PONE-D-19-33705

Beneficial effects of benfotiamine, a NADPH oxidase inhibitor, in isoproterenol-induced myocardial infarction in rats

PLOS ONE

Dear Dr Ahmed,

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 note, that lack of novelty (reviewer 2) is not a valid criticism in PLOS ONE.

We would appreciate receiving your revised manuscript by Feb 27 2020 11:59PM. When you are 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.

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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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Michael Bader

Academic Editor

PLOS ONE

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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 http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. At this time, we ask that you please provide the product number and any lot numbers provided with the Isoproterenol-HCl and benfotiamine purchased from Sigma-Aldrich in the study."

3. At this time, we request that you  please report additional details in your Methods section regarding animal care, as per our editorial guidelines:

(a) Please include the method of euthanasia  

(b) Please describe the care received by the animals, including the frequency of monitoring and the criteria used to assess animal health and well-being.

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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: Partly

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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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: No

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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: Ahmed an coauthors elegantly analysed the effects of a NADPH oxidase inhibitor, benfotiamine, on isoproterenol-induced myocardial infarction in rats. They choose an acute therapeutic model and applied benfotiamine after induction of MI, or gave benfotiamine two weeks before induction of MI. Beside measurement of classic echocardiographic outcome parameters they analyzed several plasma markers by ELISA.

Observed effects were clear and are presented in a detailed fashion. There is no need for improvement of analyses.

Figures may be improved by more concise denomination of groups eg control, Iso-A, Iso-A+BT, Iso-B, Iso-B+BT instead of "prophylactic" or "treatment". For the measurement of parameters ELISA or colorimetric assays were used. Specify and sensitivity should be given for each test, as well as number of measurements per sample (N and n).

Detailed figure legends are missing and should be provided.

The discussion may be focussed to the main observed effects, as it appears cloudy and long. Katare et al published in 2010 comparable results by applying Benfotiamine in a streptocytozin mouse model. The reviewer would appreciate a detailed discussion of the 2010 results in comparison of the presented results. I feel these data should be considered as well. Kadare R et al. JMCC, 49, 625-538.

Reviewer #2: The current study explores the potential of benfotiamine (BFT) as cardio-protective agent via direct inhibition of NADPH oxidase (NOX). Although an interesting study, the authors fail to illustrate the novelty of this work. Previous reports also show a role for BFT as cardio-protective agent with (indirect) antioxidant properties, including inhibition of NOX activity and augmentation of antioxidant systems (e.g. GSH). The novelty of the current study is therfore not clear. There are also a number of issues that the authors should address:

1) A discussion of the method of ischemia induction used here, versus Langendorff or coronary artery ligation used in other studies, should be included - specifically how the methods would impact results.

2) As NOX is a central molecule in the study, the measurement of NOX enzyme content should be supplemented by activity assays. Determination of the NOX subunits and isoforms may also add value.

3) More detailed descriptions of the methods used is needed.

4) Why was PKC used as a marker of inflammation but not one of the "conventional" markers (e.g. IL-6 or TNF alpha)? Also, which PKC isoforms play a role?

5) Were all the data normally distributed?

6) Please indicate p-values when reporting significant differences in text.

7) A measurement of NADPH levels in the cardiac tissue may add an explanation for the results observed. This may facilitate a mechanistic explanation for the link between BFT and decreased NOX activity.

8) The manuscript may benefit from language editing as some grammatical errors occur throughout the text.

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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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-19-33705R1

Beneficial effects of benfotiamine, a NADPH oxidase inhibitor, in isoproterenol-induced myocardial infarction in rats

PLOS ONE

Dear Dr Ahmed,

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 still raised by the reviewer 2.

We would appreciate receiving your revised manuscript by Apr 17 2020 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Michael Bader

Academic Editor

PLOS ONE

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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 #2: (No Response)

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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 #2: Partly

**********

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

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 #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 #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 #2: Thank you to the authors for responding to the comments from the first review. The manuscript is strengthened by the additions. However, some major concerns still persist and these points have to be addressed before the manuscript is of a high enough standard to be considered for publication. The authors should address the following points and clearly indicate in their response where in the manuscript these changes were made (line and page):

1. Please indicate in the discussion section how the simulated MI with ISO treatment differs from methods where ischemia is simulated - similarities or differences in results and parameters measured here (ECG and blood markers of cardiac damage vs. more direct measures of ischemic damage, e.g. infarct size). Also indicate how benfotiamine offers cardio-protection, making reference to previous studies (PMID: 20107192; 24286628).

2. As the authors strongly conclude that NOX activation is the primary oxidative stress-inducing mechanism, some measure of its activity is warranted. The concerns of non-specific measures may be addressed by including specific NOX and other system inhibitors in the experimental setup. Also, the premise of the article, as mentioned in line 343 as well as the title, is that benfotiamine exerts protective effects as a "NADPH oxidase inhibitor". The inhibitory effect is, however, indirect via transketolse activation. This distinction should be made clearer and all the more reason to provide stronger experimental evidence for NOX activation. Please also discuss results in context with previous studies (e.g. PMID 24747137).

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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 #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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

Beneficial effects of benfotiamine, a NADPH oxidase inhibitor, in isoproterenol-induced myocardial infarction in rats

PONE-D-19-33705R2

Dear Dr. Ahmed,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Michael Bader

Academic Editor

PLOS ONE

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