Effects of Plasma-Activated Water on Skin Wound Healing in Mice

Dehui Xu, Shuai Wang, Bing Li, Miao Qi, Rui Feng, Qiaosong Li, Hao Zhang, Hailan Chen, Michael G Kong, Dehui Xu, Shuai Wang, Bing Li, Miao Qi, Rui Feng, Qiaosong Li, Hao Zhang, Hailan Chen, Michael G Kong

Abstract

Cold atmospheric plasma (CAP) has been widely used in biomedicine during the last two decades. While direct plasma treatment has been reported to promote wound healing, its application can be uneven and inconvenient. In this study, we first activated water with a portable dielectric barrier discharge plasma device and evaluated the inactivation effect of plasma-activated water (PAW) on several kinds of bacteria that commonly infect wounds. The results show that PAW can effectively inactivate these bacteria. Then, we activated tap water and examined the efficacy of PAW on wound healing in a mouse model of full-thickness skin wounds. We found that wound healing in mice treated with PAW was significantly faster compared with the control group. Histological analysis of the skin tissue of mice wounds showed a significant reduction in the number of inflammatory cells in the PAW treatment group. To identify the possible mechanism by which PAW promotes wound healing, we analyzed changes in the profiles of wound bacteria after PAW treatment. The results show that PAW can significantly reduce the abundance of wound bacteria in the treatment group. The results of biochemical blood tests and histological analysis of major internal organs in the mice show that PAW had no obvious side effects. Taken together, these results indicate that PAW may be a new and effective method for promoting wound healing without side effects.

Keywords: bacterial inactivation; plasma-activated water; wound healing.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A) Schematic diagram of the internal connection of the portable device and (B) photograph of the portable device.
Figure 2
Figure 2
Inactivation effect of plasma-activated water (PAW) on Pseudomonas aeruginosa. The bacterial inactivation efficiency on Pseudomonas aeruginosa following incubation with PAW for 5 min after activating water for 1, 3, and 5 min in terms of (A) relative total ATP content and (B) relative total bacterial count. The results of activating water for 5 min and the effects of PAW incubation for 3, 7, and 11 min in terms of (C) relative total ATP content and (D) relative total bacterial count of Pseudomonas aeruginosa. All data are expressed as the mean ± SD of three separate experiments.
Figure 3
Figure 3
Inactivation effect of PAW on various kinds of bacteria. The results of activating water for 5 min and the effects of PAW incubating on bacteria for 5 min in (A) Pseudomonas aeruginosa, (B) Escherichia coli, (C) Staphylococcus aureus, and (D) Salmonella paratyphi-B, and (E) the statistical results of (A–D).
Figure 4
Figure 4
Timeline for animal experiments. The wounds of the mice in the treatment group had completely healed on day 17, and the wounds of the mice in the control group had completely healed on day 23.
Figure 5
Figure 5
Photographs of the wound healing process. Photographs of the mice wounds were captured at different time points (days 0, 4, 7, 10, and 17) for (A) the PAW treatment group and (B) the control group. (C) The results of the statistical analysis of wound diameters of mice in the treatment group (red lines) and the control group (blue lines) on days 0, 4, 7, 10, and 17. (D) The results of the analysis of the time required for the wounds to completely heal in the two groups of mice. Data are expressed as the mean ± SD, n = 5; **p < 0.01 (Student’s t-test).
Figure 6
Figure 6
Representative histology of skin wounds in mice on day 10. The wound skin tissues of mice from (A) the control group and (B) the PAW treatment group were stained by hematoxylin and eosin.
Figure 7
Figure 7
Differences in the types of bacterial operational taxonomic units (OTU) on skin wounds from (A) the control and (B) PAW treatment groups.
Figure 8
Figure 8
A heat map showing the differences in the bacterial abundance in (A) the control (n = 4) and (B) PAW treatment (n = 3) groups. Red indicates that the genus is more abundant in the sample than in other samples, and blue indicates that the genus is less abundant in the sample than other samples.
Figure 9
Figure 9
Relative abundance of some major bacterial operational taxonomic units (OTU) in the wound site in the control and PAW treatment group. (A) Azoarcus (Bacteria, Proteobacteria, Betaproteobacteria, Rhodocyclales, Rhodocyclaceae), (B) Enterococcus (Bacteria, Firmicutes, Bacilli, Lactobacillales, Enterococcaceae), (C) TM7_3 (Bacteria, TM7) and (D) Spirochaetes (Bacteria, Spirochaetes).
Figure 10
Figure 10
Representative histology of heart, liver, spleen, lung, and kidney stained by hematoxylin and eosin.

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