Low-Dose Non-Thermal Atmospheric Plasma Promotes the Proliferation and Migration of Human Normal Skin Cells

Round 1

Reviewer 1 Report

This is an interesting study with the fundamental observation that how Low-dose Non-thermal atmospheric plasma can regulate proliferation and migration in human normal skin cells. Overall study is well written with good experimentation, however there are some key consideration that needs to be address.

Author claims that Low dose NTP promotes proliferation and migration via activating PI3K/AKT/mTOR and WNT/Catenin pathway. However, its needs further validation by some more critical experimentation with following conditions,

1-      Author should check the phosphorylation status of these signaling protein for better validation of activation of these pathways rather than by showing only protein expression.

2-      Estimation of Phosphorylation (activation) status of PI3K/AKT/mTOR and WNT/Catenin in NTP treated with and without ROS inhibition, for more clarity of NTP induced ROS mediated signaling in the cells.

3-      Measurement of cell viability and migration after inhibition of PI3K/AKT/mTOR and WNT/Catenin signaling components with and without NTP treatment, will establish the NTP specific activation of these pathway and mediated cell viability and migration regulation.

         Minor-

1-      In method section, author should mention catalog number of reagents used, for transparency and suitability for future researchers to replicate the data.

2-      Author should mention in figure legends the number of times a experiment has been performed and with how many replicates.

Author Response

Comments from the editors and reviewers:

Reviewer: 1

 

1. Author should check the phosphorylation status of these signaling protein for better validation of activation of these pathways rather than by showing only protein expression.

Reply: Thanks a lot for the reviewer’s comments. We strongly agree with the reviewers' suggestion that the activation of phosphorylated proteins better indicates the activation of signaling pathways. Due to limited time, we were unable to detect the phosphorylation status of all the proteins detected, and several representative proteins were selected to detect their phosphorylation expression, as follows:

Figure 9: Effect of Wnt/β-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/β-catenin Pathway Inhibitor, on HaCaT Cell Migration. (B) Count of the number of migrated cells. (C) Effect of NAC on protein phosphorylation. (D) Quantification of phosphorylated protein expression. Date represent the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 , ***p < 0.001with ANOVA compared with the control.

As shown in the Figure 9 C and D, we found that NTP treatment for 15s significantly promoted the expression of P-AKT (Beyotime, China, 1:1000, AF5740) and P-β-catenin (Wuhan, China, 1:1000, AP0979). After NTP treatment and NAC pretreatment, the expression levels of these two phosphorylated proteins were significantly decreased as compared with the 15s treatment group. Moreover, the expression levels of the two phosphorylated proteins were found to be very low when pretreated with NAC alone. The above results indicated that low-dose ROS generated by NTP activated the phosphorylated AKT and β-catenin and promoted their expression, thus promoting the proliferation and migration of HaCaT cells.

 

2. Estimation of Phosphorylation (activation) status of PI3K/AKT/mTOR and WNT/Catenin in NTP treated with and without ROS inhibition, for more clarity of NTP induced ROS mediated signaling in the cells.

Reply: We greatly appreciated the reviewer’s detailed comments. In conjunction with your previous comments, we explored the assessment of the phosphorylation (activation) status of PI3K/AKT/mTOR and WNT/Catenin in the absence and presence of ROS by NTP as shown in Figure 9 C and D. In the group without NAC, we found that NTP treatment for 15s significantly increased the expression of P-AKT and P-β-catenin compared with the control group. The expression levels of P-AKT and P-β-catenin in the NTP-treated group after NAC pretreatment were significantly lower than those in the NTP-treated group without NAC pretreatment. Therefore, we believe that low-dose ROS generated by NTP treatment promoted the expression of phosphorylated AKT and β-catenin proteins.

 

3.Measurement of cell viability and migration after inhibition of PI3K/AKT/mTOR and WNT/Catenin signaling components with and without NTP treatment, will establish the NTP specific activation of these pathway and mediated cell viability and migration regulation.

Reply: We greatly appreciated the reviewer’s detailed comments.

Figure 1 C Short time NTP treatment promoted cell vitality through PI3K/AKT pathway

 

As shown in Figure 1 C, our results from the MTT cell viability assay showed increased cell viability in the NTP-treated 15 s group of HaCaT cells compared to the control group; In addition, the treatment group with the PI3K inhibitor LY294002 (Beyotime, Shanghai, China, S1737) alone had decreased cell viability compared to the control group. Furthermore, the PI3K inhibitor LY294002 pretreatment group prior to NTP showed significantly reduced cell viability compared to the 15 s NTP treatment group. Our results therefore suggest that low-dose NTP treatment promotes HaCaT cell proliferation by activating the PI3K/AKT/mTOR pathway.

In addition, the results of our Transwell experiment showed that compared with the control group in Figure 9 A and B, the number of cells passing through Transwell cells after NTP treatment with HaCaT cells for 15 s was significantly increased. However, groups pretreated with the Wnt/β-catenin inhibitor IWP-2 (Beyotime, Shanghai, China, SF6831) before NTP treatment had significantly fewer cells passing through the compartments compared with the 15 s NTP treatment group. In addition, groups pretreated with the Wnt/β-catenin inhibitor IWP-2 alone also had slightly fewer cells passing through the compartments compared to controls. Therefore, we believe that low-dose NTP treatment promotes the migration of HaCaT cells by activating the Wnt/β-catenin pathway.

4.  In method section, author should mention catalog number of reagents used, for transparency and suitability for future researchers to replicate the data. 

Reply: Thanks a lot for the reviewer’s comments. We have added the catalog numbers of the reagents used as follows: All added numbers are highlighted in blue.

Please see:

2. Materials and Methods

2.1 Cell culture

HaCaT cell was purchased from KeyGEN BioTECH (Cas: 20210513) and cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (LONSERA, Shanghai, China, S711-001S) and 1% penicillin/streptomycin (NCM BioTECH, Suzhou, China, 15140-122), which were cultured at 37℃ and 5% CO₂ incubator (Thermo Fisher Scientific, Waltham, MA, USA). Cells were grown in a single layer on 60 mm petri dishes (Biotech, Shanghai, China, QN2911) until about 80% of the bottom area was covered before NTP treatment.

2.3 Cell viability

 HaCaT cells was cultured in 60 mm petri dishes, and when the concentration required for the experiment was reached, the old medium was discarded and replaced with new medium. After NTP treatment, the cells were incubated for 24 h. The medium was discarded again, and 1.5 mL of preconfigured 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, USA, 298-93-1) working solution was added to each dish. After further incubation in the incubator for 4 h, an equal volume of dimethyl sulfoxide (DMSO, Sangon Biotechnology, Shanghai, China, 67-68-5) was added and shaken for 10 min to completely dissolve the blue-purple crystals in living cells. The culture solution was then transferred to a 96-well plate and the absorbance of each well was measured with a microplate reader at 492 nm. Cell viability was calculated from a standard curve. To investigate the effect of ROS on the viability of HaCaT cell, N-acetyl-L-cysteine (NAC, Beyotime, Shanghai, S0077), a scavenger of ROS, was used to verify the results. HaCaT cells were pretreated with 100 μL NAC (10 mM) for 1 h before NTP treatment and their cell viability was measured again.

2.4 Extracellular reactive species detection

After HaCaT cells were treated with NTP, the concentrations of ROS and RNS in the cell culture medium were detected immediately using H₂O₂ detection kit (Beyotime, Shanghai, S0038) and NO detection kit (Beyotime, Shanghai, S0021S), respectively. Incubated for 4 h after NTP treatment, and detected the concentration of ROS in the medium again. The absorbance was measured at 540 nm, and the concentration was calculated according to the standard curve.

2.5 Intracellular reactive species detection

 Intracellular ROS levels were measured by incubation for 4 h after NTP treatment. A ROS detection kit (KeyGEN BioTECH, China, S0033S) containing 2’,7’-Dichlorodihydro -fluorescein diacetate (DCFH-DA) was used as a fluorescent probe. After the fluorescent probes were mounted, the incubation was continued in the incubator for 30 minutes, and the fluorescence intensity of ROS in the cells was observed and photographed using a fluorescence microscope (Olympus, Tokyo, Japan). Image J software was used for analysis and recording.

2.6 Mitochondrial membrane potential detection

After 4 h of NTP treatment, the original medium was discarded, and then the cell was washed twice with PBS. Then 2 mL DMEM medium was added to each Petri dish, which was added the pre-prepared JC-1 staining working solution and was fully mixed according to the mitochondrial membrane potential kit (JC-1, Solarbio, Beijing, China, M8650). After incubation in the incubator for 20 min, the solution in the Petri dish was discarded, washed twice with pre-prepared JC-1 staining buffer, which was discarded afterwards. Then 2 mL DMEM medium was added to each petri dish again, and the fluorescence images were observed and recorded under a fluorescence microscope. Quantitative fluorescence analysis was performed using Image J software.

2.8 Cell migration assay

 Cell migration assay was performed in 24-well transwell chambers (Costar, Washington, DC, USA, 3415) with 8 μm Wells. Cells were treated with NTP and left for 4 h before being digested with trypsin to prepare a cell suspensions. Cells were counted so that the cell number was approximately 1×10⁵/mL. 200 μL of FBS-free DMEM cell suspension was added to the upper chamber. In the lower chamber, DMEM containing 10% fetal bovine serum (500 μL) was added as a chemical attractant. After incubation for approximately 24 hours, the cells that had crossed the membrane were washed with PBS, fixed with 4% paraformaldehyde and stained with crystal violet. The cells that did not pass through the superior ventricular membrane were gently wiped with a cotton swab, and the remaining cells were observed under a low-power microscope (100×) in five randomly selected fields.

2.9 Cell cycle analysis

Cells grown to about 80% of the bottom of the dish were subjected to NTP treatment and then incubated for 24 h at 37℃ in a 5% CO₂ incubator. The original medium was blotted dry, cells were washed with PBS, dissociated by trypsin, and then fixed by suspension in 70% cold alcohol and overnight at 4℃ temperature. The prefabricated propidium iodide staining solution was added at room temperature and incubated for an additional 30 minutes in the absence of light according to the manufacturer's instructions (KGI Biology, Nanjing, China, KGA512), then the FACs Calibur system (BD Biosciences, Franklin Lakes, NJ, USA) was used for cell cycle analysis. ModFit Version 3.1 Software (Verity Software House, Inc, Topsham, ME, USA) was used to analyze the percentage of cells in G0/G1, S, and G2/M phases.

2.10 Western Blot

WB was used to detect the proteins expression of proliferation-related PI3K-AKT signaling pathway and migration-related WNT/β-catenin signaling pathway. Briefly, proteins (20 μg) were separated by SDS-PAGE (sodium dodecyl sulfatepolyacrylamide gel electrophoresis, P0012A) and then transferred to nitrocellulose membranes. Rabbit anti-PI3K antibody (1:1,000 3011S), rabbit anti-AKT antibody (1:1,000 4685S), rabbit anti-CyclinD1 antibody (1:1,000 55506), rabbit anti-ERK antibody (1:1,000 A16686), rabbit anti-mTOR antibody (1:1,000 2983) and rabbit anti-STAT3 antibody (1:1,000 4904), all the above proteins were purchased from Cell Signaling, USA and used for incubation membranes. rabbit anti-WNT3A antibody (1:1,000 A0642), rabbit anti-β-catenin antibody (1:1,000 A19657) and rabbit anti-ACTB antibody (1:1,000 AC062) were purchased from Abclonal, China and also used for incubation membranes. Horseradish peroxidase-coupled anti-rabbit IgG secondary antibody (1:10,000 AS014) (AB Clone, China) was then incubated for 40 minutes. Chemiluminescence kit (Thermo Fisher Scientific Searle Scientific Company, USA A3855) and chemiluminescence gel imaging system (Tanon, Shanghai, China) were used to detect and quantify protein. Finally, the ratio of fluorescence intensity of each objective band to the 0 s control group was calculated.

 

5. Author should mention in figure legends the number of times a experiment has been performed and with how many replicates.

Reply: We greatly appreciated the reviewer’s detailed comments. Based on your comments, we have added the number of replicates to the legend section as follows:

 

Figure 1: Cell viability of HaCaT cells. (A) HaCaT cell viability after NTP treatment for 24 h. (B) Effect of NTP on the viability of HaCaT cell pre-incubated with NAC. C 1: cells given neither NAC nor NTP treatment ; C 2: cells treated with NAC in the absence of NTP treatment. Date represent the mean ± SD of three independent experiments. *p < 0.05, ***p < 0.001, ****p < 0.0001 with ANOVA compared with the control.

Figure 2: Extracellular NO and H₂O₂ content. (A) The concentration of NO in the cell culture medium after NTP treatment for 0, 5, 10, 15, 20 and 25 s. (B) The concentration of H₂O₂ in the cell culture medium for 0 h and 4 h after NTP treatment for 0, 5, 10, 15, 20, and 25 s. Date represent the mean ± SD of three independent experiments. ”ns” was not considered statistically significant. *p < 0.05, **p < 0.001, ****p < 0.0001 with ANOVA compared with the control.

Figure 3: Intracellular ROS levels. (A) Fluorescence images of intracellular ROS generation in HaCaT cells. (B) ­The quantification by measuring fluorescence pixel intensity using Image J software. Date represent the mean ± SD of three independent experiments. *p < 0.05, ****p < 0.0001 with ANOVA compared with the control.

Figure 4: Mitochondrial membrane potential in HaCaT cells. (A) Red fluorescence image of mitochondrial membrane potential in HaCaT cells. (B) Quantification was performed by measuring the intensity of red and green fluorescence pixels in HaCaT cells using Image J software. (C) Green fluorescence image of mitochondrial membrane potential in HaCaT cells. (D) The ratio of red to green fluorescent pixels was quantified using Image J software. Date represent the mean ± SD of three independent experiments. *p < 0.05, **p < 0.001, ***p < 0.001, ****p < 0.0001 with ANOVA compared with the control.

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 Image J software. (C) Migration of HaCaT cells treated with NTP by the Transwell assay. (D) Quantitative analysis was performed by measuring the number of HaCaT cells crossing the membrane using Image J software. Date represent the mean ± SD of three independent experiments. *p < 0.05, **p < 0.001, ***p < 0.001, ****p < 0.0001 with ANOVA compared with the control.

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. Date represent the mean ± SD of three independent experiments. ”ns” was not considered statistically significant. *p < 0.05, **p < 0.001, ****p < 0.0001 with ANOVA compared with the control.

Figure 7: NTP activated the PI3K/AKT and STAT3 pathways in HaCaT cells. (A) The expression of PI3K, AKT, mTOR, Cyclin D1, ERK and STAT3 were determined by WB. (B) Th­e quantification by measuring the protein expression with Photoshop software. Date represent the mean ± SD of three independent experiments. **p < 0.001, ****p < 0.0001 with ANOVA compared with the control.

Figure 8: NTP activated the WNT3A/β-catenin pathways in HaCaT cells. (A) The expression of WNT3A, β-cetenin and Cyclin D1 were determined by WB. (B) Th­e protein expression quantification measured with Photoshop software. Date represent the mean ± SD of three independent experiments. ****p < 0.0001 with ANOVA compared with the control.

Reviewer 2 Report

The authors have investigated the proliferation and migration of HaCaT cells after cold atmospheric pressure plasma treatment. This can be published after modification. 

1. The authors have only reported the results on cell viability, proliferation, and migration increases for low-dose NTP and decrease for high-dose NTP. The possible explanations are not mentioned anywhere in the manuscript.  Please explain. Some reasons for high dose NTP can be found in

http://dx.doi.org/10.1109/TRPMS.2023.3235358

Role of cold atmospheric plasma in microbial inactivation and the factors affecting its efficacy - ScienceDirect 

2. Why this contradiction happens in Figure 4? Please explain.

3. Figure 6 is not clear. Replace a better one.

4. Why s-phase cell decrease with plasma treatment time, but the M-phase cell increased? 

5. What are the limits of used plasma source for this kind of study? 

Author Response

Reviewer: 2

1. The authors have only reported the results on cell viability, proliferation, and migration increases for low-dose NTP and decrease for high-dose NTP. The possible explanations are not mentioned anywhere in the manuscript. Please explain.

Reply: Thanks for reviewer’s kindly comments. Studies have shown that cells show different cell states and fates to different concentrations of H₂O₂ [1, 2]. For nerve cells, at levels below 1nM H₂O₂, cell growth is slow and development and regeneration are impaired; In addition, the cells tended to be in a resting state with no tendency to proliferate and differentiate. When the nerve cells were exposed to H₂O₂ in the range of 1–10 nM, the growth of axons and dendrites was promoted. It was mainly because the cells produced oxidative stress under this H₂O₂ concentration, which stimulated the proliferation and differentiation of cells. In addition, the study also showed that a modest increase in H₂O₂ concentration (up to about 100 nM) could further promote dendritic growth. Abnormally high H₂O₂ (more than 100 nM) can lead to the death of nerve cells and tissue degradation [1].

Based on the relationship between different H₂O₂ concentrations and the state and fate of nerve cells, we believe that this property may exist in all cells, including HaCaT cells. Therefore, according to our results, a certain concentration of H₂O₂ was present in the NTP-treated medium and it increased in a dose-dependent manner with the NTP treatment time. Besides, the results of our cell viability, cell scratches, cell cycle and Western Blot experiments also showed that a short time of NTP treatment (10s or 15s) could promote cell proliferation, enhance cell viability and promote the expression of proliferation-related signaling pathway proteins. However, the results of a long time of NTP treatment (20s or 25s) were completely opposite. In addition, the results of cell viability experiments with pre-addition of NAC (reactive oxygen species H₂O₂ scavenger) showed that NTP treatment had no significant effect on cell viability at this treatment time (Figure 1). In conclusion, it is reasonable to believe that the effect of NTP treatment on the proliferation and migration of HaCaT cells is directly related to the H₂O₂ concentration produced, which is involved in metabolic regulation and stress response through the main proxy signal transduction of specific protein targets, to support cells in adapting to changing environments and pressures. Of course, different cells respond differently to different H₂O₂ concentrations, but the mechanisms are consistent.

• Sies, H.; Jones, D.P. Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat Rev Mol Cell Biol. 2020, 21(7):363-383. doi:10.1038/s41580-020-0230-3.
• Schieber, M., & Chandel, N. S. (2014). ROS function in redox signaling and oxidative stress. Current biology: CB, 24(10), R453–R462. https://doi.org/10.1016/j.cub.2014.03.034.

In addition, we have included the explanation of the response here in the discussion section of the document. Please see Page 14, Line 531-557.

 

2. Why this contradiction happens in Figure 4? Please explain.

Reply: Thanks a lot for the reviewer’s comments. The important comments are very helpful for us to significantly improve the quality of the manuscript.

After careful analysis, the contents of Figure 4 are not contradictory. Our JC-1 (Cell Mitochondrial Membrane Potential Detection Kit) test showed that the ratio of the red fluorescence to the green fluorescence of HaCaT cells firstly increased and then decreased when the NTP treatment time was from 0 s to 25 s. We know that when the mitochondrial membrane potential of cells is high, JC-1 accumulates in the mitochondrial matrix to form polymers, which can produce red fluorescence. When the mitochondrial membrane potential was low, JC-1 could not be aggregated in the mitochondrial matrix, and JC-1 as a monomer could produce green fluorescence. In addition, studies have shown that mitochondrial dysfunction caused by high-dose sodium tungstate is related to the destructive effects of mitochondrial respiratory chain [1]. It has also been demonstrated that cadmium-induced apoptosis and mitochondrial damage in BEAS-2B cells are also closely related to the increase of intracellular ROS levels [2]. Therefore, in our experiment, when NTP treated HaCaT cells for 5s, 10s or 15s, the red fluorescence in cells increased, indicating that the cells were in good condition and the mitochondrial membrane potential was high. Combined with the results of cell viability and cell cycle experiments in our 5s, 10s and 15s NTP-treated groups, the cell viability was enhanced and the cells were promoted to proliferation at this NTP treatment time. However, the green fluorescence of cells under this NTP treatment time is very weak. Therefore, it was considered that the ROS generated during this NTP treatment time promoted the function of the respiratory chain of the mitochondrial membrane in cells, so the ratio of red fluorescence to green fluorescence was gradually increased.

When the treatment time of NTP was increased to 20s and 25s, the red fluorescence in cells almost disappeared, while the green fluorescence was strong. These results indicated that ROS generated during this NTP treatment time could damage the mitochondrial respiratory chain, decrease mitochondrial membrane potential, and even lead to early apoptosis. Therefore, the ratio of the red fluorescence to the green fluorescence in this NTP treatment time gradually decreased.

• Cheraghi, G., Hajiabedi, E., Niaghi, B., Nazari, F., Naserzadeh, P., & Hosseini, M. J. (2019). High doses of sodium tungstate can promote mitochondrial dysfunction and oxidative stress in isolated mitochondria. Journal of biochemical and molecular toxicology, 33(4), e22266. https://doi.org/10.1002/jbt.22266
• Cao, X., Fu, M., Bi, R., Zheng, X., Fu, B., Tian, S., Liu, C., Li, Q., & Liu, J. (2021). Cadmium induced BEAS-2B cells apoptosis and mitochondria damage via MAPK signaling pathway. Chemosphere, 263, 128346. https://doi.org/10.1016/j.chemosphere.2020.128346

3. Figure 6 is not clear. Replace a better one

Reply: Thanks a lot for the reviewer’s detailed and thorough comments. We have replaced Figure 6 with a clearer one based on your comments.

Revised parts:

 

 

4. Why s-phase cell decrease with plasma treatment time, but the M-phase cell increased ?

Reply: Thanks for reviewer’s kindly comment and suggestion. Our results showed that when the HaCaT cells were treated with NTP for 5 s or 10 s, the number of cells in the S phase was significantly increased. The research of Choi et al. has shown that in the cell division phase, the increased number of cells evolving from G1 phase to S phase or even G2 phase indicates cell proliferation[1]. The results indicated that NTP treatment time of 5 s and 10 s promoted cell proliferation. When the NTP treatment time was 15s, the cells in the S phase were decreased significantly, but the cells in the G2 phase were increased significantly, indicating that the 15s NTP treatment time also promoted the division process and proliferation of cells.

However, when the time for NTP to treat HaCaT cells was further increased to 20s, the S-phase cells were further decreased, while the M-phase cells were further increased. Moreover, when the NTP treatment time was increased to 25s, there was no significant change in the proportion of cells in M phase compared with the 25s treatment group. Studies have shown that ROS produced by high-dose NTP can kill cancer cells by blocking them in the G2/M phase [2]. The G2/M phase arrest of cells may be due to DNA damage and the initiation of cell checkpoints to prevent mitosis [3]. Therefore, we believed that when the treatment time of NTP was increased to 20s or even 25s, the ROS content produced by high-dose NTP was too high, which blocked HaCaT cells in G2/M phase and prevented further mitosis of cells, thereby inhibiting cell proliferation. In the 10s and 15s treatment groups, low-dose NTP-produced ROS promoted the production of oxidative stress in cells, and promoted the transformation of G1-phase cells into S-phase or even G2-phase, thus promoting cell proliferation.

• Choi JH, Song YS, Song K, Lee HJ, Hong JW, Kim GC. Skin renewal activity of non-thermal plasma through the activation of β-catenin in keratinocytes. Sci Rep. 2017;7(1):6146. Published 2017 Jul 21. doi:10.1038/s41598-017-06661-7.
• Liu, T.; Zhao, X.; Song, D.; Liu, Y.; Kong, W. Anticancer activity of Eremanthin against the human cervical cancer cells is due to G2/M phase cell cycle arrest, ROS-mediated necrosis-like cell death and inhibition of PI3K/AKT signalling pathway. J BUON. 2020, 25(3): 1547-1553.
• Karki, S. B.; Gupta, T. T.; Yildirim-Ayan, E.; Eisenmann, K. M.; Ayan, H. Miniature Non-thermal Plasma Induced Cell Cycle Arrest and Apoptosis in Lung Carcinoma Cells. Plasma Chemistry and Plasma Processing 2019, 40(1): 99-117. doi: 10. 1007/s11090-019-10037-2

 

5. What are the limits of used plasma source for this kind of study?

Reply: We greatly appreciate the reviewer’s comment. There are two common devices for low temperature plasma, dielectric barrier discharge device and plasma jet device [1]. The device used in our laboratory is a dielectric barrier discharge device. This type of device has the characteristics of large discharge area, high plasma intensity, and is suitable for processing specimens with large area. However, the plasma generated by the plasma jet device is relatively soft and has a small area. Plasma jet devices are generally made as plasma needles or plasma guns for better application to smaller wound surfaces. Then there is the problem that the unity of the device cannot be guaranteed, for the use of low temperature plasma to study wound healing.

Second, various substances generated by plasma are different, so there are many uncertain factors that cannot completely determine whether plasma treatment of wounds brings side effects to cells or even human body. In addition, our current plasma device is only suitable for cell experiments and cannot be applied to clinical patients at present. A large number of animal experimental and clinical data are needed to improve and perfect the plasma device finally used for clinical application.

[1] Yan, D., Sherman, J. H., & Keidar, M. (2017). Cold atmospheric plasma, a novel promising anti-cancer treatment modality. Oncotarget, 8(9), 15977–15995. https://doi.org/10.18632/oncotarget.13304

 

In addition to the above changes, we have also made the following changes:

Please see

Page 3 Line 128-135:

In addition, in order to explore whether short-term NTP treatment (15 s) promoted the viability of HaCaT cells through the PI3K/AKT/mTOR pathway, we added PI3K inhibitor LY294002 (Beyotime, Shanghai, China, S1737) to conduct an MTT experiment, the working concentration was 25μM , and divided the cells into four groups: the control group (without NTP treatment and LY294002 pretreatment), the NTP treatment group (NTP treatment 15 s), the inhibitor group (LY294002 pretreatment only) and the inhibitor pretreatment group (LY294002 pretreatment before NTP treatment). MTT experimental method was the same as before.

 

Page 4 Line 182-189:

In addition, in order to explore whether short-term NTP treatment (15 s) promoted the migration of HaCaT cells through the Wnt/β-catenin pathway, we added IWP-2 (Beyotime, Shanghai, China, SF 6831), an inhibitor of the Wnt/β-catenin pathway to conduct the Transwell experiment, the working concentration was 25 nM. The cells were divided into four groups: control group (no NTP treatment and IWP-2 pretreatment), NTP treatment group (NTP treatment for 15 s), inhibitor group (IWP-2 pretreatment only) and inhibitor pretreatment group (IWP-2 pretreatment before NTP treatment). Transwell experimental method is the same as before.

Page 5 Line 219-225:

To further validate the effect of low-dose ROS produced by NTP treatment on the phosphorylation of protein, we supplemented the WB experiments with p-AKT (Beyotime, China, 1:1000, AF5740) and p-β-catenin (Wuhan, China, 1:1000, AP0979). According to the needs of the experiment, they were divided into four groups: control group (no NTP treatment and NAC pretreatment), NTP treatment group (NTP treatment for 15 s), NAC group (NAC pretreatment only) and NAC pretreatment group (NAC pretreatment before NTP treatment for 15 s).

 

 

 

Page 8 Line 336-341:

According to the previous research results, we believed that intracellular ROS generated by low-dose NTP promoted cell viability by increasing intracellular mitochondrial membrane potential, while ROS generated by high-dose NTP could induce early apoptosis and inhibit cell viability by reducing intracellular mitochondrial membrane potential.

Page 12 Line 493:

while ROS produced by high-dose NTP may inhibit the migration of HaCaT cells by inactivating WNT/β-catenin signaling pathway (Figure 10).

 

Page 13 Line 495-502:

 

 

Page 13 Line 504:

Figure 10: Schematic representation of the mechanism by which NTP treatment promoted proliferation and migration of HaCaT cells. ROS produced by low-dose NTP could promote the proliferation and migration of HaCaT cells by promoting the expression of PI3K, AKT, mTOR, ERK, PI3K, Cyclin D1, WNT and β-catenin.

 

 

Page 14 Line 509-530:

3.9 Low-dose ROS promotes HaCaT cell proliferation and migration by activating the PI3K/AKT and Wnt/β-catenin pathways.

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/β-catenin pathways, we again used WB experiment to detect the expressions of AKT and β-catenin phosphorylation. The results showed that the phosphorylated expressions of AKT and β-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, 9D). Besides, we also explored the effects of PI3K/AKT pathway inhibitor LY294002 on the activity of HaCaT cells and the effect of Wnt/β-catenin pathway inhibitor IWP-2 on the migration ability of HaCaT cells. 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 Transwell's 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, 9B). 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/β-catenin pathways.

 

 

Page 16 Line 632-634:

8. Sarthak, D.; Veda, P. G.; Sarita, M.; Gagandeep, S.; Satyananda, K. Role of cold atmospheric plasma in microbial inactivation and the factors affecting its efficacy. Health Sciences Review. 2022, Volume 4, Published 2022 Sep 1 , doi: 10.1016/j.hsr.2022.100037.

 

Page 19 Line 795-796:

68. Schieber, M.; Chandel, N.S. ROS function in redox signaling and oxidative stress. Curr Biol. 2014. 24(10):R453-R462. doi:10.1016/j.cub.2014.03.034

 

Round 2

Reviewer 1 Report

NA

Reviewer 2 Report

I am satisfied with the responses. Now it can be published.