Low-Intensity Extracorporeal Shock Wave Therapy Enhances Brain-Derived Neurotrophic Factor Expression through PERK/ATF4 Signaling Pathway

Bohan Wang, Hongxiu Ning, Amanda B Reed-Maldonado, Jun Zhou, Yajun Ruan, Tie Zhou, Hsun Shuan Wang, Byung Seok Oh, Lia Banie, Guiting Lin, Tom F Lue, Bohan Wang, Hongxiu Ning, Amanda B Reed-Maldonado, Jun Zhou, Yajun Ruan, Tie Zhou, Hsun Shuan Wang, Byung Seok Oh, Lia Banie, Guiting Lin, Tom F Lue

Abstract

Low-intensity extracorporeal shock wave therapy (Li-ESWT) is used in the treatment of erectile dysfunction, but its mechanisms are not well understood. Previously, we found that Li-ESWT increased the expression of brain-derived neurotrophic factor (BDNF). Here we assessed the underlying signaling pathways in Schwann cells in vitro and in penis tissue in vivo after nerve injury. The result indicated that BDNF were significantly increased by the Li-ESWT after nerve injury, as well as the expression of BDNF in Schwann cells (SCs, RT4-D6P2T) in vitro. Li-ESWT activated the protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK) pathway by increasing the phosphorylation levels of PERK and eukaryotic initiation factor 2a (eIF2α), and enhanced activating transcription factor 4 (ATF4) in an energy-dependent manner. In addition, GSK2656157-an inhibitor of PERK-effectively inhibited the effect of Li-ESWT on the phosphorylation of PERK, eIF2α, and the expression of ATF4. Furthermore, silencing ATF4 dramatically attenuated the effect of Li-ESWT on the expression of BDNF, but had no effect on hypoxia-inducible factor (HIF)1α or glial cell-derived neurotrophic factor (GDNF) in Schwann cells. In conclusion, our findings shed new light on the underlying mechanisms by which Li-ESWT may stimulate the expression of BDNF through activation of PERK/ATF4 signaling pathway. This information may help to refine the use of Li-ESWT to further improve its clinical efficacy.

Keywords: PERK/ATF4 pathway; Schwann cells; brain-derived neurotrophic factor; low-intensity extracorporeal shock wave treatment.

Conflict of interest statement

Tom F. Lue is a consultant to Acoustic Wave Cell Therapy, Inc. All others have no conflict of interest.

Figures

Figure 1
Figure 1
Expression levels of brain-derived neurotrophic factor (BDNF) in penis were significantly increased by the low-intensity extracorporeal shock wave therapy (Li-ESWT) after nerve injury. Bilateral cavernous nerve crush injury (BCNI) rats were treated with Li-ESWT (0.06 mJ/mm2, 3 Hz, 500 pulses) twice a week. (a) BDNF expression levels in penile tissues in control and Li-ESWT groups were measured by reverse transcription-polymerase chain reaction (RT-PCR) at 3, 10, 20, and 26 days (n = 6); (b) Compared to the control group, the expression levels of BDNF in penis was significantly increased at 3, 10, 20, and 26 days (p < 0.05) by the usage of Li-ESWT, and kept at a stable level until up to 26 days after the nerve injury. M: DNA molecular weight marker.
Figure 2
Figure 2
Li-ESWT treatment enhanced BDNF expression in RT4-D6P2T cells and activated intracellular signaling in a pulse-dependent fashion. (a) BDNF expression levels in SCs cells in control and Li-ESWT groups (n = 3 in triplicates); (b) BDNF expression was higher in Li-ESWT groups (p < 0.05); (c) Protein levels of p-PERK (phosphorylated protein kinase RNA-like endoplasmic reticulum (ER) kinase), PERK, p-eIF2α (phosphorylated eukaryotic initiation factor 2 α), eIF2α, ATF4, and β-actin at different pulses (0, 50, 100, 300, 500, and 1000) (n = 3 in triplicates); (d) Phosphorylation level of PERK increased significantly at pulses 50, 100, 300, 500, and 1000 (* p < 0.05). Intensity ratios depicted in corresponding bar graphs were calculated using phosphorylated and total protein expression; (e) Phosphorylation level of eIF2α increased significantly at pulses 100, 300, 500, and 1000 (* p < 0.05). Intensity ratios depicted in the corresponding bar graphs were calculated using phosphorylated (p-) and total protein expression; (f) Expression level of ATF4 increased significantly at pulses (300, 500, and 1000, * p < 0.05). Intensity ratios depicted in the corresponding bar graphs were calculated using ATF4 and β-actin expression.
Figure 3
Figure 3
GSK2656157 effectively inhibited the phosphorylation of PERK and eIF2α and the expression of ATF4 in RT4-D6P2T. (a) Expression of p-PERK, PERK, p-eIF2α, eIF2α, and ATF4 with usage of Li-ESWT and PERK inhibitor (In) GSK2656157 (n = 3 in triplicates); (b) The phosphorylation level of PERK increased significantly with Li-ESWT, and could be blocked by GSK2656157 (* p < 0.05). Intensity ratios depicted in the corresponding bar graphs were calculated using phosphorylated (p-) and total protein expression; (c) Phosphorylation level of eIF2α increased significantly with Li-ESWT, and could be blocked by GSK2656157 (* p < 0.05). Intensity ratios depicted in corresponding bar graphs were calculated using phosphorylated (p-) and total protein expression; (d) Expression of ATF4 increased significantly with Li-ESWT, and could be blocked by GSK2656157 (* p < 0.05). Intensity ratios depicted in the corresponding bar graphs were calculated using ATF4 and β-actin expression.
Figure 4
Figure 4
Silencing of ATF4 with siRNA in RT4-D6P2T cells in vitro. (a) The expression of BDNF, ATF4, hypoxia-inducible factor (HIF)1α, and glial cell-derived neurotrophic factor (GDNF) with siRNA and Li-ESWT (n = 3 in triplicates); (b) Silencing of ATF4 eliminated the effect of Li-ESWT on the expression of BDNF (p < 0.05); (c) Silencing of ATF4 eliminated the effect of Li-ESWT on the expression of ATF4 (* p < 0.05); (d) siATF4 and Li-ESWT had no effect on HIF1α (* p > 0.05); (e) siATF4 and Li-ESWT had no effect on GDNF (* p > 0.05).

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