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Additional Editor Comments (if provided):

Dear Authors:

Your manuscript PONE-D-25-27151 has been reviewed by multiple reviewers, who have provided some important comments and suggestions. This is an important study and provides some interesting mechanistic insights; but the manuscript requires significant changes. Therefore, we are inviting you to submit a revised version of the manuscript. Please address all the comments of all reviewers in a pointwise response letter. Please incorporate all suggestions of the reviewers in the manuscript and highlight the textual changes in blue font.

In addition, please address comments by the editor:

1) Please make sure that all relevant research efforts of other groups are adequately cited in the manuscript—both in favor/support and against the current research.

2) Please ensure that the methodology is detailed and all information for reagents has been included.

3) Please ensure scale bars, labels, resolution of images are appropriate and adherent to Journal guidelines.

4) Please ensure that statistical analyses are rigorously performed for all data. Please also ensure that data shown in figures are representative of at least three independent experiments performed in replicates.

5) Please avoid/remove any overstatements in the manuscript. Please ensure that the statements are based on results obtained.

[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?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Partly

Reviewer #6: Yes

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: 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

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

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Reviewer #1: 1. The author should carry out experiments to understand the chemo profile of Periplaneta americana extract. Without knowing what is contains, its not justified to consider it as therapeutic agent.

2. Dose dependent toxicity studies are required.

3. The precise molecular markers related with recurrent oral ulcers should be carried out using a model cell line as well in in vivo model study, simply apoptosis related markers is not enough.

4. There is need to select a positive control for comparison purpose.

5. The MS has been written in very poor manner.

Reviewer #2: This research is exceptionally well-designed and meticulously executed. It presents a highly engaging and valuable contribution to the therapeutic field of ulcer, employing a wide range of advanced techniques to robustly support and validate the proposed hypothesis.

Reviewer #3: The manuscript presents a well-designed study on the therapeutic effects of Periplaneta americana extract (PAD) in a rat model of recurrent oral ulcers. It provides valuable insights into PAD’s multi-target actions through the gut-oral-immune axis. The findings support PAD's potential as a natural therapeutic agent, especially in traditional Chinese medicine. The study contributes meaningfully to the field of natural compound-based therapy and mucosal immunology.

Reviewer #4: Abstract

-Some meanings of the acronyms were not expressed in this section, such as SCFAs, RT-qPCR, and WB. They should be given in their full form when they were first used in the text to increase clarity.

-I would prefer a structured abstract rather than a uniform text.

-Except for these, this section clearly summarizes the objective, methods, and results of the study.

Introduction

-While the graphical abstract well summarizes the study, the term located in the figure explanation "ameliorates" should be changed to "ameliorating" or "amelioratory" in the revised version.

-" While ROU attacks are localized and typically resolve on their own, the intense burning pain, swollen lymph nodes, general malaise, and other symptoms that come with the onset of the condition can significantly impact patients' mental well-being and quality of life"

Although I agree with the fact that the symptoms can decrease the quality of life, as a patient with ROUs, I found the term "impacting the mental health significantly" to be extravagant. For me, this disease is a mild disease without a severe, life-threatening symptoms and complications.

-"Currently, ROU is clinically treated with Western medicine, and the commonly used drugs are glucocorticoids, thalidomide, and colchicine, but they have adverse effects such as teratogenicity, dizziness, and gastrointestinal adverse reactions" In this sentence, the authors presented the ROUs similar to the clinical symptoms and treatment of Behçet's Disease. However, isolated ROUs are free from multisystemic involvement and rheumatological clinical progress. Therefore, to avoid confusion in readers, the authors should clearly define and illustrate the clinical picture of the ROUs.

-Except for these, the introduction section clearly explains the objective of the study and pathophysiological interactions between the ROUs, intestinal barrier, and Chinese traditional therapies.

Methods

-"Complete Freund’s adjuvant, butyric, propionic, butyric, and 101 pentanoic were purchased from Sigma-Aldrich (St. Louis, MO, USA)." "Butyric" is repeated in this sentence.

-"UPLC-Q-TOF-MS (Agilent, USA). Microscope (Olympus, Japan). GC-MS system 118 (Agilent, USA). QuantiFluor™-ST blue fluorescence quantitative system (Promega, 119 Madison, WI, USA). Illumina MiSeq platform (Shanghai Yuan Shen Biomedical 120 Technology Co. Ltd., Shanghai, China). Ultramicro nucleic acid and protein analyzer 121 (Thermo Fisher Scientific, Waltham, MA, USA). CFX96 Real-Time PCR Detection 122 System (Bio-Rad Laboratories, Hercules, CA, USA). Image-Pro Plus 6 software (Media 123 Cybernetics, Inc., Rockville, MD, USA). SPSS 26.0 statistical software (SPSS Inc., 124 Chicago, IL, USA)." There is no meaningful sentence in this part except brand names.

-Except for these, the methods section is very detailed and explains all of the steps performed during the study.

Results

-The authors supported their results with very detailed figures, illustrations, and demonstrations.

Discussion

-The discussion section widely explains the results of the study. Numerous references and comparisons with similar studies in the literature are available.

Conclusion

This section effectively summarizes the significance of the results and their implications for the literature and clinical practice. However, the limitations of the results should have been discussed at the end of the discussion section rather than in this section.

Generally, this is a detailed study examining the effect of PAD treatment, a component of traditional Chinese medicine, in ROUs. Several parameters related to this issue were evaluated in the study, including inflammatory cytokine levels, tissue specimens, and DNA analysis. The topic is important, and the results are novel, with the potential to impact the daily practices of clinicians. Therefore, I found this study very valuable.

Reviewer #5: Manuscript title: Periplaneta americana extract ameliorates recurrent oral ulcers in rats by enhancing the intestinal epithelial barrier and regulating gut microbiota

Manuscript ID: PONE-D-25-27151

The paper presents a comprehensive mechanism where Periplaneta americana extract (PAD) treats ROUs not just locally, but by improving gut health. It regulates the gut microbiota, increases beneficial metabolites and strengthens the intestinal barrier which in turn reduces systemic inflammation that contributes to the formation and recurrence of oral ulcers.

The manuscript shows many significant findings, while some unaddressed issues are present such as

• Discussion on the correlation between oral and gut microbiota.

• The study does not identify or isolate the bioactive compound responsible for the action which makes it difficult to standardize for therapeutic application or even just for laboratory replication.

• The results show a decrease in the Lactobacilli concentration. This is counterintuitive as Lactobacillus is generally a probiotic bacterium beneficial for gut health. The discussion does not discuss why the treatment would lead to a reduction in a beneficial genus that is abundant in the control group.

The paper provides compelling evidence for PAD’s efficacy through gut-oral axis, its primary limitation is the lack of oral microbiota data, uncharacterized crude extract and the lack of clarity whether the improved intestinal barrier is a direct result of PAD’s action or an indirect consequence of the modulated gut microbiota and increased SCFA production.

The discussion section is more like a combined version of the introduction and the results. The author has not discussed the results with other available research on ROUs or PAD as such. The discussion part could be rewritten to give a persuasive validation for the hypothesis made in the study.

The study demonstrates PAD’s effectiveness over a 7-day treatment period. A key feature of ROUs is their recurrence. While the introduction notes that Traditional Chinese medicine can inhibit recurrence rates, the short duration of the study does not allow for long term effects on preventing ROUs. Since the study is based on the hypothesis of amelioration of ROUs by enhancing gut microbiome, the sustainability of the changes in gut microbiota and intestinal barrier should be made clear. Effective arguments regarding the sustainability of the same after the treatment dosage reduction or cessation should also be mentioned to strengthen the correlations made in the study.

Queries:

1. Does the Kangfuxin liquid also work through a similar mechanism in reducing the ROUs as its raw material is also PAD and has been in use for a long time?

2. Mention the primer sequence for the internal control for the qPCR and cite a reference for the same. Also cite a reference for the RQ calculation.

3. IL-2 is seen in higher level in RAS patients and contributes to the release of TNF-α and IL-6. Why is the serum level higher in control and lower in the model group? (Section3.3, Fig. 1B)

4. Section 3.3, why IL-2 expression not determined among all the other cytokines expressed in serum? (Fig. 1C)

5. Section 3.11 & Fig 8A, SIgA significantly reduced in the model group seems not to be the right term as compared to the treatment group, the levels of SIgA are on a similar level.

6. Levamisole hydrochloride (LM), has been used as positive control in the study. Since LM is commonly used in clinical treatments of ROUs, mentioning its side effects or why PAD could be better than commercially available drugs could be included in either the introduction or the discussion section- justification required.

Major corrections:

• Periplaneta americana extract in abbreviated (PAD) form has not been mentioned prior to Line 71.

• Line 100: Butyric has been mentioned twice.

• Lines 117-124 could be written as a sentence. Mentioning each instrument separately makes it incomplete.

• Line 127: medicinal material has been mentioned twice.

Suggestion: “Briefly, 540g of Periplaneta americana (medicinal material) was crushed into a powder using a mortar and pestle.” (or whatever instrument was used to grind the insects)

• Section 2.3: The title of the section sounds vague

Suggestions: Experimental model, research model or model organism.

• Mention the diet for the model organism used in the study.

• The term model group can be misinterpreted. Standard term “negative control” can be used to indicate rats administered with saline solution.

• Fig 6A: Change “gobler cell” to “goblet cells”

• Fig legend: either mention ROUs as in Fig 1, 5, 6 or recurrent oral ulcers as mentioned in Fig 3, 4.

• Use the abbreviated form for an organism’s name throughout the manuscript after its first mention. (ex. Discussion line 477: Periplaneta americana to P. americana).

Minor corrections:

• Follow the same tense through the manuscript (ex. Line 193: sulfuric acid was added and then in the same paragraph Line 195: microporous surface is used to)

• Graphical abstract caption “ameliorates effect” to “ameliorating effects”

• Line 93: “material and methods” to materials and methods”

• Line 414: “antigen emulsifier” to “Antigen emulsifier” (Sentence case)

• Fig 1A legend: Line 635 ‘theoral mucosa’ to the oral mucosa’

• Fig 6 legend: Line 661 add spacing

Reviewer #6: Major Comments

Model justification: The antigen emulsifier-induced ROU model is not standard. The authors should provide stronger justification that this model reflects the pathophysiology of human recurrent aphthous ulcers, and discuss its limitations.

The conclusion should be tempered to emphasize association rather than causality.

Overstated conclusions: The manuscript at times suggests PAD is an “effective treatment” for ROUs. Given that the data are preclinical (rat model only), the discussion should highlight translational limitations and avoid clinical overstatements.

Clarity and redundancy: The Introduction and Discussion are long and at times repetitive. Streamlining would improve readability.

Minor Comments

• Figures and legends should be checked for clarity (some are difficult to interpret in the PDF).

• Abbreviations (PAD, ROU, SCFA, GM) should be redefined in Results for reader convenience.

• Ethical approval is mentioned, but animal welfare descriptions could be expanded (analgesia, humane endpoints).

• The limitation section briefly mentions oral flora, but further elaboration is needed (e.g., small sample size, lack of female animals).

• References: Some claims in Introduction and Discussion would benefit from more recent citations.

• Grammar and syntax require polishing for international readability.

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what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: Yes: Shweta Sharma

Reviewer #3: No

Reviewer #4: Yes: Assoc. Prof. Kubilay İşsever

Reviewer #5: Yes: S Janarthanan

Reviewer #6: No

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Attachments

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Submitted filename: reviwer comments (7).docx

Periplaneta americana extract ameliorates recurrent oral ulcers in rats by enhancing the intestinal epithelial barrier and regulating gut microbiota

PONE-D-25-27151R1

Dear Dr. Xiao,

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,

Mukul Godbole, Ph.D.

Academic Editor

PLOS One

Additional Editor Comments (optional):

Based on reviewer comments and suggestion, the manuscript can be accepted for publication.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

Reviewer #6: (No Response)

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

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #6: Yes

**********

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

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #6: Yes

**********

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

The PLOS Data policy

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #6: Yes

**********

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

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #6: Yes

**********

Reviewer #3: (No Response)

Reviewer #4: I sincerely thank all the authors for their detailed explanations and corrections to the reviewer recomendations. All my recommendations are adressed and corrected in the revised version of the manuscipt.

Reviewer #6: (No Response)

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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.

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 #3: No

Reviewer #4: Yes: Associated Professor Kubilay İşsever, MD

Reviewer #6: No

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Attachments

Attachment

Submitted filename: Decision on Manuscript IDPONE-D-25-27151R1.docx