PONE-D-25-23758

Mechanism of Huanglian Wendan Decoction in Ameliorating Non-Alcoholic Fatty Liver Disease via Modulating Gut Microbiota-Mediated Metabolic Reprogramming and Activating the LKB1/AMPK Pathway

PLOS ONE

Dear Dr. Li,

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.

Your manuscript has been reviewed by three experts in the field. My additional comment is as follows: Please provide the raw data related to the gut microbiome analysis conducted in this study. This is essential to validate the reliability and integrity of the analysis. Kindly ensure this data is included in your revised submission.

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Request from the Editorial Office: In your Methods section, please provide the following information: 1) The source of the plants used in the study, their authentication, and which parts of each plant were used to produce the decoction. 2) How was the decoction given to the animals, and what is the relevance of the in vivo assays to its traditional use? 3) What is the rationale for the concentrations of the medicinal compound used in the experiments? We would expect an acute toxicity test to have been performed. 4) How were the animals modelled?

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Please submit your revised manuscript by Aug 14 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Fahrul Nurkolis

Academic Editor

PLOS ONE

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When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

6. Thank you for stating the following financial disclosure:

 [This work was supported by Hunan Provincial Department of Education Research Project (23B0375); Hunan Provincial Administration of Traditional Chinese Medicine Research Project (C2023021); Hunan University of Traditional Chinese Medicine University-level Research Project (2024XJZC017).]. 

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.

7.  Thank you for stating the following in the Acknowledgments Section of your manuscript:

[This work was supported by Hunan Provincial Department of Education Research Project (23B0375); Hunan Provincial Administration of Traditional Chinese Medicine Research Project (C2023021); Hunan University of Traditional Chinese Medicine University-level Research Project (2024XJZC017).]

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

  [This work was supported by Hunan Provincial Department of Education Research Project (23B0375); Hunan Provincial Administration of Traditional Chinese Medicine Research Project (C2023021); Hunan University of Traditional Chinese Medicine University-level Research Project (2024XJZC017).]. 

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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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: No

Reviewer #3: Yes

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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: 1. Abstract

Strengths : Clearly presents important topic, comprehensive methodology, mechanistic insight and strong outcome summary

Suggestions for improvement :

The abstract would benefit from clearer segmentation into logical components : Background, objective, methods, key results, conclusion. Research objective need explicit statement. Too many abbreviations may overwhelm readers unfamiliar with them. The final sentence effectively summarize the results, but could emphasize what gap this study fills or how it advances current knowledge.

2. Introduction

Strengths : The introduction provides a well-structured and scientifically grounded rationale for the proposed study. It integrates current knowledge on NAFLD pathogenesis with the potential of Traditional Chinese Medicine (TCM) interventions, specifically Huanglian Wendan Decoction (HLWDD). The writing is rich in mechanistic detail, and the research hypothesis is clearly stated at the end. However, there are areas that could be improved for clarity, focus, and better alignment with the journal criteria.

Suggestion for improvement :

While a hypothesis is stated, a specific research question is not directly framed in a question format. It is recommended to clearly formulate the research aim or question (e.g., “Does HLWDD ameliorate NAFLD through modulation of the gut-liver axis and activation of the LKB1/AMPK pathway?”).

The introduction is overly dense, covering too many compounds and mechanisms, which may dilute the main focus on HLWDD. Suggested improvement: Briefly summarize the rationale for prior TCM components, then clearly narrow down the novelty and justification for choosing HLWDD.

While the text implies the gap (limited translation of probiotics, complexity of AMPK activators, etc.), it does not explicitly state what specific knowledge gap the current study addresses. A stronger opening or closing paragraph could highlight: “Despite evidence for the metabolic and microbiota-modulating effects of HLWDD constituents, the precise mechanisms through which HLWDD influences the gut-liver axis and AMPK signaling in NAFLD remain unclear.”

The contribution to scientific knowledge is implied but not explicitly declared. Clarify what novel insight this study will offer (e.g., “This study will elucidate the synergistic effects of HLWDD on microbial metabolites and hepatic energy metabolism, providing a novel therapeutic avenue for NAFLD.”)

3. Methods

Strengths : This Materials and Methods section reflects a well-designed and multifaceted experimental strategy that integrates modern systems biology with traditional medicine research.

Suggestion for improvement :

There are multiple grammatical inconsistencies, awkward phrasing, and redundancies throughout. Example: “...use used to screen...” → should be “used to screen”. Suggestion : Comprehensive language editing is needed to meet international publication standards.

The section is overly lengthy and lacks clear subsections, making it difficult to navigate.

Suggestion: Divide into well-labeled subsections such as:

Reagents and Chemicals

Preparation of HLWDD

Quantification of Core Components

Network Pharmacology and Molecular Docking

Animal Experimentation

Histological and Biochemical Analysis

Microbiome and Metabolomic Analysis

Statistical Analysis

Primer sequences are referred to in Table 3, but the actual table is not included.

Positive drug details (e.g., purity and brand of metformin) are not mentioned.

The exact number of rats used per group post-modeling isn't clearly stated after accounting for attrition.

Quality control parameters for the LC-MS/MS (e.g., resolution, scan range, ionization mode) are not fully described.

Preparation steps for HLWDD decoction and ethanol precipitation are wordy and at times unclear (e.g., “accurately measure 2 mL... take the filtrate and you have it”).

Suggestion: Use stepwise or bulleted protocol format and include specific parameters (e.g., temperature of refrigeration, type of ethanol).

While software used is listed, the rationale for choosing certain tests (e.g., LSD-t test instead of Tukey) is not explained.

Suggestion: Justify the choice of tests and confirm assumption checks (e.g., normality, homogeneity of variance).

Some materials (e.g., primer sources) are repeated unnecessarily.

Suggestion: Consolidate repeated supplier information or group by function.

Result :

Strengths : This Results section is comprehensive, well-organized, and logically structured, covering multiple layers of evidence (chemical profiling, network pharmacology, molecular docking, quantitative validation, in vivo assays, and multi-omics analysis). It successfully integrates phytochemical analysis with biological and pharmacological relevance to NAFLD, which enhances the translational impact of the work.

Suggestion for improvement :

Clarify if identification was based on MS/MS fragmentation or just database matching

Example : “A total of 58 compounds were identified based on retention times and MS/MS spectra, as referenced in ChemSpider and MassBank databases (Table S1; Supplementary Table S1).”

Consider adding a supplementary table listing all 229 shared targets for transparency.

Consider stating whether these seven compounds were chosen based on pharmacological relevance or abundance.

Metformin is mentioned but not introduced earlier—clarify its role as a positive control

Clearly separate qPCR vs. WB results (e.g., “Protein levels assessed by WB showed...”).

Use more precise statistical notation; avoid duplication (e.g., "was significantly downregulated (P < 0.05), and significantly regulated...").

Discussion :

Strengths : The discussion is well-structured, provides mechanistic insight, and draws appropriate connections between experimental findings and current literature.

Suggestions for improvement :

The text frequently suggests causal relationships (e.g., "HLWDD controls intestinal flora metabolic reprogramming") based on associative findings in an animal model.

Suggestion: Use more cautious language such as “may regulate,” “is associated with,” or “suggests potential modulation” unless causality is experimentally confirmed.

The discussion lacks acknowledgment of key limitations, such as:

Use of a single animal model

Absence of dose-response data or comparison with known therapies

Lack of functional validation (e.g., using inhibitors of AMPK or germ-free animals)

Suggestion: Add a paragraph critically acknowledging these limitations and propose future directions.

The paragraph on aspartate metabolism and its bidirectional relationship with LKB1/AMPK is dense and hard to follow. Suggestion: Break down complex mechanisms into simpler sentences.

Reviewer #2: The manuscript addresses an important and timely topic within the scope of PLOS ONE: the therapeutic potential of Huanglian Wendan Decoction (HWD) in the treatment of non-alcoholic fatty liver disease (NAFLD), with a particular focus on gut microbiota-mediated metabolic modulation and activation of the LKB1/AMPK pathway. This line of research holds substantial scientific merit given the global prevalence of NAFLD and the growing interest in gut–liver axis-based interventions. While the manuscript is largely free of major grammatical errors, the overall scientific presentation requires significant improvement. In particular, the rationale, experimental design, and mechanistic interpretations need to be better structured and supported. Some key claims are overstated and not fully justified by the data provided. Based on these concerns, I recommend a major revision prior to any further consideration for publication. The authors are encouraged to address the following critical issues in detail:

Major comments

1. Insufficient mechanistic foundation in the Introduction

The Introduction lacks a focused discussion of the mechanistic rationale behind HWD use in NAFLD. The description of prior work is superficial, and the central hypothesis is not clearly stated. Expand the background on NAFLD pathophysiology and gut–liver interactions. Elaborate on individual components of HWD and their relevance to metabolic or inflammatory regulation. Conclude with a clear and concise hypothesis.

2. Incomplete experimental design descriptions

Key experimental details are missing or vague, including dosage justification, treatment duration, and microbiome data analysis pipelines. Justify HWD dose selection in relation to existing literature or pharmacological considerations. Provide details on 16S rRNA analysis, sequencing platform, cutoff values for LDA, and quality control metrics. Include specifics about antibody sources, Western blot normalization methods, and sample sizes.

3. Overinterpretation of mechanistic claims

The role of the gut microbiota in modulating the LKB1/AMPK pathway is not causally demonstrated but presented as a confirmed mechanism. This weakens scientific rigor.

Soften causal language (e.g., “may be associated with,” “suggests,” “potentially mediates”) and clearly state limitations of the correlative nature of the findings. If no inhibitor or rescue experiments were performed, causal claims should be avoided.

4. Incomplete figure annotations and lack of visual clarity

Some figure legends lack sufficient information to be interpreted independently. In particular, Figures 3 and 4 do not include sample size (n), statistical methods, or clear indication of significance. For each figure, include full legend details—experimental groups, statistical tests used, and precise p-values. Consider including a mechanistic summary diagram (graphical abstract or final schematic) to enhance readability and highlight your proposed model.

5. Clarification and consistency in terminology and abbreviations

The manuscript includes several abbreviations that are either undefined or inconsistently used.

Ensure all abbreviations are defined at first mention and consistently used throughout the text. Consider adding an abbreviation list at the end of the manuscript.

Minor comment

1. Suggested additional analysis or visualization (optional but recommended)

Include a table summarizing the major bioactive components of HWD, their known biological targets, and relevance to NAFLD. This would strengthen the herbal pharmacology framework of your study.

Reviewer #3: Over all the manuscricpt is fine but these point must be address during revision

Weather author performed LCMS at all dilution of decoction. toxic limits of decoction any previous data

in any liver or any other disorders and author also incorporate supporting its medicinal importance in introduction section at cellular level in mammalian cell line data in support of this study .

why author choose only these cytokines but not any chemokines and also not choosing any anti inflammatory cytokines markers

Impact of decoction on healthy animals behaviour of food and water intake must be mentioned .

And author does not mentioned the grade of non alcholic fatty liver in this study which is the main target of study. this to be detail discuused in discussion part.

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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: Yes:  Saad Mustafa

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Attachments

Attachment

Submitted filename: Reviewer comments_Plos.pdf

<div>PONE-D-25-23758R1

Mechanism of Huanglian Wendan Decoction in Ameliorating Non-Alcoholic Fatty Liver Disease via Modulating Gut Microbiota-Mediated Metabolic Reprogramming and Activating the LKB1/AMPK Pathway

PLOS ONE

Dear Dr. Li,

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 Sep 12 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.

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,

Fahrul Nurkolis

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.

Additional Editor Comments:

Request from the Editorial Office: In your Methods section, please provide the following information: 1) The source of the plants used in the study, their authentication, and which parts of each plant were used to produce the decoction. 2)  What is the rationale for the concentrations of the medicinal compound used in the experiments? We would expect an acute toxicity test to have been performed. 

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

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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: Yes

Reviewer #3: Yes

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

Reviewer #2: Yes

Reviewer #3: Yes

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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

Reviewer #3: Yes

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

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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: I have thoroughly reviewed the revised manuscript and sincerely appreciate the authors’ careful attention to the reviewer’s comments. The revisions have notably enhanced the clarity, scientific rigor, and overall quality of the manuscript. I commend the authors

Reviewer #3: Discuss the decoction impact on liver enzymes and skin and insulin sensitivity in discussion section why this decoction is better than available current product and therapeutics

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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: Yes:  Moon Nyeo Park PhD, College of Korean Medicine, Kyung Hee University

Reviewer #3: Yes:  Saad Mustafa

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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.

Mechanism of Huanglian Wendan Decoction in Ameliorating Non-Alcoholic Fatty Liver Disease via Modulating Gut Microbiota-Mediated Metabolic Reprogramming and Activating the LKB1/AMPK Pathway

PONE-D-25-23758R2

Dear Dr. Li,

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

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Professor Fahrul Nurkolis

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors have revised the manuscript in accordance with the editor’s requests and the reviewers’ comments

Reviewers' comments: