PONE-D-22-05865Leonurine inhibits cardiomyocyte pyroptosis against cardiac fibrosis via the TGF-β/Smad2 signaling pathwayPLOS ONE

Dear Dr. Zhu,

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 several points raised during the review process (please, see below). Moreover, the manuscript requires significant English editing.

Please submit your revised manuscript by Jul 17 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,

Luis Eduardo M Quintas, 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. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service. 

Whilst you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://aje.com/go/plos) for a 15% discount off AJE services. To take advantage of our partnership with Editage, visit the Editage website (www.editage.com) and enter referral code PLOSEDIT for a 15% discount off Editage services.  If the PLOS editorial team finds any language issues in text that either AJE or Editage has edited, the service provider will re-edit the text for free.

Upon resubmission, please provide the following:

The name of the colleague or the details of the professional service that edited your manuscript

A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a *supporting information* file)

A clean copy of the edited manuscript (uploaded as the new *manuscript* file).

3. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information on the animal research and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering.

4. Thank you for stating the following financial disclosure: 

This work was supported by the Macau Science and Technology Development Fund (FDCT 0007/2019/AKP, 0021/2020/AGJ, 0011/2020/A1). The National Natural Science Foundation of China (Nos. 81973320).

Please state what role the funders took in the study.  If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." 

If this statement is not correct you must amend it as needed. 

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

5. Thank you for stating the following in your Competing Interests section:  

Authors declare no conflict of interest.

Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state "The authors have declared that no competing interests exist.", as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now 

 This information should be included in your cover letter; we will change the online submission form on your behalf.

6. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. 

  

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

7. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

8. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please delete it from any other section. 

[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: Partly

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: 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: No

Reviewer #2: No

Reviewer #3: No

**********

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: Dear author, your article is unpublished and has relevance in the field of study. However, it is necessary to pay attention to some questions that I put below for clarification:

Introduction

Page 9

“35 … After

36 respective inflammasomes activation, activated Caspase 1 and Cleaved

37 gasdermin D (GSDMD) to formed cell membrane pores that promote the

38 maturation and release of pro-inflammatory cytokines (IL-1β and IL-18),

39 ultimately leading to pyroptosis [4,10]...”

Sugested:

After

36 respectively inflammasomes activation, activated Caspase 1 and Cleaved

37 gasdermin D (GSDMD) to form cell membrane pores that promote the

38 maturation and release of pro-inflammatory cytokines (IL-1β and IL-18),

39 ultimately leading to pyroptosis [4,10].

Pags. 9 and 10

“39…Reportedly, in myocardial

40 fibroblasts, NOD-like receptor protein 40 3 (NLRP3) inflammasome

41 increased collagen synthesis and expressions of IL-1β, IL-18 and caspase

42 1 [11,12] which are playing an indispensable role in the pathogenesis of

43 cardiac fibrosis.”

Sugested:

“39…Reportedly, in myocardial

40 fibroblasts, NOD-like receptor protein 40 3 (NLRP3) inflammasome

41 increased collagen synthesis and expressions of IL-1β, IL-18 and caspase

42 1 [11,12], which plays an indispensable role in the pathogenesis of

43 cardiac fibrosis.”

Pags 9 and 10

“ 39…Reportedly, in myocardial

40 fibroblasts, NOD-like receptor protein 3 (NLRP3) inflammasome

41 increased collagen synthesis and expressions of IL-1β, IL-18 and caspase

42 1 [11,12] which are playing an indispensable role in the pathogenesis of

43 cardiac fibrosis. In additional, Caspase 1 activation, GSDMD cleavage and

44 cell membrane perforation and promoting secretion of IL-1β and IL-18,

45 which aggravated myocardial ischemia-reperfusion injury [13].

The text is confused. I suggest be more clear. Like:

“…According to reports, the NOD-like receptor protein 3 (NLRP3) inflammasome increased collagen synthesis and the expression of IL-1, IL-18, and caspase 1 in myocardial fibroblasts [11,12], all of which are essential in the pathogenesis of cardiac fibrosis. In addition, Caspase 1 activation, GSDMD cleavage and cell membrane perforation, and promoting secretion of IL-1 and IL-18, which aggravated myocardial ischemia-reperfusion injury…”

Pag. 10

“ …Therefore, present

55 study aims to examine whether leonurine improves cardiac fibrosis and its

56 possible mechanism in pyroptosis.”

Suggested:

“…Therefore, the present

55 study aims to examine whether leonurine improves cardiac fibrosis and its

56 possible mechanism in inhibition of pyroptosis.”

Materials and methods

Pag. 13

“ 117 followed by probed with the indicated primary antibodies including mouse…”

Sugested :

… followed by incubation with the indicated primary antibodies including mouse…”

Pag. 13

“120… rabbit anti-Smad2 phosphorylation

121 (1:1000, Cell Signaling),...”

Suggested:

“ 120… rabbit anti- phospho Smad2

121 (1:1000, Cell Signaling),…”

Pag. 14

“137…and incubated with Smad2 (1:1000, Cell Signaling)

138 antibodies at 4 ℃.”

For how long?

The experimental design is not entirely clear. When does Leonurine begin to be applied to the animal?

Another issue concerns the in vitro study. There is no reference to cell culture and treatment. It is necessary to describe the culture conditions, medium, culture time. And the experimental treatment design.

Results

Pag. 16

“164 After ISO treatment, rats were administered leonurine for eight weeks.

165 Levels of left ventricular end-diastolic pressure (LVEDP), its rising and

166 falling rate (±dP/dt max), lactic dehydrogenase (LDH) and creatine kinase

167 (CK) were significantly increased 1.53-, 2.01-, 2.31-, 1.48- and 1.41-fold,

168 and left ventricular systolic pressure (LVSP) was decreased 1.71-fold in

169 rats after ISO treatment (Fig 1 and 2).”

I suggest rewriting this section. Lots of information about the measured parameters. It could put the numerical data in an ordering closer to parameter groups. Ex: left ventricular end-diastolic pressure (LVEDP), its rising and falling rate (±dP/dt max) increased as shown respectively 1.53-, 2.01- and 2.31.

Pag. 17

“183 After leonurine treatment, expressions of 183 proteins related to

184 pyroptosis were decreased in the heart from rats induced by ISO (Fig 5).”

Sugested:

183... After leonurine treatment, the expression of proteins related to

According to the figures, it is clear that there are different answers in relation to the concentrations of leonurine used. These differences are not addressed during the discussion and there is no information for the reader to choose the concentration used in the cell culture treatment. The descriptor text of the results does not even indicate when there is a change in the use of animal tissue for cell culture. Clarity in writing is necessary for the understanding of the article to be total.

Figures:

Fig. 3 Areas of cardiac fibrosis in ISO-induced rats with leonurine treatment for eight weeks. Representative pictures of left ventricles from each group after H&E staining, Masson staining, and PSR staining (magnification, ×4, ×40). Leonurine decreased areas of collagen and fibers compared to the model group. (A) Collagen and fibers areas are pink after H&E staining. (B) Collagen and fibers areas are blue after Masson staining. (C) Collagen and fibers areas are yellow after PSR staining.

The description of figure A is interchanged with that of figure B.

In figure 8 there is an error in the first line of image A. It should be cleaved Capase 1.

Considerations:

I suggest that a rereading of the article be done in order to make the writing clearer.

The authors speak of the effect of leonurine in blocking the TGFbeta/SMAD-2 signaling pathway. An experiment in which leonurine was used and compared to a pathway inhibitor would be great.

Reviewer #2: Leonurine inhibits cardiomyocyte pyroptosis against cardiac fibrosis via the TGF-β/Smad2 signaling pathway

Reviewer Recommendation and Comments

I have had the pleasure of reading Li and colleagues’ manuscript. The research manuscript aimed to answer the question whether the alkaloid Leonurine can block the progression of cardiac fibrosis and elucidate the molecular mechanism involving pyroptosis. I would like to suggest an English review by a native English-speaking reviewer for improve the understanding of the reader. Moreover, I further have a few comments on specific points in other to improve the manuscript understanding.

Major Concern:

1. Title: “Leonurine inhibits cardiomyocyte pyroptosis against cardiac fibrosis via the TGF-β/Smad2 signaling pathway.” I think the word “against” leads to the reader a misunderstanding about the main view of the manuscript. I would suggest, “Leonurine inhibits cardiomyocyte pyroptosis via the TGF-β/Smad2 signaling pathway to prevent/block (the right word depends on the answer of question 4.c.) cardiac fibrosis.

2. Abstract: The citation (line 7): “The manuscript studied the novel mechanism…” please change for “This report…”.

3. Introduction: It is missing the working hypothesis of the work. It is confusing from line 51 through the end of the paragraph. Reference 16 mentioned in the manuscript is also about the attenuation of myocardial fibrosis. Moreover, I don´t understand what the authors means that it has a great cardioprotective effect. Are the authors related this sentence with cardiac function (LEVDP, LVSP, Dp/Dt) preservation? If the answer is yes, what is the new point of this manuscript? Please clarify the working hypothesis enhancing the differences from previous publications.

4. Materials and Methods:

a. Leuronine was obtained from Fudan University; to me it seems that the substance is not commercially available. Is it as extraction? The authors should provide more information.

b. The authors should provide the total n number and mention if the treatment induced any loss of the animals.

c. Treatment protocol is confusing. To all rats, ISO was injected during 48 days. When exactly Leuronine was orally administrated? During ISO administration or after? For how long, 8 days? This may change the interpretation of the data. If the Leuronine was treated after ISO administration for 8 days, the substance reverts cardiac fibrosis. This issue should be discussed.

5. Results: The quality of Fig 3 is poor. The yellow mentioned in the text can´t be seen.

6. Discussion:

a. Paragraphs 1 and 2 says the same things, please be concise.

b. ISO is a beta-adrenergic agonist. Do the ISO-induced pyroposis mediated by beta-adrenergic receptor or is a consequence of the sustained augmentation of cardiac haemodinamic that may stimulate others mediators? If the pyroposis is mediated beta-adrenergic receptor, what is the difference of the treatments with beta adrenergic antagonist (such as atenolol) and Leuronine. Are co-treatment with both drugs synergic? The authors should provide the data or discuss this issue.

c. Leuronine is an alkaloid, if ISO was administered in concomitance to Leuronine, the physically interaction of both drugs is possible? Or, is Leuronine able to bind to beta adrenergic receptor? Another possibility is ROS chelation? These points as missed in the discussion.

d. At the end of the discussion, the authors mentioned “some limitations”, the authors should cite and discuss it better.

Minor Concern:

1. Please, provide the p values of each result in order for a better conclusion of the reader.

2. Please, revise Figure legends 1, 2, 3 and 4. The letters that label’s the panels are exchanged.´

3. It is encourage that the authors draw a picture underling the proposed molecular mechanism of Leuronide.

Reviewer #3: The authors studied the novel mechanism of leonurine on cardiac fibrosis with its cardioprotective effects. They used both in vivo animal model and experiments on H9c2 cells were conducted. The manuscript is well written. The topic is interesting, and the results presented enhancing our existing knowledge. Even though the findings are important the authors must improve the manuscript. In this regard, the results section must be rewritten. It is not clear the experiments performed and if the results described were in the animal model or in the cells. In addition, the English needs to be improved.

**********

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

Reviewer #3: 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.

Attachments

Attachment

Submitted filename: Review PONE-D-22-05865 LSLM.pdf

Leonurine inhibits cardiomyocyte pyroptosis to attenuate cardiac fibrosis via the TGF-β/Smad2 signalling pathway

PONE-D-22-05865R1

Dear Dr. Zhu,

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,

Luis Eduardo M Quintas, 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 #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 #2: (No Response)

**********

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

Reviewer #2: (No Response)

**********

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: (No Response)

**********

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: (No Response)

**********

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: (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 #2: No

**********