or image-guided location and lessens risks by reflecting the pathology zone 32 . Also, rESWT has many transmitters with various penetration depths, which might allow rESWT to satisfy various clinical needs 33 . In recent years, fESWT has aroused the interest of more and more cell researchers who have reported that fESWT enhances the differentiation of human tendon-derived stem cells 29 and stemness of rat and human adipose-derived stem cells 34 . However, few studies have yet explored the effects of rESWT on NSCs. Due to the complicated culture 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 < 0.01, ***P < 0.001) and suppressed by DAPT (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). and differentiation of NSCs in vitro 8 and potential usefulness of rESWT, exploration of rESWT is imperative. Consequently, this study investigated the effect of rESWT on NSCs in vitro.
An appropriate rESWT dose had a powerful effect on NE-4C proliferation within 72 h. The rESWT quantity dependently increased the self-renewal of NSCs within a certain range (below 2.5 bar, 500 impulses, and 2 Hz), which is similar to the report that exposure to rESWT resulted in a number of rESWT-dependent alterations after 48 and 72 h on human fetal foreskin fibroblasts and that the effect may attribute to the mechanical stimulation of rESWT 30 . However, higher energy rESWT induced cell damage and apoptosis. This result is also found using fESWT, which may be related to increasing severity of the shock wave conditions that can increase apoptosis 35 .
NSCs survival in vitro involves the activation of the PI3K/AKT pathway 36 . PI3K is a family of enzymes that phosphorylate the 3'-OH of the inositol ring of phosphatidylinositol 37 . The action of PI3K leads to phosphorylation of AKT to p-AKT. p-AKT could then induce the phosphorylation of the transcription factor FOXO3a, leading to upregulation of Cyclin D1, which increases the number of NSCs 38,39 . Moreover, past studies have shown the inhibition of PI3K/AKT pathway reduces cell divison 36 . These studies emphasize that the PI3K/AKT pathway is vital to proliferation of NSCs. Consistent with previous studies, our data indicate that the PI3K/AKT pathway was activated when NE-4C cells number increased. Compared with the control group, rESWT promoted the proliferation of NSCs, while greatly upregulating PI3K and AKT expression within 72 h in vitro. These results demonstrate that rESWT augmentation of the number of NSCs may be associated with the PI3K/AKT pathway in the early period.
In addition to proliferation, differentiation of NSCs also has a certain role in neurogenesis. In our research, rESWT can not only affect the augment of NSCs, but also boost the regulation to neurons. The neurons markers NSE and β-tubulin III, were, measured to characterize neuronal differentiation. NSE and β-tubulin III expressions were higher on week 8 after rESWT than those in the control group, which indicates that rESWT enhanced the differentiation of NE-4C into neuron-like cells. In fewer than 8 weeks, however, the expression of NSE and β-tubulin III was not increased, which may be due to a long-term need for formation of neurons. With respect to neural development, Wnt/β-catenin signaling is critically required for NSCs differentiation 40 . Wnt3a, a member of Wnt family, has been shown to promote NSCs maturity 41 . Upon Wnt3a binding to the Frizzled receptor, adenomatous polyposis coli (APC) is dissociated and Axin is removed from the Axin/GSK3β/APC complex. Axin's interaction with the Wnt co-receptor LRP-6 causes APC to dissociate from Axin. As a consequence, β-catenin is stabilized, translocates to the nucleus, and interacts with transcription factors, which in turn induces gene expression and causes cells to differentiate [41][42][43] . Previous studies showed that the β-catenin activation in NSCs induces improvement of neuronal number in the adult forebrain 44 . To explore whether the powerful effect of rESWT on NSCs differentiation is related to the Wnt/β-catenin pathway, Wnt3a, β-catenin, and other key points of Wnt/β-catenin pathway, such as LRP-6, Axin, Frizzled, and GSK3β expression, were detected. Our investigation indicates that rESWT enhanced cell differentiation on week 8 with the activation of the Wnt/β-catenin signal. With the application of rESWT, Wnt3a and β-catenin increased significantly after 8 weeks. Moreover, inhibition of Wnt/β-catenin signal pathway with Dkk-1 45 significantly reduced the neuronal maturation of NSCs as evidenced by decreased NSE and β-tubulin III expression. These findings provide further evidence that the rESWT treatment modulates NSCs differentiation via the Wnt/β-catenin signal. Although there are a large number of studies regarding the Wnt/β-catenin pathway, our work is the first that reveals a new strong role of rESWT in NSCs differentiation in vitro that is indicated by the upregulated Wnt/β-catenin pathway.
Recent research of regulating maintenance and neuronal development of NSCs has identified additional pathways including the PI3K/AKT pathway and Wnt/β-catenin signal, Notch pathway 46 . In our study, Notch1, Jagged1, and Hes1 expression were augmented on week 8 and week 12 after rESWT compared with the control group. Notch1 plays a critical role in the development of the CNS 47 , and higher levels of in vitro neuronal differentiation in human NSCs have been observed 48 . Notch1, when activated by transmembrane ligands Jagged1, is cleaved by the Presenilin γ-secretase complex, which liberates Notch1 intracellular domain (NICD) fragments. Then, NICD is released and translocated to the nucleus where it induces target gene expression (in particular, Hes1) 49 . The key point of an increase in Notch pathway expression, which is consistent with the expression of the neuron marker NSE, is that Notch1 signaling can also be considered to be a potent modulator of NSCs maturation and the Wnt/β-catenin signal, as reported in previous studies that NSCs differentiation is driven by increased Notch pathway 50 . NSE expression stimulated by rESWT was significantly suppressed by using the Notch 1 signaling inhibitor DAPT. All these data suggest that the Notch1 signal is involved in inducing the differentiation of NSCs by rESWT. Similar to the Notch1 signal, expression of the NSCs marker nestin was higher in the rESWT group than the control group on weeks 8 and 12, and was reduced by DAPT. In light of the known effect of Notch1 signaling maintaining t NSCs self-renewal 51 , one of the mechanisms of rESWT-induced NSC proliferation at a later period in vitro is Notch1 signaling.
The results of the present study illustrate for the first time that rESWT improved the proliferation and differentiation of NSCs in vitro. This effect was likely caused, at least in part, by an upregulated PI3K/AKT pathway, Wnt/β-catenin signaling, and Notch1 signaling. Although not discussed in this research, crosstalk between these three pathways is also a pivotal factor for the fate of NSCs 52 . Our data provide novel insight into the molecular mechanisms of rEWST on NSCs and important preclinical references supporting the basis for further development of neurological function recovery promoted by NSCs recruitment.

Material and Methods
Cell culture. NE-4C (CRL-2925; ATCC, Manassas, VA) mouse NSCs were grown in Dulbecco's minimal essential medium with high glucose (DMEM, Gibco), supplemented with 10% fetal bovine serum. Cells were cultured according to ATCC culture method guidelines and incubated at 37 °C, 5% CO 2 . NE-4C cells were dissociated with 0.05% trypsin-EDTA and resuspended in the incubator supplemented DMEM described above. The plated cells were then cultured in the incubator, adding DMEM at 37 °C and 5% CO 2 for 24 h before treatment SCIEnTIFIC REPoRTS | 7: 15321 | DOI:10.1038/s41598-017-15662-5 with 20 ng/mL Dkk-1 or DAPT cells, which incubated in designated treatments for 24 h before being lysed for collection. Cells from passage levels 4-5 were used in the present study. The cell culture medium was refreshed every 3 days.

Cell proliferation test. To investigate the possible impact of rESWT on the viability/proliferation of NE-4C
cells, different doses of rESWT (from 1.0 bar to 1.5 bar to 2.0 bar to 2.5 bar to 3.0 bar to 3.5 bar, 500 impulses, and 2 Hz) were used in the cultures. The proliferation of NE-4C cells was analyzed by MTT cell (Promega, Japan) proliferation assay. After being treated with rESWT, 5 × 10 3 cells were seeded into 96-well plates to grow for 12,24,36,48,60, and 72 h. Then, 20 µl of MTT was added to each well, and culture plates were incubated at 37 °C for 2 h. The absorbance was measured by photometry at 490 nm. We chose 2.5 bar, 500 impulses, 2 Hz as a therapeutic dose.
rESWT treatment. To evaluate the influence on cell differentiation in vitro, rESWT (STORZ MEDICAL AG, Switzerland) was applied to the cultures at a dose of 2.5 bar, 500 impulses, and 2 Hz, which maximized the therapeutic effects without significant reduction of the cell viability. Coupling was used to minimize the loss of shock wave energy at the interface between the transmitter and culture bottle. The surface of the culture bottle was evenly coated with the coupling agent, and the culture medium was filled with the culture solution. The transmitter of rESWT acted vertically on the upper part, and the processing time interval was 7 days. The control group was maintained under the same culture conditions, but without rESWT exposure.
Immunofluorescence. Cells grown on coverslips were fixed with 4% paraformaldehyde, followed by treat- , and Jagged1 (1:1000, Abcam) followed by goat anti-rabbit secondary antibody (1:10,000, Abcam, Cambridge, MA). The ECL Prime Western blotting detection reagent (GE Healthcare, Piscataway, NJ) was used for visualization with the Reliance 600 imaging system (Perkin Elmer, Waltham, MA). Blot images were captured and band intensities were quantified using GeneSnap and GeneTools (Perkin Elmer), respectively. Band intensities for proteins of interest were first normalized to GAPDH and then compared with the control group for each experiment.
SCIEnTIFIC REPoRTS | 7: 15321 | DOI:10.1038/s41598-017-15662-5 Statistical analysis. Quantitative data are expressed as the mean SD. All analyses were conducted using SPSS statistical software. Statistical comparisons were performed by one-way ANOVA followed by the Least Significant Difference test to compare the differences at each time. A probability value < 0.05 was considered significant.
Data Availability. All data generated or analysed during this study are included in this published article.