## 3 Results and Discussion
### NTP Activated the WNT3A/\(\beta\)-catenin Pathways in HaCaT Cells  
In addition to the scratch test and transwell test, we also used WB test to further study the mechanism of NTP promoting cell migration. Results showed that the expression of WNT3A and \(\beta\)-catenin reached the peak in the 15 s NTP treatment group, while the expression of Cyclin D1 reached the peak in the 10 s NTP treatment group, the expression of all proteins gradually decreased with further extension of NTP treatment time (Figure 8A,B). Therefore, we also concluded that low-dose NTP might promote HaCaT cell migration by activating the WNT/\(\beta\)-catenin pathway, while high-dose NTP inhibits it by inhibiting this pathway. The WNT/\(\beta\)-catenin signaling pathway was an important intracellular activation pathway for cell migration, and studies had shown that the expression levels of protein related to this signaling pathway were also significantly increased in HaCaT cells with promoted migration levels [36, 59, 60]. In this study, the results showed increased WNT3A and \(\beta\)-catenin protein expression in the NTP low-dose treatment group and decreased WNT3A and \(\beta\)-catenin protein expression in the NTP high-dose treatment group. In addition, it has been shown that ROS were involved in activating the WNT/\(\beta\)-catenin pathway [61]. Therefore, we can conclude that ROS produced by low-dose NTP may promote the migration of HaCaT cells by activating the WNT/\(\beta\)-catenin signaling pathway, while ROS produced by high-dose NTP may inhibit the migration of HaCaT cells by inactivating the WNT/\(\beta\)-catenin signaling pathway. Low-Dose ROS Promotes HaCaT Cell Proliferation and Migration by Activating the PI3K/AKT and Wnt/\(\beta\)-catenin Pathways  
We also explored the effects of the PI3K/AKT pathway inhibitor LY294002 on the activity of HaCaT cells and the effect of the Wnt/\(\beta\)-catenin pathway inhibitor IWP-2 on the migration ability of HaCaT cells. An MTT assay showed that the cell viability of the NTP-treated group was significantly higher than that of the control group. The inhibitor LY294002-pretreated group showed significantly reduced cell viability compared with the NTP-treated group (Figure 1C). In addition, the results of the Transwell experiment showed that the number of migrated cells in the NTP treatment group was significantly higher than that in the control group. The number of migrated cells was significantly reduced in the inhibitor IWP-2-pretreated group when compared with the NTP-treated group (Figure 9A,B). Figure 8: NTP activated the WNT3A/\(\beta\)-catenin pathways in HaCaT cells. (**A**) The expression of WNT3A, \(\beta\)-catenin, and Cyclin D1 were determined by WB. (**B**) The protein expression quantification measured with Photoshop software. Data represent the mean \(\pm\) SD of three independent experiments. *** \(p\) < 0.0001 with ANOVA compared with the control. To further explore whether short-term NTP treatment (15 s) promoted the proliferation and migration of HaCaT cells through the low-dose ROS-generated activation of PI3K/AKT and Wnt/\(\beta\)-catenin pathways, expressions of AKT and \(\beta\)-catenin phosphorylation were also detected by using a WB experiment. The results showed that the phosphorylated expressions of AKT and \(\beta\)-catenin in the NTP treatment group were significantly higher than those in the control group. In addition, phosphorylation was significantly reduced in the NAC-pretreated group when compared with the NTP-treated group (Figure 9C,D). In conclusion, the above results indicated that short-term NTP treatment (15 s) promoted the proliferation and migration of HaCaT cells through the generation of low-dose ROS that activated PI3K/AKT and Wnt/\(\beta\)-catenin pathways (Figure 10). Figure 9: Effect of Wnt/\(\beta\)-catenin pathway inhibitor IWP-2 on HaCaT cell migration and effect of NAC on protein phosphorylation. (**A**) Picture of effects of IWP-2, a Wnt/\(\beta\)-catenin pathway inhibitor, on HaCaT cell migration.