Liquid plasma as a treatment for cutaneous wound healing through regulation of redox metabolism

Abstract

The skin functions as the outermost protective barrier to the internal organs and major vessels; thus, delayed regeneration from acute injury could induce serious clinical complications. For rapid recovery of skin wounds, promoting re-epithelialization of the epidermis at the initial stage of injury is essential, wherein epithelial keratinocytes act as leading cells via migration. This study applied plasma technology, which has been known to enable wound healing in the medical field. Through in vitro and in vivo experiments, the study elucidated the effect and molecular mechanism of the liquid plasma (LP) manufactured by our microwave plasma system, which was found to improve the applicability of existing gas-type plasma on skin cell migration for re-epithelialization. LP treatment promoted the cytoskeletal transformation of keratinocytes and migration owing to changes in the expression of integrin-dependent focal adhesion molecules and matrix metalloproteinases (MMPs). This study also identified the role of increased levels of intracellular reactive oxygen species (ROS) as a driving force for cell migration activation, which was regulated by changes in NADPH oxidases and mitochondrial membrane potential. In an in vivo experiment using a murine dorsal full-thickness acute skin wound model, LP treatment helped improve the re-epithelialization rate, reaffirming the activation of the underlying intracellular ROS-dependent integrin-dependent signaling molecules. These findings indicate that LP could be a valuable wound management material that can improve the regeneration potential of the skin via the activation of migration-related molecular signaling within the epithelial cell itself with plasma-driven oxidative eustress.

Conflict of interest statement

The authors declare no competing interests.

© 2023. The Author(s).

Figures

Fig. 1. Effects of LP treatment on cell migration.

A Scratch wound migration assay after LP treatment on HaCaT (human keratinocyte) cells. The cells were plated in six-well plates, grown to 90% confluency, and with a monolayer denuded with a sterile pipette tip. Scratch wound migrations (scale bars = 500 μm) were documented by photography after 24 h of incubation. The data graph presents the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01, and ***P < .001. B SEM analysis (scale bars = 10 μm) of morphological changes in cytoskeletal (red indicated) structure in HaCaT cells that were either left untreated or treated with various diluted LP for 24 h. C Gelatin zymography for matrix metalloproteinase (MMP) in HaCaT cells. LP increased MMP-2 enzyme activity. Bar graph presents the mean ± standard deviation of three independent experiments. ***P < 0.001. D Quantitative PCR analysis was used for the expression level of MMP-2 in HaCaT cells. The mRNA expression of MMP-2 was significantly increased in the group treated with LP. Asterisks indicate statistically significant differences (***P < 0.001).

Fig. 2. LP increases the expression of proteins involved in extracellular matrix (ECM) signaling.

A Expression of proteins involved in ECM signaling in HaCaT cells was confirmed by western blot. The degree of protein expression of integrin β1, p-FAK, and p-paxillin was significantly increased with LP (1/4) treatment time than the control group. All the western blotting experiments were performed under the same condition. After transferring the blots onto PVDF membranes, we immediately cropped the targeted blots according to referenced indicating markers. Targeted proteins were immunoblotted with the specific antibody for protein normalization. B Integrin β1 protein levels were determined by immunocytochemistry after LP (1/4) treatment for 24 h. Representative fluorescence microscopic images of HaCaT cells are shown. Scale bars = 50 μm. The increase in the expression of ECM molecule after LP treatment was confirmed using immunofluorescence assay. The green-stained stock in the cytoplasm represents p-FAK (C), and p-paxillin (D). The amount of green-stained stock increased in the LP-treated group. We performed Texas-Red conjugated phalloidin to visualize the cytoskeleton (F-actin) and used Hoechst 33258 to label cell nuclei. Each figure is representative of three experiments with triplicates.

Fig. 3. Treatment of LP increases cellular oxidative stress and mitochondrial membrane potential (∆Ψm).

A The HaCaT cells treated with LP for 24 h were analyzed using flow cytometry, with dihydroethidium and 5-(and-6)-carboxy-2’,7’-dichlorodihydrofluorescein diacetate staining. B For the measurement of mitochondrial superoxide, the cells were incubated with 2.5 mM of MitoSOX and then stained and analyzed using flow cytometry. In (A) and (B), the data graph presents the mean ± standard deviation of three independent experiments. ***P < 0.001. C Measurement of ∆Ψm with JC-1. The JC-1 fluorescence shifted from red-orange to green, indicating depolarization of the ∆Ψm. The ∆Ψm change was measured objectively using a fluorescence microscope and flow cytometry. Bar graph presents the mean ± standard deviation of three independent experiments, calculated as a percentage of the control. **P < 0.01, ***P < 0.001. D Intracellular ROS stimulated with LP induces expression of NOX3 protein in HaCaT cells. Western blotting analysis for NOX3 antibody. GAPDH was used as the loading control.

Fig. 4. Correlation among LP-induced cellular ROS generation and ECM signaling activation.

The HaCaT cells were incubated for 1 h before treatment with LP in the presence or absence of NAC (10 mM). A Scratch wound migration assay after LP treatment on HaCaT (human keratinocyte) cells. LP significantly increased cell migration across the cell stripped area, but significantly attenuated it when treated with the ROS scavenger, NAC. B Gelatin zymography for MMP-2. NAC attenuated the enzymatic activity of MMP-2 increased by LP. Bar graphs represent the mean ± standard deviation of three independent experiments. ***P < 0.001. C Quantitative PCR for MMP-2. The mRNA levels of MMP-2 decreased after NAC treatment. Each figure is representative of three experiments with triplicates. **P < 0.01, ***P < 0.001. D, B SEM images confirmed the effect of NTP on cell morphology. The cytoplasm of LP-treated HaCaT cells showed an increase in horizontal polarization and contraction and cytoplasmic protrusion compared with the control group (red arrow), but it was significantly inhibited in NAC-treated cells. E Western blot analysis. Integrin β1,3,5, p-FAK, and p-paxillin expression were suppressed significantly after NAC treatment to HaCaT cells. F–H Immunofluorescence analysis confirmed whether NAC could attenuate the expression of integrin β1 (F) and ECM molecules (p-FAK (G), and p-paxillin (H)) increased by LP. We performed Texas-Red conjugated phalloidin to visualize the cytoskeleton (F-actin); we used Hoechst 33258 to label cell nuclei. Each figure is representative of three experiments with triplicates. Scale bars = 50 μm.

Fig. 5. LP treatment promoted wound regeneration of murine dorsal acute skin wounds by activating intracellular ROS and epithelial cell adhesion molecules.

Twenty rats were separated into untreated control (n = 10) or LP treatment (n = 10) groups. A Images represent control and LP-treated wounds in rat dorsum. Two full-thickness wounds, 8 mm in diameter, were created on both sides of a rat’s upper back. Left: photographs of both sides of control and LP-treated (upper) and PBS (control) or LP-soaked gauze applied wounds (lower). Right: photograph of initially created wound on day 1 and its cross-section histologic image. Scale bar = 3000 μm. B Photographs of dorsal skin wound closures (left), which were controlled and treated with LP on days 1, 3, 7, 11, and 14. Quantification of the relative wound area (%) to the initial wound in control and LP-treated wounds (right). Mean ± SEM, *P < 0.05, **P < 0.01. C Left: On days 7 and 14 after wound creation, the wounds’ cross-sections stained by H&E staining showed decreased wound diameter in the LP-treated group. Ki-67 expressions were detected by IHC in each wound epithelium (black squares) and demonstrated increased expression in the basal layer of the epithelium in the LP-treated group. Scale bar = 3000 μm (H&E images)/100 μm (IHC images). Right: Positive immunostaining area of Ki-67 in both control and LP-treated groups on days 7 and 14 represented graphically. Mean ± SEM. D Left: To evaluate the effect of LP treatment on wound tissues, we performed NOX3, integrin β1, p-FAK, and p-paxillin IHC staining in both control and LP-treated groups on days 7 and 14. The IHC images in each box are 10× the magnification of the star-marked area in inserted H&E images of each box with an original magnification of 40×. Scale bar = 100 μm (IHC images), 3000 μm (H&E images). Right: Positive immunostaining area of each marker in both control and LP-treated groups on days 7 and 14 represented graphically. Mean ± SEM, *P < 0.05, **P < 0.01, and ***P < 0.001. E Schematic of LP treatment on keratinocytes and intracellular ROS mediated adhesion molecule activation as a subsequent process for promoting cell migration and wound regeneration.