-->PONE-D-25-58627-->-->Sodium nitrite promotes atherosclerosis via IL-1β: Network toxicology and machine learning insights-->-->PLOS One

Dear Dr. Yang,

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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Wenxing Li

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PLOS One

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Natural Science Foundation of Hunan Province (Grant No. 2024JJ9466). URL: http://kjt.hunan.gov.cn/

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-->Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Partly

**********

-->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?

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Reviewer #1: Yes

Reviewer #2: Yes

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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: Dear Editor and Authors,

Thank you for inviting me to review a manuscript for this esteemed journal.

My review comments on the requested manuscript (PONE-D-25-58627) are as follows.

# Overall Comments

This manuscript employs network toxicology and multifaceted bioinformatics methodologies to elucidate the mechanisms by which sodium nitrite contributes to atherosclerosis development.

This represents a valuable approach for unravelling complex toxicological mechanisms, and the scientific merit of both the subject matter and the findings is considerable.

However, to fully meet PLOS ONE's publication standards, methodological rigour should be enhanced in the following aspects.

# suggestions

1. The manuscript's methodology describes that the authors collected targets for sodium nitrite via ChEMBL, STITCH, and CTD, and AS-related targets via OMIM and GeneCards.

However, the GeneCards database returns thousands of genes based on their relevance score. This manuscript merely states that ‘duplicate entries were removed’, without specifying a concrete cut-off value for the Relevance Score.

Simply including all genes risks incorporating false positive targets, which could undermine the reliability of the results.

Therefore, please specify the exact filtering criteria applied to the GeneCards database and provide the rationale for selecting these criteria.

Furthermore, for the STITCH database, the confidence score settings should also be reported.

2. The finding that approximately 70% of sodium nitrite's targets (34 in total) overlap with disease targets suggests a close association between sodium nitrite and AS.

However, conversely, the possibility that the initial target collection was overly broad cannot be ruled out.

I recommend that the consideration section thoroughly address whether the 24 targets represent direct binding targets of sodium nitrite or indirect effects via downstream signalling mechanisms.

3. The abstract and main text claim that ‘multi-omics bioinformatics’ was performed.

However, the actual analyses conducted were limited to Transcriptomics (GEO datasets).

Direct integrated analysis of Metabolomics or Proteomics data was not performed; it remains confined to a literature review.

The term ‘multi-omics’ should be revised to something like ‘Transcriptomics integrated with Network Toxicology’, or results from the actual integrated analysis of other omics data should be added. The current phrasing may mislead readers.

4. In this manuscript, the machine learning techniques LASSO and SVM-RFE were employed to select four key genes.

However, it is unclear what the input data (Feature Matrix) used to train the machine learning models actually is.

Contextually, it is presumed to utilise expression levels from the GEO dataset (GSE28829); however, this pertains to ‘AS patient samples’ rather than ‘samples exposed to sodium nitrite’.

If machine learning was performed using AS patient data, it is difficult to definitively conclude that the selected genes are biomarkers for AS rather than toxicity targets for sodium nitrite.

The causal relationship that sodium nitrite regulates these genes is not proven by the machine learning results alone.

Clearly describe in the Methods section what the machine learning training data comprised (number of samples, group information, etc.).

Furthermore, to substantiate that these results represent “targets of sodium nitrite”, it would seem necessary to demonstrate that these genes either match the sodium nitrite target list (initial 34) or at least possess a significant connection to it.

5. When performing both feature selection and validation (ROC curve) on a single GEO dataset with a limited number of samples, is there not a significant risk of data leakage and overfitting?

Secure an external validation dataset (e.g., another GEO dataset) to revalidate the diagnostic capability of the derived four genes.

If this proves difficult, explicitly state in the manuscript whether the training/test set separation was strictly adhered to during the internal validation process to enhance reader confidence.

6. Regarding the LASSO and SVM results presented in Fig 3, the Kernel function (Linear, RBF, etc.) and specific parameter settings used when performing SVM-RFE must be explicitly stated.

Furthermore, additional performance metrics such as Accuracy, Sensitivity, and Specificity for the final selected model should also be presented.

7. In molecular docking, results such as -6.9 kcal/mol (IL-1β) using AutoDock Vina have been reported.

However, as this binding affinity is difficult to objectively consider strong, it would be advisable to present docking results with a Positive Control (an existing known IL-1β inhibitor or binding ligand) alongside these findings to demonstrate sodium nitrite's relative binding affinity to readers.

Furthermore, regarding the description in the Results section, it is considered more appropriate in scientific literature to temper the expression ‘strong binding affinities’ to something like ‘moderate to strong’ or to present the values relative to the control group.

8. In Fig 8A, the protein backbone RMSD shows an initial increase followed by stabilization, whereas the Ligand RMSD (Fig 8B) exhibits greater variability.

It would be beneficial to visually supplement this explanation using trajectory snapshots to clarify whether the ligand is stable within the binding pocket or exhibits tumbling motion within it.

9. This manuscript utilizes ssGSEA to confirm increased M0 macrophages, consistent with the pathology of AS.

However, interpreting the results as ‘sodium nitrite induces immune infiltration’ is excessive.

The current findings merely support that M0 levels are elevated in AS tissue and correlate with key genes.

As this is not sodium nitrite exposure data, a more appropriate explanation would be along the lines of: ‘The target genes of sodium nitrite are closely associated with changes in the immune microenvironment of AS, suggesting a potential role in mediating toxicity.’

10. While the Introduction section effectively outlines the risks of sodium nitrite, it would be beneficial to provide a more detailed explanation of why previous studies failed to elucidate the molecular mechanism.

Furthermore, the statement ‘Sodium nitrite is generally considered protective...’ mentions the positive effects of low doses.

To clearly delineate this from the study's focus on “toxicity”, it would be advisable to cite specific numerical values from prior research regarding the concentrations or conditions under which toxicity manifests.

11. In the Discussion section, it would be beneficial to discuss the causal relationship between sodium nitrite → IL-1β binding → inflammatory response → AS induction in greater depth and detail, focusing on the AOP framework introduced by the authors and comparing it with prior literature.

12. Throughout the manuscript, ‘Sodium nitrite’ and ‘sodium nitrite’ are used interchangeably.

It is standard practice to use lowercase letters for chemical names unless they are at the beginning of a sentence.

13. This study relies entirely on computer simulations and public database analysis; biological validation through in vitro or in vivo experiments has not been performed.

Experimental data verifying changes in mRNA or protein expression of key targets derived from sodium nitrite-treated cell models should be added.

If this is not feasible, the limitations of the paper should be clearly stated, and the tone of the title or conclusion should be moderated to ‘Prediction’ or ‘In silico insights’.

Reviewer #2: Comments

This study established an adverse outcome pathway (AOP) framework linking sodium nitrite exposure to the development of atherosclerosis. These findings provide new mechanistic insights and offer a theoretical foundation for assessing nitrite-associated cardiovascular risks. Before publication, the major revision is suggested to address the questions and comments below:

1. Sodium nitrite is used for cyanide poisoning, and can be used for patients with cardiovascular and arteriosclerosis, but the dosage should be reduced and the injection rate should be slowed down. However, the results of this study showed that sodium nitrite could promote atherosclerosis, which is inconsistent with the previous view, please explain the reason.

2. The relationship between sodium nitrite and atherosclerosis is complex, whether it ultimately plays a protective role or risk, whether there is epidemiological survey analysis or clinical verification, please supplement the relevant content.

3. Univariate and multivariate Cox regression analysis were used to study the relationship between survival time and potential risk factors for the disease. It is suggested that univariate and Cox regression analysis should be supplemented to verify the relationship between core genes, age, gender, race and other factors and the survival time of patients, and to clarify whether it is independent prognostic factor.

4. Sodium nitrite showed strong binding affinities with IL-1β, PTK2, IL6, and NOS3, with binding energies of –6.9, –6.7, –6.3, and –6.8 kcal/mol, respectively. The binding energies of the four targets were not significantly, especially the binding energies of IL-1β and NOS3 were basically close, why only IL-1β was selected for the following experiments?

5. This research screened out four core targets: IL-1β, PTK2, IL6 and NOS3. The characteristics of these four targets and research progress in the field of atherosclerosis should be elaborated and analyzed in the discussion section.

6. While LASSO regression was used, details about model robustness (e.g., external validation, overfitting control, data splitting strategy) are missing. Reporting performance on an independent test set and including more ML performance metrics (AUC, precision, recall, F1-score) is required.

**********

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Reviewer #1: No

Reviewer #2: No

**********

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-->PONE-D-25-58627R1-->-->Sodium nitrite promotes atherosclerosis via IL-1β: Network toxicology and machine learning insights-->-->PLOS One

Dear Dr. Yang,

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 Mar 08 2026 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.. 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 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 . 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,

Wenxing Li

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.

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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: (No Response)

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 #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.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

**********

-->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: Dear editor and authors,

After reviewing the revised manuscript, I find that my suggestions have been well incorporated in many areas, resulting in significant improvements.

However, there are a few minor concerns that require additional revisions.

Once these are addressed, I believe there will be no further issues preventing publication.

1. In the response letter, the authors specified GeneCards' criteria as “Top 2000 genes... with a minimum correlation score threshold of 1.26.” However, the actual manuscript only states “the top 2000 genes based on relevance scores were selected,” omitting the specific score. Therefore, a brief supplementary description is needed in the manuscript.

2. Regarding the MD Simulation, it seems necessary to add a snapshot figure showing the actual ligand movement.

3. This concerns the suggestion to perform comparative docking simulations using known inhibitors to compare binding affinity. While the authors reinforced their explanation by citing prior literature, the actual comparative docking data does not appear to have been added.

Reviewer #2: This study established an adverse outcome pathway (AOP) framework linking sodium nitrite exposure to the development of atherosclerosis. These findings provide new mechanistic insights and offer a theoretical foundation for assessing nitrite-associated cardiovascular risks.

**********

-->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 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 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 ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures

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NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

-->

-->PONE-D-25-58627R2

Sodium nitrite promotes atherosclerosis via IL-1β: Network toxicology and machine learning insights

PLOS One

Dear Dr. Yang,

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 Mar 27 2026 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.. 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 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 . 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,

Wenxing Li

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.]

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

Reviewer #3: All comments have been addressed

Reviewer #4: 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: Yes

Reviewer #3: Partly

Reviewer #4: Yes

**********

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

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: 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.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

Reviewer #3: Yes

Reviewer #4: 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

Reviewer #3: Yes

Reviewer #4: 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: (No Response)

Reviewer #3: Title: Sodium nitrite promotes atherosclerosis via IL-1β: Network toxicology and machine learning insights. Manuscript Number: PONE-D-25-58627R2

Major Comments

1. The manuscript still refers to the “top 2000 genes,” an issue previously raised by the reviewer and not addressed by the authors (Line 91).

2. The authors used AMBER for molecular dynamics simulations; why was CHARMM not used.

3. The authors state that a ‘de-identified public dataset’ was used; however, the reason for de-identification should be clearly explained.

4. The machine learning methodology is unclear, particularly regarding the target size; if only 24 targets were used, the dataset may be too small for reliable model training.

5. The hyperparameters used in the machine learning models are not reported and must be explicitly stated.

6. Although SVM & LASSO is mentioned, it is unclear whether other classifiers were tested; a comparative table reporting accuracy, RMSE, and correlation for all classifiers is needed to justify the choice of SVM.

7. The features used in the machine learning model and their total number are not clearly described, and any feature selection method applied should be specified.

8. Details of the machine learning validation strategy are missing, including the validation dataset and the training–testing split ratio.

9. The immune infiltration correlation analysis requires further explanation, particularly how the results demonstrate a significant increase in M0 macrophage infiltration in AS tissues compared to normal tissues.

10. A 2D interaction diagram for the molecular docking results should be provided, as conclusions based solely on docking scores are insufficient.

11. Figure 9 shows large RMSD fluctuations at multiple intervals, suggesting unstable docking; therefore, the molecular dynamics simulations should be reassessed or repeated.

Minor Comments

1. The Results section is brief and would benefit from more detailed explanation and interpretation.

2. Molecular dynamics plots should use nanoseconds (ns) as the time unit for consistency and clarity.

3. The peak observed in Figure 10B is unclear and requires a better viewing angle or improved representation.

Reviewer #4: Please correct a factual error regarding the CANTOS trial: the manuscript currently states “targeting IL-1β with kanamycin” , which should be revised to the correct intervention (canakinumab).

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Reviewer #2: No

Reviewer #3: Yes: Dr. Sanket BapatDr. Sanket Bapat

Reviewer #4: No

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Submitted filename: Reviewer Comments_PONE-D-25-58627R2.docx

<p>Sodium nitrite promotes atherosclerosis via IL-1β: Network toxicology and machine learning insights

PONE-D-25-58627R3

Dear Dr. Yang,

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.

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Wenxing Li

Academic Editor

PLOS One

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Reviewer #4: All comments have been addressed

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Reviewer #4: Yes

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Reviewer #4: Yes

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Reviewer #4: Yes

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Reviewer #4: Yes

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Reviewer #4: The author answered all the questions and presented a clear and logical argument. I agree to accept this article.

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Reviewer #4: No

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