## 3 Results and Discussion
### Effects of NTP Treatment on Cell Cycle  
To further elucidate the mechanism of NTP stimulating cell proliferation, flow cytometry was used to examine the cell cycle. Compared with the control group, the results showed that the number of S-phase cell in the 10 s NTP treatment group (25.25%) was significantly higher than that in the control group (18.23%), and the result from the 5 s NTP treatment group (19.38%) was also slightly higher than that of the control group. With increased in NTP treatment time, the cycle phase of treated cells was gradually increased. The number of S-phase cell in the 15 s treatment group (4.72%) suddenly decreased, and M-phase cells increased significantly from the 15 s treatment group (54.6%) to the 25 s treatment group (83.5%) in a dose-dependent manner (Figure 6A,B), which showed that low-dose NTP treatment (10 s) promoted the proliferation of HaCaT cells by increasing the proportion of S-phase cells, while high-dose NTP advanced the cell cycle progress, arrested cells in the G2/M phase, and inhibited cell proliferation.  
Figure 5: Effect of NTP treatment on HaCaT cell migration. (**A**) Migration of HaCaT cells treated with NTP by the scratch wound healing assay. (**B**) Quantitative analysis was performed by measuring the scratch area of HaCaT cells using ImageJ software. (**C**) Migration of HaCaT cells treated with NTP by the transwell assay, the picture scale size was the same as (**A**). (**D**) Quantitative analysis was performed by measuring the number of HaCaT cells crossing the membrane using ImageJ software. Data represent the mean \(\pm\) SD of three independent experiments.* \(p<0.05\),** \(p<0.001\),** \(p<0.001\),** \(***p<0.0001\), *** \(p<0.0001\) with ANOVA compared with the control.  
Moreover, ROS produced by low-dose NTP promoted the proliferation of fibroblasts by promoting the transition from G1 to S phase [38], while ROS produced by high-dose NTP tended to arrest cells in the G2/M phase and fought cancer [54]. The G2/M phase arrest of cells might be due to DNA damage, which initiated cell cycle checkpoints to prevent mitosis [55]. In our study, the results showed that the number of cells int the S phase gradually increased in the 5 s and 10 s treatment groups compared with the control group. However, when the NTP treatment time increased to 15 s, the number of S phase cells suddenly decreased, and the number of G2/M phase cells gradually increased with the increase in the treatment time. The decrease in S phase cells after NTP treatment of HaCaT cells for 15 s seemed to be inconsistent with the overall result in the article. However, we could see that although the number of cells in the G2/M phase increased sharply after NTP treatment of 15 s, the increase in cells in the G2 phase was more significant, suggesting that NTP treatment of cells for 15 s might promote the process of cell division and thus cell proliferation, which is consistent with the overall result in the article. In addition, compared with the NTP 15 s treatment group, the M-stage cells in the 20 s treatment group and the 25 s treatment group were significantly increased (Figure 6), suggesting that the cells might be blocked in the cell division stage at this time, and the cells stayed in the cell division stage and did not continue to divide, thus cell proliferation was inhibited. Therefore, ROS generated by low-dose NTP promoted the proliferation of HaCaT cells, while ROS generated by high-dose NTP blocked the cells in the G2/M phase and inhibited cell proliferation.  
Figure 6: Effect of NTP treatment on HaCaT cell cycle. (**A**) The cell cycle distribution of HaCaT cells after NTP treatment was detected by flow cytometry. (**B**) The cell population at each stage was expressed as a percentage of the total number of cells. Data represent the mean \(\pm\) SD of three independent experiments. \({}^{n}\)ns” was not considered statistically significant. \({}^{*}\)\(p<\) 0.05, \({}^{**}\)\(p<\) 0.001, \({}^{***}\)\(p<\) 0.0001 with ANOVA compared with the control.