Negative air ions through the action of antioxidation, anti-inflammation, anti-apoptosis and angiogenesis ameliorate lipopolysaccharide induced acute lung injury and promote diabetic wound healing in rat

Yu-Hsuan Cheng; Hung-Keng Li; Chien-An Yao; Jing-Ying Huang; Yi-Ting Sung; Shiu-Dong Chung; Chiang-Ting Chien

doi:10.1371/journal.pone.0275748

Peer Review History

Original SubmissionMay 5, 2022
19 Jul 2022 Decision Letter - Y. Peter Di, Editor PONE-D-22-13242Negative Air Ions through the Action of Antioxidation, Anti-inflammation, Anti-apoptosis and Angiogenesis Ameliorate Lipopolysaccharide Induced Acute Lung Injury and Promote Diabetic Wound Healing in RatPLOS ONE Dear Dr. Chien, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== Please make sure to address the reviewers' comments especially regarding the clarity of the experiments (details, reagents, and designs), updated references, and in-depth discussion. ============================== Please submit your revised manuscript by Aug 26 2022 11:59PM. 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[Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In the present manuscript entitled: Negative Air Ions through the Action of Antioxidation, Anti-inflammation, Anti-apoptosis and Angiogenesis Ameliorate Lipopolysaccharide Induced Acute Lung Injury and Promote Diabetic Wound Healing in Rat, the authors focused to investigate the impact of NAI on LPS-induced acute lung injury and on the healing process of skin wounds in STZ-induced diabetic rats. Several points should be seriously taken in consideration for the following raisons: 1- The authors should alphabetically arrange the key words. 2- The authors should respect the use of abbreviations throughout the manuscript. The abbreviation must be defined at the first appear in the text and then the authors should use the abbreviations throughout the manuscript. 3- In the materials and methods section, the authors should add the information about the primary and secondary antibodies used for IHC analysis. 4- The discussion section must be improved, and the authors must add more recent references. 5- In the figure 1B &C and Figure 2B & C, the authors should add symbol for significance. 6- In the figure 5, the authors must normalize the phosphor-p65 to the total p65 not to beta-actin. Similarly, the authors must normalize the phosphor-IkB to the total IkB not to beta-actin. 7- In the figure 3A, the expression levels of cleaved caspase-3 must be normalized to the total caspase-3 not to beta-actin. 8- In figure 9 the authors used Masson’s trichrome stain to monitor collagen expression in the wounded skin tissues, but I think Sirius red stain is more suitable for this purpose. Reviewer #2: The authors explored the benefits of negative air ions on rats in terms of acute inflammatory injury and chronic skin recovery, respectively. Although the experiments look interesting, some logical flaws and unclear expressions were presented in the manuscript. Thus, I would recommend the authors address the following comments before further consideration: 1. Figure 1B-C, the authors are trying to detect ROS level by using chemiluminescence analyzer, and the positive control group and/or negative control group of ROS level should be presented to show the accuracy of this test. For example, recognized antioxidants are recommended to use. Why did the saline group show very high levels of ROS? 2. Line 86, please make sure the detail of data from reference is correct. It is “320-350 000 ions/cm2” in the paper (Sirota et al. 2008) instead of “320-35 million ions/cm3”. 3. Line 90-99, the authors seem to want to explore COVID-19-induced ALI, so why not use SARS-CoV-2 spike protein instead of bacterial LPS to stimulate rats? 4. The authors did not mention the time of infection in the acute lung injury experiment, which is very confusing. Figure 3-5, the time frame is not shown for the changes in the protein levels following the LPS infection. 5. In Figure 5, it is very arbitrary for the authors to determine the level of inflammation in a lung by quantifying the number of cells in the pathology images. Instead of doing this, the authors should have collected alveolar lavage fluid and then quantified the level of inflammation in the lungs by the number of inflammatory cells. 6. In Figure 6, the expression of Beclin-1 was not significantly suppressed in the LPS group vs. Con group, which is contrary to the author's description (Line 294-295). 7. The pathway proteins in this study are superficial and part of the conclusions cannot be supported by the current data. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes: Gamal Badr Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0275748.r001
Revision 1
8 Sep 2022 Author Response Response to the Reviewer #1: In the present manuscript entitled: Negative Air Ions through the Action of Antioxidation, Anti-inflammation, Anti-apoptosis and Angiogenesis Ameliorate Lipopolysaccharide Induced Acute Lung Injury and Promote Diabetic Wound Healing in Rat, the authors focused to investigate the impact of NAI on LPS-induced acute lung injury and on the healing process of skin wounds in STZ-induced diabetic rats. Several points should be seriously taken in consideration for the following raisons: 1- The authors should alphabetically arrange the key words. Ans: Thanks for your comments. We have rearranged the key words alphabetically. 2- The authors should respect the use of abbreviations throughout the manuscript. The abbreviation must be defined at the first appear in the text and then the authors should use the abbreviations throughout the manuscript. Ans: Thanks for your comments. We have carefully used the abbreviations throughout the revised manuscript. 3- In the materials and methods section, the authors should add the information about the primary and secondary antibodies used for IHC analysis. Ans: Thanks for your comments. We have added the information about the primary and secondary antibodies used for IHC analysis. These messages have been included in the revised methods as following: “We assessed the degree of apoptosis by evaluating the expression of caspase 3 and PARP and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay (DNA Fragmentation Kit; BioVision, Milpitas, California, USA) and autophagy by Beclin-1 (Cell Signaling Technology, Inc.) and LC3-II (Cell Signaling Technology, Inc.) staining in the rat lungs. The determined techniques have been followed as reported previously [Yang et al., 2022]. In brief, tissue sections from 10% formalin fixation and paraffin embedding were deparaffinized, rehydrated, and stained with H&E or immunohistochemically. For autophagy or apoptosis related proteins staining, the 5-µm tissue sections (Leica RM 2145, Nussloch, Germany) were incubated overnight at 4°C with mouse anti-rat Beclin-1 antibody (BD Biosciences, San Jose, CA, 1:100) or LC3-II (Cell Signaling Technology, Inc., 1:100), Caspase 3 (CPP32/Yama/Apopain, Upstate Biotechnology, Lake Placid, NY, 1:100), PARP (Cell Signaling Technology, Inc., 1:100). Subsequently, biotinylated secondary antibodies (Dako, Botany, NSW, Australia) were applied, followed by incubation with streptavidin-conjugated horseradish-peroxidase (Dako). The chromogen used in this study was Dako Liquid diaminobenzidine. Twenty high-power (�400) fields were randomly selected from each gastric section, and the value of brown deposits/total section area for Beclin-1, LC3-II, caspase-3 or PARP stain was analyzed with Adobe Photoshop 7.0.1 image software. TUNEL was performed according to a previously described method [Yang et al., 2008]. Briefly, 5-µm-thick sections of gastric tissues were prepared, deparaffinized, and stained using the TUNEL assay kit.” (Line) “In brief, tissue sections from 10% formalin fixation and paraffin embedding were deparaffinized, rehydrated, and stained with VEGFR-2 or TGF-β1. For VEGFR-2 or TGF-β1 staining, the 5-µm tissue sections were incubated overnight at 4°C with mouse anti-rat VEGFR-2 antibody (VEGF #9698 Cell Signaling Technology, Danvers, MA, USA, 1:100) or TGF-β1 (sc-146 Santa Cruz, Dallas, Texas, USA, 1:100). Next, biotinylated secondary antibodies (Dako, Botany, NSW, Australia) were applied, followed by incubation with streptavidin-conjugated horseradish-peroxidase (Dako). The chromogen used in this study was Dako Liquid diaminobenzidine. Twenty high-power (�400) fields were randomly selected from each section, and the value of brown deposits/total section area for VEGFR-2 or TGF-β1 stain was analyzed with Adobe Photoshop 7.0.1 image software as described above.” (Lines 197-210) 4- The discussion section must be improved, and the authors must add more recent references. Ans: Thanks for your comments. We have rewritten the discussion section with red color and included more recent references. 5- In the figure 1B &C and Figure 2B & C, the authors should add symbol for significance. Ans: Thanks for your comment. The Figure 1B is an original graph, therefore, no statistic symbol could be added. We have added the symbol for significance in the Figure 1C and Figures 2B & 2C. 6- In the figure 5, the authors must normalize the phosphor-p65 to the total p65 not to beta-actin. Similarly, the authors must normalize the phosphor-IkB to the total IkB not to beta-actin. Ans: Thanks for your comment. We have no data involving phosphor-p65 and phosphor-IkB in this experiment. 7- In the figure 3A, the expression levels of cleaved caspase-3 must be normalized to the total caspase-3 not to beta-actin. Ans: Thanks for your comment. We have used the total caspase-3 to replace �-actin in the new Figure 3A. 8- In figure 9 the authors used Masson’s trichrome stain to monitor collagen expression in the wounded skin tissues, but I think Sirius red stain is more suitable for this purpose. Ans: Thanks for your comments. We have included the Sirius red stain in new Figure 10. The information of Sirius red stain has been indicated in the Methods, Results and Discussion sections of the revised manuscript. Response to the Reviewer #2: Reviewer #2: The authors explored the benefits of negative air ions on rats in terms of acute inflammatory injury and chronic skin recovery, respectively. Although the experiments look interesting, some logical flaws and unclear expressions were presented in the manuscript. Thus, I would recommend the authors address the following comments before further consideration: 1. Figure 1B-C, the authors are trying to detect ROS level by using chemiluminescence analyzer, and the positive control group and/or negative control group of ROS level should be presented to show the accuracy of this test. For example, recognized antioxidants are recommended to use. Why did the saline group show very high levels of ROS? Ans: Thanks for your comments. We used saline containing H2O2 as positive control and saline with vitamin C containing H2O2 as negative control in the revised Figure 1. In our methods, 200 µL of saline with or without NAIs treatment was mixed with 0.5 mL of 0.1 mmol/L luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, Sigma, Chemical Co., St. Louis, MO, USA) and 0.1 mL of H2O2 (0.03%) and was analyzed with a chemiluminescence analyzing system (CLA-ID3, Tohoku Electronic Inc. Co., Sendai, Japan). The chemiluminescence signals emitted from the mixture of saline, H2O2 and luminol, which represented the hydrogen peroxide content in the mixture, were recorded. The increased chemiluminescent signals from the sample–luminol–H2O2 mixture were determined for 300 s. Therefore the saline group showed a very high level of ROS for the containing H2O2 (0.03%). 2. Line 86, please make sure the detail of data from reference is correct. It is “320-350 000 ions/cm2” in the paper (Sirota et al. 2008) instead of “320-35 million ions/cm3”. Ans: Thanks for your comment. We have corrected the data “320-350 000 ions/cm2” in the revised text. 3. Line 90-99, the authors seem to want to explore COVID-19-induced ALI, so why not use SARS-CoV-2 spike protein instead of bacterial LPS to stimulate rats? Ans: Thanks for your comments. We have included new reference to state that “SARS-CoV and SARS-CoV-2 could induce acute lung injury (ALI) and inflammation, which is mimicked by lipopolysaccharide (LPS), a bacterial endotoxin present on the outer membrane of Gram-negative bacteria [Pooladanda et al., 2021], implicating SARS-CoV-2 and LPS through similar pathways of binding toll-like receptor-4 (TLR4) to activate angiotensin converting enzyme 2 (ACE2), to induce ALI and hyperinflammation [Aboudounya et al., 2021; Yang et al., 2021].” in the Introduction section. 4. The authors did not mention the time of infection in the acute lung injury experiment, which is very confusing. Figure 3-5, the time frame is not shown for the changes in the protein levels following the LPS infection. Ans: Thanks for your comment. The methods for the time of infection were rewritten as following. “During the stabilization period of 30 min after anesthesia, 200 μg/kg of LPS stimulation was immediately infused into trachea at 200 μl for 210 min. Physiological functions were continuously recorded for 30 min of baseline period and 210 min of experimental period, and arterial blood gases and respiratory mechanics were recorded at the end of the experiment. The NAIs treatment to the rats by 10-cm distance of NAIs exposure were performed for 210 min. Control rats were also placed indoors, but are only exposed to the air in the environmental chamber. We determined the respiratory rate, flow rate and breathing depth in these animals in total 240 min.” The protocol was also indicated in the new Figure 2A. The time frame of Figures 3-5 has also indicated in each figure legend. 5. In Figure 5, it is very arbitrary for the authors to determine the level of inflammation in a lung by quantifying the number of cells in the pathology images. Instead of doing this, the authors should have collected alveolar lavage fluid and then quantified the level of inflammation in the lungs by the number of inflammatory cells. Ans: Thanks for your comment. We have done the larvage neutrophils and reactive oxygen species amount according to your suggestion in the new Figures 5E& 5F. 6. In Figure 6, the expression of Beclin-1 was not significantly suppressed in the LPS group vs. Con group, which is contrary to the author's description (Line 294-295). Ans: Thanks for your comments. We have reworked a new western blot of Beclin-1 in the revised text as shown in new Figure 6. This data now is consistent to the description on Line 294-295. 7. The pathway proteins in this study are superficial and part of the conclusions cannot be supported by the current data. Ans: Thanks for your comments. We have modified the conclusions for consistency and clarification according to the current data. https://doi.org/10.1371/journal.pone.0275748.r002
22 Sep 2022 Decision Letter - Y. Peter Di, Editor Negative Air Ions through the Action of Antioxidation, Anti-inflammation, Anti-apoptosis and Angiogenesis Ameliorate Lipopolysaccharide Induced Acute Lung Injury and Promote Diabetic Wound Healing in Rat PONE-D-22-13242R1 Dear Dr. Chien, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Y. Peter Di, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors properly answered all the raised comments and the revised manuscript is now suitable for publication. Reviewer #2: (No Response) ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes: Gamal Badr Reviewer #2: Yes: Zhonghui Zhu ******** https://doi.org/10.1371/journal.pone.0275748.r003
Formally Accepted
13 Oct 2022 Acceptance Letter - Y. Peter Di, Editor PONE-D-22-13242R1 Negative Air Ions through the Action of Antioxidation, Anti-inflammation, Anti-apoptosis and Angiogenesis Ameliorate Lipopolysaccharide Induced Acute Lung Injury and Promote Diabetic Wound Healing in Rat Dear Dr. Chien: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Y. Peter Di Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0275748.r004

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