Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article entitled “Yin-dan-ping-gan Capsule mitigates CCL4-induced liver fibrosis via activating PPAR γ/GPX4 signaling and suppressing ferroptosis” written by Xue Jiang and co-workers, is a good scientific effort. The overall manuscript has generated some useful information. However, to improve the quality of manuscript and to help readers following are the comments need to be addressed. 

1. The title and abstract claim the mechanism is “via activating PPARγ/GPX4 signaling and suppressing ferroptosis.” Meanwhile, the data indicate correlation, not direct activation.
2. In the results section, the narrative is somehow confusing as in the current form the text is often a description of the figure rather than an interpretation of the result. Additionally, some key terms are undefined.
3. The central proposed mechanism (YDPG → PPARγ → GPX4 → inhibits ferroptosis) is not tested. It is only an observation from separate experiments. So, the authors need to modify the discussion and explicitly state this as a limitation and proposed model.
4. The authors need to incorporate some pf the key details such as what is the vehicle for YDPG and Silymarin? How was it prepared?, loading control also need to be mentioned for the blot images.
5. The figures quality as well as labeling need to be cross checked for improvement.
6. The discussion section needs proper modification as it jumps from the data to broad literature-based mechanisms without clearly separating the authors findings from speculation.
7. The discussion needs proper modification. Some of the very important and recent articles which the authors need to study and include in the discussion section in order to strengthen the mechanistic basis of this study are the recent work on the GPX4-ferroptosis axis in liver injury (https://doi.org/10.1007/s00204-025-04198-7), the role of TGF-β/Smad signaling in fibrosis (https://doi.org/10.1038/s41467-025-56922-7), and the precedent of natural products targeting this pathway to treat liver fibrosis (https://doi.org/10.1080/21691401.2020.1717507).
8. There are typos and formatting errors which need to be corrected.

Author Response

We appreciate the reviewers' overall positive assessment and detailed suggestions. Each of your recommendations has been immensely valuable and significantly contributed to improving the manuscript. We have addressed each comment as follows:

1. The title and abstract claim the mechanism is “via activating PPARγ/GPX4 signaling and suppressing ferroptosis.” Meanwhile, the data indicate correlation, not direct activation.

  Response: Our results indeed lack key evidence directly validating the association between PPARγ/GPX4 and ferroptosis. Following your advice, we replaced “activating” with “regulating” in the title and abstract. We also added a limitation statement in the final paragraph of the Discussion section.

2. In the results section, the narrative is somewhat confusing as the current text often describes figures rather than interpreting results. Additionally, some key terms remain undefined.

   Response: We have rewritten subsection 2.1 (page 5, lines 13-27), of the results and rearranged some results and figure order to enhance overall logic and readability.

3. The central proposed mechanism (YDPG → PPARγ → GPX4 → inhibits ferroptosis) is not tested. It is only an observation from separate experiments. Therefore, the authors need to modify the discussion and explicitly state this as a limitation and proposed model.  

   Response: A separate “Limitations” subsection (page 19, lines 22-26) has been added to the discussion, acknowledging that the PPAR-γ/GPX4 axis remains a putative, not proven, master route for YDPG’s anti-ferroptotic effect in liver fibrosis, and downstream components need to be functionally validated before a complete mechanism can be claimed.

4. The authors need to incorporate some key details such as: What is the vehicle for YDPG and Silymarin? How was it prepared? Loading controls also need to be mentioned for the blot images.

   Response: Section 4.3 now explicitly states that both YDPG and silymarin were suspended in 0.5% CMC-Na solution (page 20, line 28). All Western blot images (Figures 4 and 7) include GADPH as a loading control.

5. Figure quality and labeling require cross-checking for improvement.

    Response: Figures 4, 5, and 7 have been redrawn and adjusted. All images were re-exported in 600 dpi TIFF format.

6. The discussion section requires proper modification as it jumps from the data to broad literature-based mechanisms without clearly separating the authors' findings from speculation.  

   Response: The first paragraph (page 17, lines 6-14), third paragraph (page 18, lines 1-16), and fifth paragraph (page 19, lines 15-23) of the discussion section have been rewritten to clarify our findings and conclusions.

7. The discussion requires significant revision. To strengthen the mechanistic basis of this study, authors should review and incorporate key recent publications, including: - Recent work on the GPX4-ferroptosis axis in liver injury (https://doi.org/10.1007/s00204-025-04198-7), the role of TGF-β/Smad signaling in fibrosis (https://doi.org/10.1038/s41467-025-56922-7), and the precedent of natural products targeting this pathway to treat liver fibrosis (https://doi.org/10.1080/21691401. 2020.1717507).

   Response: Thank you very much for recommending these references. They are highly valuable for discussing our results. The references have been incorporated into the discussion and cited at appropriate locations (page 17, lines 18 and 23; page 18, line 16).

8. Typos and Formatting Corrections  

  Response: Over 7 spelling errors have been corrected; Latin binomial names are now uniformly italicized; the chemical formula “CCl₄” has been standardized as a subscript.

All changes are highlighted (yellow) in the revised manuscript. We hope these revisions address the reviewers' concerns. We are deeply grateful for your high-quality suggestions, which have significantly enhanced the manuscript's quality.

Thank you once again for your constructive feedback.

Sincerely,  

Prof Xianyu Li 

Corresponding Author

phd_xianyuli@foxmail.com

E-China Academy of Chinese Medical Sciences, Beijing, 100700, China.

Tel: +86-010-64015130

Reviewer 2 Report

Comments and Suggestions for Authors

Manuscript Title

Yin-dan-ping-gan Capsule mitigates CCl₄-induced liver fibrosis via activating PPARγ/GPX4 signaling and suppressing ferroptosis

 

General Assessment

The manuscript presents a comprehensive and mechanistically insightful study investigating the anti-fibrotic effects of Yin-dan-ping-gan Capsule (YDPG) in a CCl₄-induced liver fibrosis model. The integration of in vivo pharmacological validation, network pharmacology, proteomics, and deep-learning–based drug–target interaction prediction is a notable strength and reflects a modern, systems-level research approach.Overall, the study addresses an important biomedical problem and has clear relevance to the field of hepatic fibrosis and traditional medicine–derived therapeutics. I recommend the article to be published after a minor revision on the below said points.

 

Specific Comments

1. The manuscript is generally understandable; however, several sections—particularly the Introduction and Discussion—contain long sentences, minor grammatical errors, and stylistic inconsistencies. Professional English language editing is recommended to improve clarity and readability.
2. The compound carbon tetrachloride is inconsistently written as “CCl₄” and “CCL4” across the manuscript, figures, and captions. Please use single and clear chemical formula for that or you can give full form for the same throughout the article.
3. Minor inconsistencies are observed  in the reference list regarding journal name formatting and capitalization. Please revise them carefully in accordance with journals format.
4. Some descriptive passages related to ferroptosis, oxidative stress, and NRF2 signaling closely resemble standard review literature. Even though citations are provided, I suggest you to further paraphrasing to minimize similarity index concerns.
5. Several figures indicate n = 3. Please clarify whether these represent biological replicates or technical replicates, particularly for proteomics and Western blot experiments.
6. The Deep Purpose-based drug–target interaction analysis would benefit from additional methodological details, including model selection, parameter settings, and criteria used to select high-confidence interactions.
7. While network pharmacology, proteomics, and deep learning converge on similar pathways, the manuscript would be strengthened by a brief explanatory paragraph flexibly describing how overlapping targets were prioritized across these platforms.
8. Supplementary Tables (S1–S3) are appropriate and informative; however, some information is unnecessarily described in the main text. Please revise it
9. If relevant to the discussion, the authors are requested  cite recent studies involving nanofiber-based systems or mesoporous silica platforms in modern drug delivery tools. For that I had given some list of DOIs . There is no need that you should strictly bind with reference given below you can cite even more recent reference related to your filed of study

https://doi.org/10.1016/j.jiec.2011.11.121

 

 

1. The proposed schematic mechanism is informative but could be visually simplified. Clear differentiation needed between upregulated and downregulated pathways to enhance the readers understandability.

 

Recommendation: Minor Revision is expected in the above said comments

All the best

 

Comments on the Quality of English Language

little refinement is needed in introduction and methodology.

Author Response

We appreciate the positive overall evaluation and the helpful suggestions. We have revised the manuscript point-by-point as follows:

1. The manuscript is generally understandable; however, several sections—particularly the Introduction and Discussion—contain long sentences, minor grammatical errors, and stylistic inconsistencies. Professional English language editing is recommended to improve clarity and readability.  

   Response: The entire text was professionally edited again. Long sentences in the Introduction and Discussion were shortened and converted to active voice where appropriate.

2. The compound carbon tetrachloride is inconsistently written as “CCl₄” and “CCL4” across the manuscript, figures, and captions. Please use a single and clear chemical formula for that or you can give the full form for the same throughout the article.  

   Response: “CCl₄” (with subscript 4) is now used uniformly throughout the manuscript, figures, and captions.

3. Minor inconsistencies are observed in the reference list regarding journal name formatting and capitalization. Please revise them carefully in accordance with the journal’s format.  

   Response: All 36 references were reformatted according to the MDPI style (correct journal abbreviations, capitalization, and punctuation).

4. Some descriptive passages related to ferroptosis, oxidative stress, and NRF2 signaling closely resemble standard review literature. Even though citations are provided, I suggest you further paraphrase to minimize similarity-index concerns.  

   Response: Paragraphs describing ferroptosis, oxidative stress, and NRF2 signaling were paraphrased (page 18, lines 1–16; page 19, lines 1–9).

5. Several figures indicate n = 3. Please clarify whether these represent biological replicates or technical replicates, particularly for proteomics and Western blot experiments.  

   Response: Figure legends now state: “n = 3 biologically independent animals; each sample was measured in technical duplicate.” The same wording was added to the proteomics and Western blot sections in Methods.

6. The Deep Purpose-based drug–target interaction analysis would benefit from additional methodological details, including model selection, parameter settings, and criteria used to select high-confidence interactions.  

   Response: A new subsection (Methods 4.7, page 23, lines 7–22) details the model (Graph-CNN-CPI), hyperparameters (learning rate 0.001, batch 256, epochs 100), and high-confidence cut-off (score ≥ 0.90).

7. While network pharmacology, proteomics, and deep learning converge on similar pathways, the manuscript would be strengthened by a brief explanatory paragraph flexibly describing how overlapping targets were prioritized across these platforms.  

   Response: Thank you for this suggestion. A new paragraph explaining how overlapping targets were prioritized across network pharmacology, proteomics, and deep-learning platforms has been added to the Discussion (page 19, lines 15–23).

8. Supplementary Tables (S1–S3) are appropriate and informative; however, some information is unnecessarily described in the main text. Please revise it.  

   Response: Three lengthy lists in the main text were shortened to one sentence each, directing readers to Supplementary Tables S1–S3.

9. If relevant to the discussion, the authors are requested to cite recent studies involving nanofiber-based systems or mesoporous silica platforms in modern drug-delivery tools. For that I had given some list of DOIs. There is no need that you should strictly bind with reference given below; you can cite even more recent references related to your field of study (https://doi.org/10.1016/j.jiec.2011.11.121). The proposed schematic mechanism is informative but could be visually simplified. Clear differentiation is needed between up-regulated and down-regulated pathways to enhance reader understandability.  

   Response: Thank you very much for the recommended literature; the relevant paper has been added and cited appropriately in the Discussion (page 17, reference 8). The mechanistic diagram has been redrawn: red arrows now highlight the multi-target actions of YDPG, and non-essential elements were removed to improve clarity.

 

All changes are highlighted (blue) in the revised manuscript.We hope that these revisions fully address your concerns and improve the clarity and impact of our manuscript.  

Thank you again for your constructive feedback.

 

Sincerely,  

Prof Xianyu Li 

Corresponding Author

phd_xianyuli@foxmail.com

E-China Academy of Chinese Medical Sciences, Beijing, 100700, China.

Tel: +86-010-64015130

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Manuscript ID: pharmaceuticals-4089263

The manuscript with the title „Yin-dan-ping-gan Capsule mitigates CCL4-induced liver fibrosis via activating PPAR γ/GPX4 signalling and suppressing ferroptosis” described the results of investigating the impact of YDPG on liver fibrosis, a mouse model of liver damage caused by carbon tetrachloride (CCL4).

Following YDPG treatment, the authors observed a decrease in the fibrosis index and an improvement in liver function. Network pharmacology, deep learning, and proteomics collectively identified the ferroptosis and PPAR signalling pathways as pivotal in the anti-fibrosis effects of YDPG on the liver. In addition, the authors found that YDPG inhibited MDA and Fe2+ content and increased GSH activity in fibrotic liver. Based on their results the authors finally concluded that YDPG limits the progression of liver fibrosis by regulating the PPARγ-GPX4-ferroptosis pathway. These results indicate that YDPG could be a potential medication for hepatic fibrosis.

The introduction section provides a general overview and impact and root causes of hepatic fibrosis. The involvement of the PPAR γ)- pathway and the role of ferroptosis is shortly reviewed. The information provides a high level overview of the current disease, whereas the relationship between ferroptosis and the PPAR signalling pathway in liver fibrosis is gradually being elucidated.

In the second part of the introduction the role of Chinese medicines in the treatment of liver diseases is described. To confirm the therapeutic impact of YDPG on liver injury, the authors created a mouse model of liver fibrosis caused by CCL4. The presented  findings revealed the mechanism landscape of YDPG and provided potential targets for the treatment of liver fibrosis.

The “Materials and Method” section is divided in 11 sections, und of them (Network Pharmacology Research) is further structured in sub-sections:

• Chemical and material
• Animals
• Experimental design
• Evaluation of blood and liver parameters
• Histological Analysis
• Network Pharmacology Research
  • Prediction of YDPG Component Targets
  • Prediction of Liver Fibrosis Targets
  • Construction of Protein-Protein Interaction (PPI) Network
  • Construction of Target-Pathway Network Diagrams
  • Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Enrichment Analysis 175 and Gene Ontology (GO) Function
• Drug Target Interaction Prediction
• Proteome analysis
• Antioxidant enzyme activities and Fe2+ levels detection
• Western blot assay
• Statistical analysis

The chosen structure allows the reader to move step by step through their findings. Experimental results and their respective interpretation are clearly separated. The experimental design of the mouse study is well justified. All experimental details applied for analysis are presented in a state of the art manner.

The Ishak scoring system for liver fibrosis is provided in a tabulated form. More detailed information is enclosed in the supplementary document (in tabulated form as well)

The chemical components of YDPG were investigated using a hybrid quadrupole or-bitrap high-resolution mass spectrometer combined with ultra-performance liquid chromatography (UHPLC-Q-Orbitrap HRMS). Mass spectrometry data were analysed using Progenesis QI 3.0 (Waters Corp., MA, USA). The methodology and the type of data bases used for prediction of Liver Fibrosis Targets are well described. The construction of Protein-Protein Interaction (PPI) Network and Target-Pathway Network Diagrams are documented in separate sub-chapters.

For the Drug Target Interaction Prediction the prediction framework offered by DeepPurpose is used. All details of the Proteome analysis and Antioxidant enzyme activities and Fe2+ levels detection are described in a separate sub-section as well. All details of the Western blot assays (including more details of the applied antibodies in the supplement) and the statistical analysis are perfectly summarized as well.

The “Results” section consists of 5 subchapters and several illustrating figures for visualizing the complex data packages:

• YDPG alleviates CCL4-induced liver fibrosis
• Identification of chemical constituents in YDPG
• Analysis of targets of YDPG for improving hepatic fibrosis
• Proteomic changes in liver tissue of CCL4-treated mice after YDPG intervention
• YDPG activated the PPAR γ/GPX4 pathway in the liver of CCL4-treated mice
• YDPG prevents CCL4-induced hepatocyte ferroptosis

The alleviation of CCL4-induced liver fibrosis by ADPG is well documented and supported by a figure. In the Identification of chemical constituents in YDPG section it is stated that a total of 275 compounds were identified in the seven YDPG herbs. The component analysis of YDPG is supported by a quite complex figure. Here I see a real weak point of the study. To discuss only the numbers of compounds is definitely not sufficient. At least some more information about the functionalities of the structures and some comments about the diversity of chemical constituents is recommended. Are there any compounds which have the main activity of this complex mixture(s)?

The ” Analysis of targets of YDPG for improving hepatic fibrosis” is based on network pharmacology analysis. The results of the KEGG pathway enrichment analysis indicated that the pathway included the oxidative phosphorylation, PPAR signaling pathway, mTOR signaling path-way, and other signaling pathways. The Analysis of targets of YDPG is further supported by illustrative figures. The “Proteomic changes in liver tissue of CCL4-treated mice after YDPG intervention” are discussed in a separate sub-chapter including illustrative figures.

YDPG activated the PPAR γ/GPX4 pathway in the liver of CCL4-treated mice. The functional enrichment analysis of the proteomics results is discussed in this section, again supported by illustrative figures. The final sub section of the “Results” is describing how YDPG prevents CCL4-induced hepatocyte ferroptosis. In present study, the authors utilized a drug-target interaction (DTI) prediction framework provided by DeepPurpose to further reconfirm potential targets and pathways for YDPG treatment of CCl4-stimulated liver injury. Since this chapter provides the main findings of the study and is the longest part  supported by two illustrative figures.

The ”Discussion” section is nicely summarizing the complex data sets and the taken conclusions are nicely supported by the presented data. Therefore, the “conclusion” section is short and conclusive: The studies illustrate the therapeutic efficacy of YDPG in liver fibrosis. The consistent upregulation of GPX4 and downregulation of Fe²⁺/MDA following YDPG treatment strongly suggest that  this axis contributes to the anti-fibrotic effects observed.

Overall, a very well written manuscript with a proper and valid investigation. The biochemical and in vivo assessment part is well written and the data are comprehensively summarized. Only some more details of the chemical composition should be amended by some more structural information of the relevant chemical compound classes.

Author Response

We sincerely appreciate your positive evaluation and constructive feedback on our research. Our detailed responses are as follows:

We fully agree that “listing only the numerical values of chemical constituents” is insufficient. Therefore, we have adjusted the placement of Figure 2 to logically link it with the results in Figure 6. This demonstrates that after identifying the chemical constituents of YDPG, we further validated its potential targets and action pathways for treating carbon tetrachloride-induced liver injury using the Drug-Target Interaction (DTI) prediction framework provided by DeepPurpose.

Additionally, we have incorporated a pie chart into Figure 5 (formerly Figure 2) to provide an in-depth analysis of the identified compound analogues and their respective proportions. Corresponding descriptions for this new pie chart have been added to the Results section (pages 11-12, lines 13-17 and 1-5).

All changes are highlighted (red) in the revised manuscript.We hope these revisions address your concerns. We sincerely appreciate your feedback on this manuscript, which has been invaluable in refining our research.  

Sincerely,  

Prof Xianyu Li 

Corresponding Author

phd_xianyuli@foxmail.com

E-China Academy of Chinese Medical Sciences, Beijing, 100700, China.

Tel: +86-010-64015130

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I have reviewed the revised manuscript. The authors have adequately addressed most of my previous comments and concerns. The manuscript has been improved and is now methodologically sound and clearly presented. I find the current version acceptable and recommend it for publication.