PONE-D-22-24419The Role of Magnesium Ions in the Improvement of Vascular Function in Exhausted RatsPLOS ONE

Dear Dr. Wang,

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. It is advised that results on the effect of moderate exercise on the mediators analyzed with that of the observations made in this study are to be included. Bibliography included should be as latest as possible. Statistical analyses are to be redone for better clarity. Provide strong rationale for the study in the introduction. The limitations of the study should be mentioned in the discussion.

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Suresh Yenugu

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PLOS ONE

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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

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

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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

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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

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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: The manuscript is technically sound and presents scientifically relevant data. However, some points need to be addressed. The authors compare control rats to EEE rats and show an increase in interleukin-1 β (IL-1β), tumor necrosis factor –α (TNF-α), malondialdehyde (MDA), reactive oxygen species (ROS) in serum. A decrease in reported in the nitric oxide (NO) and endothelin 1 (ET-1) ratio. However, there is no comparison to moderate exercise, which the authors mention has been inversely linked to mortality. Therefore, the concentrations of these mediators in moderate exercise conditions need to mentioned at least in discussion for a clear understanding of this study.

Minor changes:

1. Page 7: change 'scientifical regular physical activity'.

2. Page 7: 'exercise time exceeding' to 'exercise time exceed'.

3. Page 7: 'making the body in a state of' to 'triggering'

and similar language changes in rest of the text are required.

Reviewer #2: This study aimed to evaluate the effects of Mg2+ on vascular function damage caused by a long-term exhaustive exercise.

According to the authors, the long-term exhaustive exercise induced an increase on oxidative stress, leading to inflammation, while Mg2+ can significantly improve the vasomotor function of exhaustive exercised rats.

The study is well organized and written. The methods used are all well-accepted and described. Results are presented in a logical manner, which facilitates the comprehension of the paper. The finds will certainly contribute to the academic area.

Please find below some suggestions and comments in the attempt to contribute to the quality of the manuscript:

1- In my opinion the title should be more specific, addressing directly the main find of the manuscript;

2- The references used in the manuscript are appropriate. However, only 21 from all 61 references used (~34%) are from the last 5 years;

3- A clear stated hypothesis would benefit the manuscript. I would suggest authors include a hypothesis at the end of the Introduction, and discuss if it was confirmed or not at the beginning of the Conclusion section;

4- The discussion section should start by presenting the main find of the study;

5- Regarding statistical analysis, I understand that the use of independent t-test is appropriate, however, the one-way ANOVA (described in methods) is not. Considering the results presented on figures 5E and 6E, we can observe the presence of 2 factors (groups and Mg2+ concentration), what makes necessary the use of a two-way ANOVA;

6- Also, report magnitude regarding statistical significance is not recommended (“P < 0.05 means significant difference and P < 0.01 means extremely significant difference”; page 12). I would recommend authors to calculate and provide the effect sizes (Cohen’s d or Hedges’ g) of each comparison, which can provide a greater value for understanding the magnitude of the observed changes;

7- Authors have shown an increase of IL-1beta and TNF-alfa serum concentration. A balance between pro- and anti-inflammatory cytokines is essential to understand the real inflammatory state induced by a specific treatment. Is it possible to evaluate some anti-inflammatory cytokines? If not, please consider use the literature to discuss this topic more detailed in the discussion;

8- Another topic that could be considered in the discussion is the known reaction that occurs between anion superoxide and nitric oxide (forming peroxynitrite), which is much faster than the dismutation of superoxide by the antioxidant enzyme SOD. Considering the participation of peroxynitrite in oxidative stress and inflammation, combined to the results obtained in this study, I would recommend authors to consider add some discussion regarding this process;

9- Does this study have any limitations? It would be good to have it described in the discussion. Some “directions” for further studies should be also added.

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Reviewer #1: No

Reviewer #2: No

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Magnesium Ions Improve Vasomotor Function in Exhausted Rats

PONE-D-22-24419R1

Dear Dr. Wang,

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.

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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,

Suresh Yenugu

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

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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

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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: My comments and criticisms have all been well addressed. I recommend the acceptance of this manuscript.

Reviewer #2: I congratulate the authors for this revised version of the manuscript. All my suggestions and comments have been adequately addressed.

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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: No

Reviewer #2: Yes: Rafael Herling Lambertucci

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