Effects of low-intensity pulsed ultrasound on nerve growth factor-induced neurite outgrowth mainly through ERK-CREB pathway in PC12 cells

Nerve growth factor (NGF) therapy has been considered as potential therapeutic approach for the treatment of neural injury and degeneration. However, the high concentration of NGF may exert adverse effects and long period treatment of NGF raises an issue of medical cost. Thus, to investigate combination of NGF therapy with other approach to promote neurite outgrowth is of great interest. Here, we intend to explore the possibility and associated molecular mechanisms of combining NGF and low-intensity pulsed ultrasound stimulation (LIPUS) in promoting neurite outgrowth, which is an essential process in neuronal regeneration. PC12 cells, which will differentiate into neurons upon NGF stimulation as the in vitro model. LIPUS (1MHz) was applied to PC12 cells for 10 min every other day with spatial average intensity 50 mW/cm², 20% duty cycle, 100 Hz pulse repetition frequency, and then incubated with NGF. It is found that LIPUS alone could not significantly induce neurite outgrowth in PC12 cells. However, LIPUS could significantly enhance the neurite length in the presence of NGF. In addition, LIPUS alone could increase the phosphorylation of ERK1/2, Akt and CREB. However, LIPUS combined with NGF could only increase the levels of p-ERK1/2 and p-CREB, but not change the level of p-Akt. Taken together, these results provide important insights that LIPUS synergistically enhances NGF-induced ERK1/2-CREB pathway to promote neurite outgrowth.