PONE-D-22-09036AICAR attenuates postoperative abdominal adhesion formation by inhibiting oxidative stress and promoting mesothelial cell repairPLOS ONE

Dear Dr. Xue,

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 submit your revised manuscript by Jul 24 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Md Ekhtear Hossain, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf  and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. As part of your revision, please complete and submit a copy of the Full ARRIVE 2.0 Guidelines checklist, a document that aims to improve experimental reporting and reproducibility of animal studies for purposes of post-publication data analysis and reproducibility: https://arriveguidelines.org/sites/arrive/files/Author%20Checklist%20-%20Full.pdf (PDF). Please include your completed checklist as a Supporting Information file. Note that if your paper is accepted for publication, this checklist will be published as part of your article.

3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

4. Thank you for stating the following in the Acknowledgments Section of your manuscript:

“This study was supported by 2021 Science and technology Talents Support project of Shaanxi Provincial People's Hospital (2021JY-08) and (2021JY-15).”

We note that you have provided additional information within the Acknowledgements Section that is not currently declared in your Funding Statement. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“Fei Xue received 2021 Science and technology Talents Support project of Shaanxi Provincial People's Hospital (2021JY-08), Yunhua Wu received 2021 Science and technology Talents Support project of Shaanxi Provincial People's Hospital (2021JY-15),The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

[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: Yes

**********

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: No

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 this manuscript the authors investigated the role of AMPK agonist AICAR in inhibiting post-operative abdominal adhesion formation. The authors showed that AICAR can reduce adhesion formation by inhibiting oxidative stress and facilitate peritoneal repair in a rat model.

Here’s my comments on the manuscript:

Major Comments:

1.Multiple studies indicated that p-eNOS expression and nitric oxide (NO) production were increased by in vivo administration of AICAR; please include results of NO and p-eNOS levels in your in vivo study groups. This data will further justify the effect of AICAR on oxidative stress.

2.As the authors indicated in the discussion section, “Peritoneal mesothelial cell mesenchymal transition (MMT) is an important process in fibrosis” please include the results of α-SMA and vimentin expression levels in your in vivo study groups (like you did for Nrf2).

3.In Figure 5D, the number of visible cells in the TGF-β group is way less than in other groups, which dismantled the validity of the quantitative data; please replace this image with the better one and recalculate the graph accordingly. 

Minor comments:

1.Indicate the full name of AICIR (5-aminoimidazole-4-carboxyamide ribonucleoside) in the Material and Methods section and the purchasing information (company, purity, etc.).

2.Please include brief information about Nair’s scoring system and add a reference.

3.Please includes the purchasing information for the MDA detection kit.

4.In Figure 5D, the authors measured the transcriptional (mRNA) levels of E-cadherin, a-SMA, and Vimentin. Please indicate it in the figure, e.g., Relative mRNA expression of E-cadherin.

5.Please italicize the words in vivo and in vitro in the manuscript. The statistically significant symbol “P” should always indicate by a small letter (and italicize it); please correct it in the manuscript.

6.In Figure 3A, please replace “Relative ROS expression (%)” with “Relative ROS levels (%)” and subsequently correct the figure legend.

Reviewer #2: The manuscript by Xue F et al presented data linking AMPK agonist AICAR with post-operative abdominal adhesion formation. The experimental design was well constructed and showed the protective effect of AICAR on adhesion formation by inhibiting oxidative stress and peritoneal repair in the post-operative rats. Although the results are interesting, the reviewer has couple of comments.

1. In the abstract-back ground section should describe solely context of the research rather than describing results and concluding remark.

2. The authors conclude that AMPK activator AICAR inhibits inflammation and oxidative stress in the adhesion tissue. Thus, the author should first confirm AMPK and inflammatory signaling (i.e TGF-β and NF-kb) activation in the adhesion tissue by western blotting. All they evaluated to confirm anti-oxidative effects of AICAR were ROS and MDA. Thus reviewer is interested to see Sod, catalase and Gpx expression in the adhesion tissue.

3. They have shown inhibitory effects of AICAR on fibrosis and adhesion formation. Therefore, it would be interesting to see the protein abundance of collagen1, α-SMA, integrins and E-cadherin in the adhesion tissue of the post-operative rats.

**********

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: No

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.

AICAR attenuates postoperative abdominal adhesion formation by inhibiting oxidative stress and promoting mesothelial cell repair

PONE-D-22-09036R1

Dear Dr. Xue,

We’re pleased to inform you that your revised 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,

Md Ekhtear Hossain, Ph.D.

Academic Editor

PLOS ONE