Dear Dr. Jin,

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.

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We look forward to receiving your revised manuscript.

Kind regards,

Junzheng Yang

Academic Editor

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?

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: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Dear Authors,

Thank you for the opportunity to review your manuscript entitled “β-sitosterol alleviated HFD-induced atherosclerosis by regulating the MAPK/Nrf2/NLRP3 pathway in ApoE−/− mice.”

Your work tackles a highly relevant issue in cardiovascular research, namely the search for natural and safe agents to counteract diet-induced atherosclerosis. Overall, the manuscript is clearly structured, the results are compelling, and the study adds valuable mechanistic insight into the potential of β-sitosterol as a therapeutic agent.

Strengths:

The experimental design is solid and appropriate.

Use of ApoE−/− mice and standard models of atherosclerosis makes your study relevant and comparable to other preclinical research.

The exploration of the MAPK/Nrf2/NLRP3 axis is both novel and well supported by the presented data.

Figures and data presentation are clear and informative.

Areas for improvement:

Sample size justification:

While using 6 mice per group is common, it would strengthen the study to briefly justify this choice. Was any power analysis performed?

Statistical reporting:

The use of ANOVA is appropriate, but I recommend reporting exact p-values where possible, instead of ranges (e.g., “p < 0.05”), to improve transparency and reproducibility.

NF-κB interpretation:

You mention that β-sitosterol had no significant effect on NF-κB levels. Given the central role of NF-κB in inflammation, a short paragraph discussing this finding—and how it fits with the MAPK/Nrf2/NLRP3 interplay—would be appreciated.

Mechanistic depth:

The proposed mechanism is biologically plausible and compelling. However, it would be good to acknowledge the limitations of relying on expression data alone. For future work, pathway inhibition or knockout studies would help confirm causal links between β-sitosterol and the signaling cascades.

English and grammar:

The manuscript is generally well written. Some minor grammar issues should be corrected during revision. For example:

“...the mechanisms underlying the pathogenesis of AS have not fully elucidate...” → should read: “...have not been fully elucidated.”

“...treatment mechanisms of β-sitosterol...” → consider rephrasing as “...mechanisms underlying the therapeutic effect of β-sitosterol.”

Ethical approval:

You clearly mention ethical approval by the China Pharmaceutical University Animal Care Committee. If available, please include the approval number.

The study is technically sound and offers promising evidence that β-sitosterol could be a useful anti-atherosclerotic agent by modulating oxidative stress and inflammation. With some minor clarifications and careful proofreading, I believe this manuscript will be a valuable contribution to the field.

Best regards,

Reviewer #2: 1.The expression patterns of MAPK/Nrf2/NLRP3 signaling pathway and MAPK/Nrf2/NLRP3 pathway remain consistent.

2. p38 MAPK: On line 167, it is written as p38 MAPK, while elsewhere (such as line 124, Figure 2B) it is p38 MAPK. Should be unified as p38 MAPK (with spaces)

3. Figures 1F-G: The caption (line 160) states (n=3). However, the results section (line 155) describes the aortic root plaques, and the methods section (lines 109-113) also describes section staining of the aortic root. Each group has 6 animals, and usually aortic root section analysis is performed on multiple sections, but the final statistical unit is usually the number of animals per group. The caption (n=3) contradicts the animal grouping of n=6.

4. Figure 3I: The result description (lines 193-194) mentions scale bar=100 μ m, but the scale is not shown on the figure. A ruler must be added to the diagram.

5. Figures 1, 2, and 3: The statistical significance markers (*, *, * * *, * * *, ns) in all the captions cannot be seen on the corresponding data points or whether there are markers on the bar chart. It is necessary to confirm whether these significant markers have been correctly labeled at the corresponding comparison positions in the graph. Reviewers need to be able to directly see which comparisons are significant from the graph.

6. Line 229� the sentence, “For example, Camosic acid downregulated Nrf2, resulting in a decrease expression of antioxidant enzymes such as CAT”, should be added in here and its reference, https://doi.org/10.26599/FMH.2025.9420021added in here.

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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.

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Reviewer #1: Yes:  Oussama Bekkouch

Reviewer #2: No

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[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.

Dear Dr. Jin,

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 Oct 06 2025 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.

• 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 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,

Junzheng Yang

Academic Editor

PLOS ONE

Journal Requirements:

1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 

2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

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

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: (No Response)

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Reviewer #1: This revised version shows significant improvement. The objectives are now clearly stated, statistical reporting has been strengthened, and histological data presentation is more robust. The addition of recent references (2020–2024) increases the relevance of the work.

Strengths:

Comprehensive evaluation of Hibiscus sabdariffa across antioxidant, antidiabetic, and topical anti-inflammatory models.

Clearer abstract and inclusion of numerical outcomes enhance the scientific rigor.

Histological figures and the addition of Table 7 (inflammatory scoring) improve accessibility for readers.

Discussion of mechanisms (AMPK, GLUT4, β-cell protection) enriches the translational value.

Suggestions for minor improvement:

Ensure all histological figures include scale bars for clarity.

In the Discussion, explicitly mention the limitation regarding the absence of release/penetration profile studies in the topical formulation.

A final proofreading pass could further refine fluency and eliminate residual awkward phrasing.

Reviewer #2: (No Response)

**********

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:  Oussama Bekkouch

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

β-sitosterol alleviated HFD-induced atherosclerosis by regulating the MAPK/Nrf2/NLRP3 pathway in ApoE−/− mice

PONE-D-25-25290R2

Dear Dr. Jin,

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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support .

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,

Junzheng Yang

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewer #1:

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

**********

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

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

**********

Reviewer #1: The authors have satisfactorily addressed all reviewer comments and substantially improved the manuscript. The experimental design is robust, and the results are clearly presented and supported by appropriate statistical analysis. The inclusion of scale bars in histological figures and clarification of funding/ethics/data availability statements strengthen the transparency of the work.

The conclusions are well supported, and the study provides new insights into the role of β-sitosterol in modulating MAPK/Nrf2/NLRP3 pathways during atherosclerosis progression. The discussion appropriately highlights the therapeutic potential as well as limitations (preclinical model, need for clinical validation).

Overall, I find the manuscript acceptable in its current form and recommend it for publication in PLOS ONE.

**********

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:  Oussama Bekkouch

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