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  • Xiaojin Liu 1,2,*,
  • Shuyue Chen 1,2 and
  • Yefan Gu 1,2
  • et al.

Reviewer 1: Anonymous Reviewer 2: Rafael Carvalho Reviewer 3: Anonymous

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

  1. The potential cytotoxic effects of EUE on B16 cells should be evaluated to exclude the possibility that the observed biological effects are attributable to cytotoxicity rather than specific pharmacological activity.

  2. A positive control for antioxidant activity, such as ascorbic acid, should be included to enable a more meaningful assessment of the antioxidant efficacy of EUE.

  3. The concentrations of H2O2 and EUE reported in Figures 1 and 2 are inconsistent and require clarification. Specifically, the concentration of H2O2 is indicated as either 0.5 mg/mL or 0.05 mg/mL, while the serial concentrations of EUE are reported as 0.1–0.8 mg/mL in some sections and 0.02–0.16 mg/mL in others. The authors should carefully verify and correct these discrepancies throughout the manuscript.

  4. To further elucidate the underlying mechanisms by which EUE promotes HDPC proliferation, the protein expression levels of key signaling molecules, including VEGF and components of the Wnt/β-catenin pathway, should be assessed. Evaluation of mRNA expression alone is insufficient to substantiate the proposed mechanisms of action.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

I read the study with genuine interest. The multi-target rationale, combining antioxidant defense, folliculogenic signaling, and antiandrogenic activity within a single botanical preparation, is timely, and the integrative design spanning chemical characterization, cell-based assays, a cell-free enzymatic model, permeation testing, and an in vivo model is a real strength. The explicit HPLC-DAD characterization and the candid Limitations section are also appreciated.

At the same time, I identified a number of points that, in my view, should be addressed to strengthen the work, chiefly regarding the alignment of the central mechanistic claims with the evidence actually presented, together with several internal inconsistencies in doses, figures, and statistical reporting that would benefit from clarification.

My complete, point-by-point assessment is provided in the attached file, organized as (1) a brief summary of the manuscript and its contributions, (2) a detailed, section-by-section evaluation of the methodology, analyses, and conclusions, and (3) constructive, prioritized suggestions for improvement. 


Comments for author File: Comments.pdf

Comments on the Quality of English Language

The manuscript is generally clear and well organized but would benefit from light English editing and the standardization of units and notation. Typographical anomalies (e.g., in “attenuates,” “Committee,” and “Wetzlar”) are likely PDF font-encoding artifacts; please verify the source file. Furthermore, panel labels and repeated captions (Figures 2 and 3) should be revised and corrected.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

         

The submitted manuscript for review, titled „Eucommia ulmoides Bark Extract Mitigates Oxidative Damage and Promotes Hair Follicle Regeneration by Activating Nrf2/HO-1 and Wnt/β-Catenin Signaling “ (molecules-4403988), presents a study on the antioxidant, melanoprotective, and hair-regenerative potential of Eucommia ulmoides using in vitro and in vivo approaches. I think the article needs major revision. I have the following recommendations and questions for the authors:

  1. In the authors' names, put a comma between 1 and 2.
  2. “In vivo” and “in vitro” should be in italic.
  3. In the text of the results section, always explain the results in which figures a, b, c... are shown.
  4. In Fig. 1b, indicate better the treatment with H2O2 in groups 0.02-0.08 EUE. The same for Fig. 2b for groups 0.1 and 0.2 EUE.
  5. If necessary, correct the p-value in the sentence “MDA levels were concurrently and progressively reduced following EUE treatment, with the most pronounced reduction observed at 0.08 mg/mL (p < 0.01) (Figure 1).” In Fig. 1a, p appears to be < 0.001.
  6. Correct the sentence “Hydrogen peroxide exposure significantly downregulated Nrf2 and HO-1 expression, whereas EUE treatment restored and enhanced their mRNA and protein levels in a dose-dependent manner.” because the results do not show a dose-dependent effect.
  7. „The highest induction was observed at 0.8 mg/mL for mRNA expression, with Nrf2 and HO-1 transcripts increased by 2.5- and 3.2-fold, respectively, compared with the oxidative model (p < 0.01).“ – for Nrf2, the highest results are not in group 0.8 mg/mL EUE. Correct the results.
  8. Correct the sentence “Based on the initial screening, EUE showed no significant cytotoxicity at concentrations up to 0.16 mg/mL (Figure 3a)”. Fig. 3a shows these results up to 0.32 mg/mL EUE.
  9. Why didn’t you add the concentration of 0.32 mg/mL EUE in the experiments?
  10. Correct the sentence “CCK-8 assays demonstrated that EUE significantly increased HDPC viability in a dose-dependent manner, with the highest concentration (0.16 mg/mL) promoting a 22% increase over untreated controls (p < 0.01)” because the results do not show a dose-dependent effect and the biggest increase is in the 0.08 mg/mL EUE group.
  11. In the legend of Fig.3, add the value of ***p.
  12. In the legend of Fig.3, what does " vs. untreated control " mean? Untreated with what?
  13. “RT-qPCR analysis revealed that EUE markedly upregulated the mRNA expression of vascular endothelial growth factor (VEGF), β-catenin, and Wnt5a by 1.8-, 2.1-, and 1.7-fold, respectively, compared with control cells (Figure 3).” - indicate for which groups these results are.
  14. „In the initial evaluation, 40 ng/mL DHT reduced cell viability to approximately 65% of the control (Figure 4a), establishing this concentration for subsequent evaluation.“ - These percentages are not visible in the figure.
  15. „EUE treatment significantly antagonized this androgen-induced suppression; specifically, at 0.16 mg/mL, EUE successfully restored cell viability to 92.41% (Figure 4b)“ – you have higher results for other concentrations of EUE.
  16. Check the concentrations of EUE in all figures, their legends, and in the text – is it 0.04 or 0.4; 0.08 or 0.8….. etc.
  17. Add “ns” in the legends of all figures where it appears.
  18. To be chronological, give the results with the direct inhibition of 5α-reductase activity by EUE in vitro in Fig. 4a.
  19. Write the significance value in the legend of Fig. 5.
  20. To be chronological, give the results of Fig. 5C in 5A and after that the results from the histological analysis.
  21. Explain why you used doses of 0.5, 1 and 2 mg/mL EUE in the in vivo experiments.
  22. Explain in the discussion why the observed effects are not most pronounced at the highest concentrations.
  23. In the discussion, the results obtained should be compared with those of other authors.
  24. How will the results obtained be explained with the chemical composition of the extract? Provide data from other authors on the main components in the extract for which there are results in the effects studied.
  25. What kind of extract was used? The technology of extract preparation must be presented.
  26. Results should be presented as mean ± SEM, not SD.
  27. An analysis should be performed to determine the normality of the distribution of the results.
  28. Provide the number of permits for working with experimental animals at the university and state levels.

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for the authors' revisions. The manuscript has been adequately improved and is suitable for acceptance.

Author Response

Thank you very much for your positive evaluation and recommendation for acceptance. We sincerely appreciate the time and care you devoted to reviewing our revised manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

I thank the authors for a careful and responsive revision. The central scientific concern raised in the first round, namely that the title and conclusions overstated the mechanistic evidence, has been addressed convincingly. The revised title, Abstract, Results, Discussion and Conclusions now describe attenuation of H2O2-induced follicular suppression and modulation of Wnt/beta-catenin-associated signaling, rather than promotion of regeneration and pathway activation. The clarification that Figures 3b to 3d correspond to H2O2-challenged HDPCs, the reframing of the 5-alpha-reductase comparison, the repositioning of the Franz diffusion experiment as a qualitative screening assay, the correction of the H2O2 labeling in the B16 panels, and the completion of the ethics statement are all satisfactory. The disclosure of the quantification wavelengths (240 nm and 325 nm) resolves the internal discrepancy between chlorogenic acid content and apparent peak intensity, and the reporting of integrated peak areas exceeds what was offered in the response letter.

However, some points still require clarification or improvement. Please refer to the attached document.

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors When responding to reviewers' comments, the lines where the changes were made must be indicated. The way the changes were made, and especially the fact that each response does not describe where the correction was made, makes the reviewer's work extremely difficult. Comment 2: Not made throughout the text. Comment 3: Тhere are still places where it is not mentioned which panel of the figure is being discussed. Comment 5: Instead of better understanding, there is a lack of information about what the p-value is and which figure reflects the results. Comment 7: The description is not detailed; there is no comment on which concentration has the strongest result. Comment 9: Most of your arguments are assumptions; the higher concentration should have been studied. It may be possible to obtain better effects with it. Comment 11: Add “vs. control” Comment 14: Not present in the results description. Comment 15: Where are the changes made? At least you should have pointed out the corrected sentences in a comment! Comment 16: Explain the use of different concentrations in the different experiments and why you used these exact concentrations. Comment 19: vs what is the significance? Comment 21: Where is the justification? It should be supported by references or your previous research. Comments 23 and 24: Тhe discussion still needs revision based on the recommendations made. Comment 26: Both parameters, SEM and SD, cannot be used. Comment 27: But if the distribution of the results is not normal, then the ANOVA test is not applied.  

Author Response

Please see the attachment.

Author Response File: Author Response.docx