Exploring the Efficacy and Potential Mechanisms of Topical Periplaneta americana (L.) Extract in Treating Androgenetic Alopecia in a Mouse Model: A Systems Pharmacology and Skin Microbiome Analysis

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

Thanks for incorporating the comments.

Author Response

Author Response：

Dear Reviewer,

On behalf of the research team, we extend our sincerest gratitude for your professional evaluation and meticulous guidance throughout the review process. Every suggestion you provided has offered invaluable direction for enhancing the manuscript’s quality, from data validation and logical structuring to textual refinement. Your rigorous approach and expert insights have been profoundly inspiring. We particularly appreciate your precise attention to research details—whether recommendations on figure clarity, improvements to literature citations, or deepening of theoretical logic—all of which have significantly elevated the manuscript’s academic rigor and readability. The professional perspective you demonstrated during the review not only helped resolve existing issues but also inspired directions for improving our future research. The manuscript’s successful acceptance today is inseparable from your patient guidance and constructive feedback. Your pursuit of academic rigor sets an exemplary standard for us. Going forward, we remain committed to refining our research to the highest standards, staying true to your professional advice. Once again, we express our heartfelt thanks! We hope to have the opportunity to seek your expertise in the future and wish you every success in your scientific endeavors.

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

PA-011 exhibits promising therapeutic potential in AGA mouse models. The mechanism and safety of PA-011 were thoroughly investigated in this study I suggested acceptance after major revision.

 

1. In page 10, the section of 2.5.9 and 2.5.10 are same.
2. In page 25, Figure 9, TUNEL immunofluorescence staining can’t be clearly seen.
3. Some relevant literature should be cited, such as https://doi.org/10.1016/j.ijpx.2025.100334; https://doi.org/10.1002/anie.202505669.
4. The font size in some of the picture was to small to be seen. For example, Figure 15, Figure 16, Figure 17, Figure and so on.

Author Response

Author Response：

We are sincerely grateful to the reviewer for conducting such a meticulous and comprehensive review of our manuscript and for providing us with so many precious and valuable suggestions. Your feedback has made us aware of the various shortcomings in our manuscript writing. We would be very grateful if you could take a moment to review our newly uploaded manuscript and provide us with your new insights, which will surely assist us in further refining the manuscript.

Q1. In page 10, the section of 2.5.9 and 2.5.10 are same.

Response: Thank you very much for your meticulous and rigorous review! We fully acknowledge that the duplication between Section 2.5.9 and Section 2.5.10, as pointed out by you, is indeed a significant oversight in our manuscript collation and proofreading process, for which we sincerely apologize. We have promptly merged and streamlined the repetitive content, and reorganized the logical flow of the sections to ensure clarity and avoid redundancy. In the revised manuscript, the relevant content has been consolidated into a single independent section, with optimized paragraph structures and language expressions, making the description of research methods more concise and coherent. This error primarily resulted from improper version management during multiple rounds of manuscript revisions, coupled with insufficiently thorough final proofreading, which led to the failure to detect the duplicate content in a timely manner.Thank you again for your correction, which has greatly helped improve the quality of our manuscript. We look forward to your further guidance on the revised version!

Q2. In page 25, Figure 9, TUNEL immunofluorescence staining can’t be clearly seen.

Response: Thank you for your meticulous review and valuable feedback on our study. Regarding the visibility issue of the TUNEL immunofluorescence staining in Figure 9 on page 25, we have conducted a thorough investigation.

Upon confirmation, the original images showed low positive rates of TUNEL immunofluorescence staining in the blank group and PA-011 group, which indeed appeared unclear after figure reduction. To avoid misinterpretation of results due to visualization issues, we apologize for the inconvenience and have completed the following improvements: We have replaced the images with those showing higher TUNEL staining positive rates in the female blank group, female PA-011L group, male blank group, and male PA-011H group, and updated the high-resolution original figures as attachments. The green fluorescence-labeled apoptotic cells in the new images can be clearly observed at higher magnification, and we have supplemented the magnification of the stained areas in the figure legend. Please feel free to let us know if further modifications are needed, and we will fully cooperate to refine the figures. Thank you again for your professional guidance, and we look forward to your further suggestions.

Q3. Some relevant literature should be cited, such as https://doi.org/10.1016/j.ijpx.2025.100334; https://doi.org/10.1002/anie.202505669.

Response: Thank you for recommending the crucial references [https://doi.org/10.1016/j.ijpx.2025.100334; https://doi.org/10.1002/anie.2025056691 - 2] during the review process. Your professional insights have provided valuable academic references for our research. The following are our specific feedbacks regarding the two papers:

Value Elaboration of [https://doi.org/10.1016/j.ijpx.2025.100334]

This study focuses on the preparation of ultra - sensitive pH - responsive hydrogels and their applications in drug controlled release. Its core innovation - constructing an intelligent delivery system with injectable and self - healing properties through benzylideneimine bonds, offers new ideas for solving the problems of insufficient drug release accuracy and high systemic toxicity in traditional chemotherapy. Specifically, the paper points out that the high drug release rate of traditional PVA - based hydrogels at physiological pH can be significantly improved by benzylideneimine bonds. This finding provides empirical support for enhancing the precision of targeted delivery systems. In addition, the self - healing and injectable properties of the hydrogel make it suitable for local administration in minimally invasive surgery, providing a theoretical basis for clinical translation. We have added the citation and analysis of this paper in Line 1049 of the discussion section.

Value Elaboration of [https://doi.org/10.1002/anie.2025056691 - 2]

The functional dendritic nanogels designed in this study integrate o - hydroxyamine units (enhancing DNA compression efficiency), temperature responsiveness (stable during in - vivo circulation / triggering drug release at the target site), and pH - dependent charge conversion characteristics (increased charge density in an acidic environment). The matching strategy between its cross - linking density and degradation rate provides a quantitative reference for optimizing the biocompatibility and transfection efficiency of the vector. Notably, the "stimulus - response - degradation - drug release" synergistic mechanism of G3 - NGs, which degrades completely within 48 hours at pH 6.8 and releases DNA simultaneously, provides a design paradigm for the development of similar vectors. We have added the citation of this paper in Line 1050 of the discussion section.

The papers you recommended, from material design strategies to in - vivo verification data, form an important complement to our research, greatly enhancing the academic depth of this paper. Currently, the citations in the paper have been completed in accordance with the journal's format specifications (see References [59 - 60]), and the theoretical connection points have been clearly marked in the relevant discussions.

Thank you again for your careful guidance. If you have any further suggestions for modification, please feel free to let us know!

59.  Yang, Y.; Zhao, Y.L.; Zou, Y.J.; Lu, C.Y.; Li, N.; Shi, Z.Y.; Li, X.; Lai, X.X. Ultra-sensitive pH-responsive hydrogels with injectable and self-healing performance for controlled drug delivery. International Journal of Pharmaceutics: X 2025, 9, 100334. https://doi.org/10.1016/j.ijpx.2025.100334.
60.  Li, X.; Ouyang, Z.; Hetjens, L.; Ni, M.; Lin, K.; Hu, Y.; Shi, X.; Pich, A. Functional Dendrimer Nanogels for DNA Delivery and Gene Therapy of Tumors. Angewandte Chemie International Edition 2025, e202505669. https://doi.org/10.1002/anie.2025056691-2.

Q4. The font size in some of the picture was to small to be seen. For example, Figure 15, Figure 16, Figure 17, Figure and so on.

Response：Thank you for your meticulous review and valuable feedback on our study! Regarding the issue of excessively small font sizes in the figures, we have attached great importance to this and conducted a comprehensive review.

Upon confirmation, the blurriness of text in the composite figures is attributed to the reduction of original large figures containing dense text. We deeply apologize for this inconvenience, though the text can be clearly viewed when the figures are zoomed in. To ensure review clarity and reader accessibility, we have implemented the following improvements:

Font optimization: All figure fonts have been standardized to an optimal size. For figures 10, 11, 12, 13, 15, 16, 17, 21, and 22, critical information potentially obscured by scaling is relabeled in tabular format in Supplementary Table S1. 

High-resolution resources: The original figures (≥300 dpi) are provided as a compressed package in Supplementary Figure S1.

A guidance note is added above each figure: "If you cannot see the content of the picture clearly, you can refer to Supplementary Material Table S1 for detailed reading." These revisions are integrated into the updated manuscript, with complete supplementary materials attached. Should further adjustments be needed—such as figure segmentation, layout modification, or additional labeling—please let us know. We remain fully committed to refining the manuscript based on your feedback. Thank you again for your expert guidance.

Round 2

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

There is a formatting error in Ref. 60. Please change "2025, e202505669" to "2025, 64, e202505669"

Author Response

Author Response：

We are sincerely grateful to the reviewer for conducting such a meticulous and comprehensive review of our manuscript and for providing us with so many precious and valuable suggestions. Your feedback has made us aware of the various shortcomings in our manuscript writing. We would be very grateful if you could take a moment to review our newly uploaded manuscript and provide us with your new insights, which will surely assist us in further refining the manuscript.

Q1. There is a formatting error in Ref. 60. Please change "2025, e202505669" to "2025, 64, e202505669".

Response: Dear Reviewer, Thank you for your meticulous review of the reference formatting during the review process. Your suggestions are crucial for enhancing the paper's standardization, and we have attached great importance to this issue and completed the rectification. Regarding the formatting error you pointed out (There is a formatting error in Ref. 60. Please change "2025, e202505669" to "2025, 64, e202505669"), we have strictly corrected it according to your requirements. The revised reference format is as follows:

60. Li, X.; Ouyang, Z.; Hetjens, L.; Ni, M.; Lin, K.; Hu, Y.; Shi, X.; Pich, A. Functional Dendrimer Nanogels for DNA Delivery and Gene Therapy of Tumors. Angewandte Chemie International Edition 2025, 64, e202505669. https://doi.org/10.1002/anie.202505669.

This revision has made us realize the importance of rigorous reference formatting for academic standards. In the future, we will use tools such as EndNote/NoteExpress to verify the formatting before submission to avoid similar issues. Thank you again for your valuable comments! We look forward to your continued guidance on the revised version.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. PA-011 is described as a complex extract; however, no quantification or standardization of key active compounds (e.g., 17α-estradiol, dehydroepiandrosterone) is provided. To ensure reproducibility, it is recommended to include quantification data (e.g., LC-MS peak area or concentration in ppm) for the major bioactive components.
2. A total of 16,515 peptides were identified, but their origin—whether insect-derived or potential mammalian contaminants and biological relevance remain unclear. Please clarify whether these peptides are endogenous to Periplaneta americana and validate a subset for bioactivity.
3. The study includes hundreds of predicted targets derived from multiple databases without accompanying in vitro validation, which may weaken the mechanistic claims. It is advised to focus on 2–3 key targets (e.g., AKT1, MAPK1) and validate their expression using qRT-PCR or Western blot analysis.
4. Molecular docking results are presented without experimental support. To validate ligand-target interactions, confirmatory assays such as luciferase reporter assays or surface plasmon resonance (SPR) are suggested.
5. Several enriched pathways (e.g., those associated with Alzheimer’s disease) appear biologically irrelevant or speculative in the context of AGA. The interpretation should focus on pathways directly implicated in AGA pathogenesis, such as Wnt/β-catenin signaling, MAPK pathway, and androgen receptor signaling.
6. The claim that PA-011 improves skin and gut microbiota lacks mechanistic evidence. To establish causality, consider using gnotobiotic mice or antibiotic-depleted microbiota models to determine whether microbiota changes mediate hair growth effects.
7. Sections 2.5.7 and 2.5.8 are identical and should be consolidated to remove redundancy.
8. The abstract should clearly state that this is a preclinical animal (mouse) study, not a clinical trial.
9. Several grammatical and stylistic issues are present throughout the manuscript. For instance, phrases such as “reduce inflammation and oxidative stress, enhance hair follicle vitality” should be restructured for clarity and parallelism. A thorough language edit is advised.
10. Some figures' descriptions (e.g., Figures 9, 12, and 20) are vague or incomplete. The figure legends should clearly include specific details such as the statistical tests used, significance levels, and scale bars.
11. Specific compound names, such as “AKTI-17a,” are unclear or ambiguous. Please revise to ensure accurate and consistent chemical nomenclature throughout the manuscript.
12. How was the dosage of PA-011 (1% and 4%) determined? Was it based on preliminary dose-response or toxicity data?
13. Is there any known mechanism or evidence supporting the transdermal penetration of PA-011 and its delivery to dermal papilla cells?
14. Were any adverse effects or toxicity symptoms observed in PA-011-treated mice during the study?
15. What is the rationale behind using different concentrations of minoxidil in male (5%) and female (2%) groups? Is it based on pharmacokinetic data or precedent from existing literature?

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Reviewer 2 Report

Comments and Suggestions for Authors

The American cockroach extract has been evaluated by the authors to treat AGA. The following queries are posed to the authors for clarification:

1. The method of the experiment was unclear. The authors mentioned five groups in line 191 and eight groups again in line 195. How do they support their claims?
2. The experiments' end points were not specifically stated by the authors.
3. There is no animal ethical approval number. Was the study approved?
4. The aim is not clear and so are the endpoints.
5. Why male and female mice used in the study. What are the parameters observed in these sexes.
6. There is no toxicity study conducted for the PA-011 extract—why? The cockroach extract may exert allergic reactions in skin, as per the previous literature. Did they conduct the skin irritation test?
7. Whether the authors used both male and female sexes for the extraction? The authors need to explain this context for the extraction. Since the LCMS analysis showed the traces of 17a-Estradiol and Acetylcholine in the sample. 17a. Estradiol may interfere with the reproductive functions and Acetylcholine may cause allergic reactions - Need to justify this context.
8. The heading 2.5.3 must be explained in detail. You cannot homogenize skin tissue without the enzyme collagenase because the skin contains collagen. This procedure needs some serious detailing.
9. Why the authors performed gut microbiota studies here - This section tends to deviate from the aim of the study. 
10. No concrete evidence was presented to support the aim of the study.
11. Network pharmacology is not considered concrete evidence. We need some experimental proof for hair growth activity.

1.  

Comments on the Quality of English Language

Quality needs improvement