Phloretin Protects Goat Adipose-Derived Mesenchymal Stem Cells Against Ferroptosis by Regulating the Nrf2/HO-1/GPX4 Signaling Pathway

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article submitted to Animals, entitled “Phloretin Suppresses Ferroptosis in Goat Adipose-Derived Mesenchymal Stem Cells Via the Nrf2/HO-1/GPX4 Pathway: Implications for Ruminant Breeding”, presents novel research on the ferroptosis model. The manuscript is well written and provides sufficient methodological information for readers to understand and replicate the experiments. The topic is interesting and may contribute to the development of future tools for ruminant breeding.

I did not identify major issues in the manuscript. However, I have a few minor suggestions:

• Materials and Methods: Instead of writing “was purchased”, I suggest that the authors simply include the name of the company supplying the reagents in parentheses after each product.
• Line 203 – “samples were observation” should be corrected (e.g., “samples were observed”).
• Line 400 – “a series of experiments were conducted”.
• Line 405 – A reference supporting that Fer-1 is a classic ferroptosis inhibitor should be included.

Overall, the manuscript is clear and scientifically sound, and I believe it is suitable for publication after these minor revisions.

Author Response

评论 1: 材料和方法：我建议作者在每个试剂后面括号内简单地包含提供该试剂的公司名称，而不是写“被购买”。

第203行 – “samples were observation” 应该更正为 (例如, “samples were observed”).

第400行 – “进行了一系列的实验”。

第405行 – 应该包含一个支持Fer-1是经典铁死亡抑制剂的参考文献。

总体而言，手稿清晰且具有科学性，我相信在进行这些小的修改后，它是适合发表的。

回复1：我们诚挚感谢审稿人对正面评论和建设性建议。我们已经通过在括号中添加供应商信息来修订试剂来源的表达，而不是使用“购买”。手稿中的语法错误已被更正，包括将“samples were observation”更改为“samples were observed”（第269行），并且诸如“a series of experiments were conducted”之类的不恰当表达已被优化。此外，已添加相关参考文献以支持Fer-1是经典的铁死亡抑制剂。手稿中已完成所有次要修订（第475行和第681行）。

所有修改已在修订的手稿中注明。我们再次对评审员的仔细评审和建设性建议表示衷心的感谢，这极大地提高了我们手稿的质量。

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript submitted for review presents a study using caprine (goat) mesenchymal stem cells derived from adipose tissue (AD-MSCs/ADSCs). The authors test the effect of phloretin on cells subjected to ferroptosis (iron-dependent, oxidative cell death) using RSL3 (a direct inhibitor of glutathione peroxidase 4). The entire study is conducted in an elegant manner: the authors expose MSCs to RSL3 with and without pretreatment with phloretin and subsequently perform numerous analyses. These include the assessment of markers of oxidative stress, ferroptosis, and factors counteracting this form of cell death through the analysis of the expression of several proteins involved in mechanisms protecting cells against oxidative stress, as well as functional assays such as the wound-healing assay (migration) and the evaluation of MSC proliferation under the tested conditions. Overall, the study is well designed and fairly well presented. The results presented are coherent and indicate a protective effect of phloretin on MSCs under conditions of induced ferroptosis.

Major comments

1. The weaker aspect of the manuscript is the presentation of the rationale for the experiments performed, which is currently unclear, somewhat inconsistent, and not entirely convincing. The authors state that their observations regarding the effects of phloretin on MSCs have a potential to improve “ruminant breeding efficiency.” It is not clear how the authors intend to use MSCs in the process of improving ruminant breeding efficiency. The authors mix issues related to the activity of endogenous MSCs with their therapeutic potential following transplantation. Indeed, it is generally considered that transplanted MSCs are exposed to oxidative stress, free radicals, and thus to the risk of ferroptosis. In this context, the search for factors that could serve as protective agents for MSCs prior to transplantation is indeed very interesting. In contrast, attempts to influence endogenous MSCs in a similar manner are not entirely justified. However, at the end of the Introduction the authors indicate that they rather view the investigated phloretin as a “green and safe feed additive/regulatory product.” They do not mention that it could be used as a substance for conditioning MSCs prior to potential transplantation. A somewhat different approach appears in the Discussion, where the authors explain that they see phloretin as a factor for preconditioning (fragment from line 477). However, in the Conclusions section the proposal appears again to treat phloretin as a “natural protective agent,” which once more suggests its use as a potential dietary supplement. This approach could be unified throughout the manuscript so that the reader has a clear understanding of the underlying assumptions of the conducted research.
2. There is a significant discrepancy between the title and the discussion section. While the title asserts a definitive mechanism (Via the Nrf2/HO-1/GPX4 Pathway), the Discussion remains speculative, stating only that phloretin “may promote” this pathway. In my opinion the title should be modified to be less definitive.
3. MSCs isolated from tissues are characterized by inter-individual variability. It is not clear how many “populations,” understood as MSCs derived from different donors, were used in the study. This information must be provided. Without it, it is difficult to assess the reliability of the results.

Other comments:

The “simple summary” is not simple. It contains detailed information that is difficult to understand for readers who are not well acquainted with the topic. It should be revised.

Introduction

The authors attach too little importance to the precise citation of the work of other researchers. They do not always indicate which of the cited findings are based exclusively on in vitro studies, which refer to the effects of MSC transplantation, and which concern endogenous processes studied in vivo. This creates some confusion and makes it more difficult for the reader to follow the content.

The terms “biological breeding” and “anti-stress breeding” are unclear and appear somewhat unprofessional.

Line 61: MSCs are not pluripotent. They are multipotent. Please correct.

The introduction of abbreviations requires correction—some are not introduced (such as HO-1 and GPX4), while others are introduced multiple times (e.g., ADSCs, although it was not expanded in the abstract).

The authors should consider changing the abbreviation used for the studied cell population. The International Society for Cell & Gene Therapy recommends the abbreviation AD-MSC, which emphasizes the mesenchymal origin of this population, whereas ADSCs, although frequently used, is less informative.

Please clarify the ethical aspects of the study. Were the animals subjected to a fat-collection procedure under anesthesia, or the tissue was collected after euthanesia?

Methods

Please provide information on how many donors the cells were isolated from and whether cells from different donors were pooled.

For each method (preferably in the figure legend), please indicate how many populations (in terms of the number of donors) were used for a given test. For example: cells from three donors were used, each with three technical replicates; or cells from one donor were used and three independent experiments were performed.

Chondrogenic differentiation: the composition of medium used is very simple and does not include TGF-β, which is commonly used for this type of differentiation. Was there a particular reason for using such composition?

An explanation of the mechanism of action of the RSL3 reagent appears only in the Discussion, whereas it would be good to provide in the Introduction or in the Methods section.

The authors should clarify information related to percentage composition—when the symbol % is used, it should be specified whether this refers to w/v or v/v and in relation to what. For example, the authors write “10% CCK-8,” whereas it should be specified as “10% v/v in growth medium” (if this is indeed the case).

The authors should add information about the origin of the CCK-8 assay.

Please add information about the culture conditions used for individual assays. In some cases such information is provided, while in others it is not. For example, on which plates were the cells cultured for fluorescence assays? Were plates with black walls used? From what surface were the cells collected for the Western blot assay?

Migration assay: how did the authors ensure that the same field of view was imaged at subsequent time points? Did they use a microscope equipped with an automated stage?

What are “double antibodies”? Please explain and justify their use.

Please provide information on the antibodies used (both in immunocytochemistry and Western blot): clone, manufacturer, and dilution (concentration).

Please specify the version of the Tecan plate reader used.

Results

“Impurities” is a term rather not used in reference to cell cultures. It would be better replaced with terms such as contaminating cells, cell debris, or tissue debris, depending on the observed contaminants.

Line 247 – should be “bottom” instead of “wall”

Lines 253-258: this part should be at the end of this paragraph to keep the sequence of citing the figures (first 1b, than 1c)

Figure 1

Please add scale bars to the images in panels 1(a) and 1(b)—it is clear that different magnifications were used. An explanation of the left and middle columns in panel 1(b) is missing in the legend. Should be “Alizarin Red”, not Red S.

Figure 2

Specify in the legend which statistical method was used. ANOVA? If so, what type of post-hoc test was applied? Cells from how many donors were used?

Figure 3

Please specify what is presented in graph 3(a). These are relative values, but it is not clear whether the analyzed parameter was fluorescence measured with a plate reader or fluorescence measured from images obtained using a fluorescence microscope (this information should also be included in the Methods section). The Y-axis description should be corrected. The term “expression of ROS” is inappropriate.

Similarly in 3(b), it is unclear what was measured and what is presented. Was only green fluorescence measured? Or the ratio of green to red? Was the measurement performed using a plate reader or based on microscopic images? Please also add this information in the Methods section.

In 3(c) and 3(d), please correct the Y-axis description—the term “expression” is conventionally used in relation to genes or proteins, not to the presence of ions or other chemical compounds such as MDA.

Specify in the legend which statistical method was used. ANOVA? If so, what type of post-hoc test was applied? Cells from how many donors were used?

Line 307: the description of mitochondria appears inconsistent with the images presented in Figure 3(f). The mitochondria appear rather swollen and enlarged rather than shrunk.

Figure 4

Please supplement the information in the figure legend regarding the statistical test used (if ANOVA, then which post-hoc test), analogous to the comments for the previous figures. Please also include information regarding the number of donors from whom the cells used in this test were derived.

Figure 5

Migration: As above: please specify which statistical tests were used and from how many donors the cells were derived.

Growth: the presented data do not include standard deviations. Does this mean that only a single experiment was performed? If so, please clarify this and comment on it appropriately. In such a case, these data should be interpreted with great caution. If more data were included in the group, please indicate the standard deviations and specify the number of biological replicates “n”. The Y-axis label should also be corrected. These values appear rather to indicate the number of cells per well (according to the description in the Methods) rather than cell density per ml.

Discussion

Lines 421–442: this part of the Discussion is highly speculative. Expressions such as “preliminary study,” “we speculate,” phloretin “may promote,” and “the Nrf2-GPX4 axis is a highly likely mechanism” appear here. This represents a significant discrepancy with the title, which contains a strong statement. In light of such inconsistencies, the title should be moderated.

 

Comments on the Quality of English Language

I am not a native speaker. Some sentences seems to be too convoluted and not very clear. Some expression sound not very scientific (I pointed it in the main review part). But generally in my opinion language requires minor corrections.

Author Response

Comments 1: The weaker aspect of the manuscript is the presentation of the rationale for the experiments performed, which is currently unclear, somewhat inconsistent, and not entirely convincing. The authors state that their observations regarding the effects of phloretin on MSCs have a potential to improve “ruminant breeding efficiency.” It is not clear how the authors intend to use MSCs in the process of improving ruminant breeding efficiency. The authors mix issues related to the activity of endogenous MSCs with their therapeutic potential following transplantation. Indeed, it is generally considered that transplanted MSCs are exposed to oxidative stress, free radicals, and thus to the risk of ferroptosis. In this context, the search for factors that could serve as protective agents for MSCs prior to transplantation is indeed very interesting. In contrast, attempts to influence endogenous MSCs in a similar manner are not entirely justified. However, at the end of the Introduction the authors indicate that they rather view the investigated phloretin as a “green and safe feed additive/regulatory product.” They do not mention that it could be used as a substance for conditioning MSCs prior to potential transplantation. A somewhat different approach appears in the Discussion, where the authors explain that they see phloretin as a factor for preconditioning (fragment from line 477). However, in the Conclusions section the proposal appears again to treat phloretin as a “natural protective agent,” which once more suggests its use as a potential dietary supplement. This approach could be unified throughout the manuscript so that the reader has a clear understanding of the underlying assumptions of the conducted research.

Response 1: We sincerely appreciate the reviewer’s insightful comment. The entire manuscript has been thoroughly revised to focus exclusively on phloretin as a preconditioning agent for in vitro transplantation of goat AD-MSCs, with all feed additive-related descriptions deleted to clarify the research boundaries between endogenous MSCs activity and post-transplantation therapeutic potential. The Introduction now clearly proposes the core hypothesis of screening efficient ferroptosis inhibitors for AD-MSCs pre-transplantation to improve stem cell-assisted breeding efficiency (Lines 244-246). The Discussion fully elaborates on the molecular mechanism, experimental verification and breeding application value of phloretin as a preconditioning agent, and the conclusion focuses on providing theoretical and experimental support for its development.

Comments 2: There is a significant discrepancy between the title and the discussion section. While the title asserts a definitive mechanism (Via the Nrf2/HO-1/GPX4 Pathway), the Discussion remains speculative, stating only that phloretin “may promote” this pathway. In my opinion the title should be modified to be less definitive.

Response 2: We thank the reviewer for the rigorous correction. The title has been revised to "Phloretin Protects Goat Adipose-Derived Mesenchymal Stem Cells Against Ferroptosis by Regulating the Nrf2/HO-1/GPX4 Signaling Pathway", with definitive expressions weakened to match the in vitro cellular level conclusions. The Discussion has been standardized: affirmative statements are used for conclusions supported by experimental data; reasonable speculations for mechanism extension are retained with a clear annotation of "in vitro cellular level verification".

 

Comments 3: MSCs isolated from tissues are characterized by inter-individual variability. It is not clear how many “populations,” understood as MSCs derived from different donors, were used in the study. This information must be provided. Without it, it is difficult to assess the reliability of the results.

Response 3: We are grateful for the reviewer’s critical reminder. Information on donor number, experimental repeats and statistical analysis has been supplemented in the Materials and Methods section to provide a solid experimental basis for the conclusions. (Lines 316–320)

 

Comments 4: The “simple summary” is not simple. It contains detailed information that is difficult to understand for readers who are not well acquainted with the topic. It should be revised.

Response 4: Thank you very much for your valuable comments on the Simple Summary. We agree that the original version contained excessive technical details and specialized terms, which made it difficult for general readers to understand. According to your suggestion, we have revised the Simple Summary to be more concise and readable, removing complex professional descriptions while retaining the core research content and significance. (Lines 13–19)

 

Introduction

Comments 5: The authors attach too little importance to the precise citation of the work of other researchers. They do not always indicate which of the cited findings are based exclusively on in vitro studies, which refer to the effects of MSC transplantation, and which concern endogenous processes studied in vivo. This creates some confusion and makes it more difficult for the reader to follow the content.

Response 5: We appreciate the reviewer’s detailed suggestion. All cited literatures in the Introduction have been sorted out, with descriptions including "in vitro study", "in vivo study" and "in vitro transplantation-related study" naturally integrated into the text to clarify the application scenarios of different research conclusions and avoid content confusion.

 

Comments 6: ① The terms “biological breeding” and “anti-stress breeding” are unclear and appear somewhat unprofessional. ② MSCs are not pluripotent. They are multipotent. Please correct. ③The authors should consider changing the abbreviation used for the studied cell population. The International Society for Cell & Gene Therapy recommends the abbreviation AD-MSC, which emphasizes the mesenchymal origin of this population, whereas ADSCs, although frequently used, is less informative. ④ The introduction of abbreviations requires correction—some are not introduced (such as HO-1 and GPX4), while others are introduced multiple times (e.g., ADSCs, although it was not expanded in the abstract).

Response 6: We thank the reviewer for the professional guidance. All terms, abbreviations and expression errors have been comprehensively revised: ① "Biological breeding" and "anti-stress breeding" revised to "molecular breeding and stem cell-assisted breeding"(Lines 127, 659-660); ② The term "pluripotent stem cells" deleted, with AD-MSCs clearly defined as “multipotent stem cells” (Lines 130); ③ "ADSCs" uniformly revised to "AD-MSC (adipose-derived mesenchymal stem cells)" throughout the manuscript in accordance with ISCT recommendations, with full names of HO-1, GPX4 marked at their first appearance; ④ Redundant abbreviations deleted to ensure consistent expression.

 

Comments 7: Please clarify the ethical aspects of the study. Were the animals subjected to a fat-collection procedure under anesthesia, or the tissue was collected after euthanesia?

Response 7: We appreciate the reviewer’s attention to experimental animal ethics. All animal operations were approved by Inspection Form of Experimental Animal Welfare and Ethical of Institute of Animal Science, Chinese Academy of Agricultural Sciences (Ethics Approval No.: IAS2026-110). Adipose tissue was collected aseptically under intravenous anesthesia with pentobarbital sodium. All operations strictly followed the Guidelines for the Welfare and Ethical Review of Experimental Animals (GB/T 35892-2018). "Experimental Animal Ethics" has been added to the Materials and Methods section to supplement all relevant information (2.1 Materials Lines 314-316).

 

Materials and Methods

Comments 8: Please provide information on how many donors the cells were isolated from and whether cells from different donors were pooled. For each method (preferably in the figure legend), please indicate how many populations (in terms of the number of donors) were used for a given test. For example: cells from three donors were used, each with three technical replicates; or cells from one donor were used and three independent experiments were performed.

Response 8: We appreciate the reviewer’s detailed suggestion. In this study, AD-MSCs were isolated from a single 9-day-old male Leizhou goat without pooling cells from multiple donors. This design eliminated genetic background variations among individuals and reduced experimental errors, which was suitable for our focus on phloretin's regulatory mechanism on RSL3-induced ferroptosis in goat ADSCs. Moreover, all experiments included three biological and three technical replicates, ensuring the reliability and reproducibility of the results.（Lines 316-320）

 

Comments 9: Chondrogenic differentiation: the composition of medium used is very simple and does not include TGF-β, which is commonly used for this type of differentiation. Was there a particular reason for using such composition?

Response 9: We sincerely appreciate the reviewer’s valuable comment on the chondrogenic differentiation medium. We apologize for the omission of key components in the original manuscript and have supplemented the complete formulation of the chondrogenic induction medium in the revised version (Lines 347), including the addition of 10 ng·mL⁻¹ TGF-β3, 10 μg·mL⁻¹ IGF-1, 0.5 mM sodium pyruvate and 50 μg·mL⁻¹ L-proline. The complete formula is as follows: basic medium + FBS + 0.1 mg/mL double antibodies + 0.2 μM dexamethasone + 40 μg·mL⁻¹ ascorbic acid + 1% ITS+10 ng^.mL^-1TGF-β3 +10 μg^.mL^-1 IGF-1 + 0.5 mM Sodium pyruvate + 50 μg/mL L-Proline.

 

Comments 10: An explanation of the mechanism of action of the RSL3 reagent appears only in the Discussion, whereas it would be good to provide in the Introduction or in the Methods section.

Response 10: We thank the reviewer for this valuable comment. To better contextualize the study design, we have added a description of the mechanism by which RSL3 induces ferroptosis in the Introduction section, as suggested. The relevant content has been revised and supplemented accordingly. (Lines 256-259)

 

Comments 11: The authors should clarify information related to percentage composition—when the symbol % is used, it should be specified whether this refers to w/v or v/v and in relation to what. For example, the authors write “10% CCK-8,” whereas it should be specified as “10% v/v in growth medium” (if this is indeed the case)

Response 11: We sincerely appreciate the reviewer’s valuable comment. All percentage expressions in the manuscript have been standardized and annotated. The type of percentage and the corresponding solvent/matrix are clearly indicated for all reagents. No ambiguous percentage descriptions remain in the revised manuscript.

 

Comments 12: The authors should add information about the origin of the CCK-8 assay.

Response 12: We thank the reviewer for the professional guidance. The manufacturer information of the CCK-8 kit has been clearly supplemented in Section 2.3(Cell Viability Assay): Cell Counting Kit-8 (CCK-8) was purchased from Shanghai Biyuntian Biotechnology Co., Ltd., China. This information is now included in the relevant part of the Materials and Methods section.

 

Comment 13: Please add information about the culture conditions used for individual assays. In some cases such information is provided, while in others it is not. For example, on which plates were the cells cultured for fluorescence assays? Were plates with black walls used? From what surface were the cells collected for the Western blot assay?

Response 13: We thank the reviewer for the professional guidance. ① The standard cell culture conditions (37 °C, 5% CO₂, humidified atmosphere) have been uniformly labeled for all assays throughout the Materials and Methods section (Lines 393-394); ② Fluorescence assays are specified to use black-walled clear-bottom 96-well plates to reduce background fluorescence interference (Lines419-421); ③ For Western blot, AD-MSCs were scraped from the bottom of 6-well culture plates with a cell scraper for collection. All missing culture conditions and consumable details have been supplemented.

 

Comment 14: Migration assay: how did the authors ensure that the same field of view was imaged at subsequent time points? Did they use a microscope equipped with an automated stage?

Response 14: We appreciate the reviewer’s detailed suggestion. This detail has been supplemented in Section 2.8. (Cell Migration Assay): an inverted microscope equipped with an automated stage (Olympus, Tokyo, Japan, Model: IX73) was used in the migration assay. The same marked fields of view were photographed immediately after scratching and re-photographed after 12 h of incubation to ensure consistent imaging of the same field.

 

Comment 15: What are “double antibodies”? Please explain and justify their use.

Response 15: We thank the reviewer for the professional guidance. "double antibodies" refers to a mixed solution of penicillin and streptomycin (100 μg/mL penicillin + 100 μg/mL streptomycin), a broad-spectrum antibacterial reagent commonly used in cell culture. Its use is to effectively prevent bacterial contamination during in vitro culture and induced differentiation of AD-MSCs, ensuring the purity of the cell culture system, and this justification has been clearly stated in the revised manuscript.

 

Comment 16: Please provide information on the antibodies used (both in immunocytochemistry and Western blot): clone, manufacturer, and dilution (concentration).

Response 16: We thank the reviewer for the professional guidance. The complete information of all antibodies, including clone number, manufacturer, and specific dilution ratios for immunocytochemistry (ICC) and Western blotting (WB). Table 1

Table 1

Antibody name	Manufacturer	Catalog No.	MW（kDa）	Species	Dilution ratio
Nrf2	HUABIO	HA723302	100	rab	1:2000
HO-1	Proteintech	10701-1-AP	33	rab	1:2000
SLC7A11	Abways	CY7046	55	rab	1:50000
ACSL4	Abways	DY1198	79	rab	1:2000
GPX4	Abways	CY6959	17	rab	1:2000
β-actin	Servicebio	GB11001	42	rab	1: 15000
Goat anti-Rabbit IgG(H+L)	Sera care	5220-0336			1：15000

 

Comment 17: Please specify the version of the Tecan plate reader used.

Response 17: We are grateful for the reviewer’s critical reminder. The exact model of the Tecan plate reader has been uniformly labeled in all relevant sections of the Materials and Methods section: Tecan Infinite M200 Pro, Austria. This instrument model information is now clearly and consistently presented in the revised manuscript.

 

Result

Comment 18: “Impurities” is a term rather not used in reference to cell cultures. It would be better replaced with terms such as contaminating cells, cell debris, or tissue debris, depending on the observed contaminants. Line 247 – should be “bottom” instead of “wall”

Response 18: We thank the reviewer for the rigorous correction. "Impurities" was uniformly revised to “cell debris”; "wall" was corrected to the bottom of the culture dish in the text, consistent with the actual cell adhesion characteristics.

 

Comment 19: Lines 253-258: this part should be at the end of this paragraph to keep the sequence of citing the figures (first 1b, than 1c)

Response 19: We appreciate the reviewer’s detailed suggestion. The description of immunofluorescence detection (Figure 1c) was moved to the end of Section 3.1. The narrative logic was reorganized to introduce the multi-differentiation potential (Figure 1b) first, then the cell surface marker detection (Figure 1c), ensuring the consistency of figure citation sequence.

 

Comment 20: Figure 1. Please add scale bars to the images in panels 1(a) and 1(b)—it is clear that different magnifications were used. An explanation of the left and middle columns in panel 1(b) is missing in the legend. Should be “Alizarin Red”, not Red S.

Response 20: We thank the reviewer for the rigorous correction. ① Scale bars were added to all microscopic images (100 μm for Figure 1a, 50 μm for Figure 1b); ② Detailed explanations for the induction control group (left column) and uninduced cells before staining (middle column) were supplemented in the legend of Figure 1b; ③ "Alizarin Red S" was uniformly corrected to Alizarin Red in the whole manuscript.

 

Comment 21: Figure 2. Specify in the legend which statistical method was used. ANOVA? If so, what type of post-hoc test was applied? Cells from how many donors were used?

Response 21: We appreciate the reviewer’s detailed suggestion. ① one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test, which was marked in the legend of Figure 2; ② The donor and replicate information was added, which was marked in the legend of 2.1. Materials.

 

Comment 22: Figure 3. Please specify what is presented in graph 3(a). These are relative values, but it is not clear whether the analyzed parameter was fluorescence measured with a plate reader or fluorescence measured from images obtained using a fluorescence microscope (this information should also be included in the Methods section). The Y-axis description should be corrected. The term “expression of ROS” is inappropriate.

Similarly in 3(b), it is unclear what was measured and what is presented. Was only green fluorescence measured? Or the ratio of green to red? Was the measurement performed using a plate reader or based on microscopic images? Please also add this information in the Methods section.

In 3(c) and 3(d), please correct the Y-axis description—the term “expression” is conventionally used in relation to genes or proteins, not to the presence of ions or other chemical compounds such as MDA.

Response 22: We thank the reviewer for the valuable comments.

Accordingly, we have clarified the detection methods for Figure 3(a) and 3(b) in both the figure legends and Methods section, and corrected the inappropriate “expression” labels on the y-axes in Figure 3(a), 3(c), and 3(d). All revisions have been updated in the manuscript Figure 3.

 

Comment 23: Line 307: the description of mitochondria appears inconsistent with the images presented in Figure 3(f). The mitochondria appear rather swollen and enlarged rather than shrunk.

Response 23: We thank the reviewer for the careful observation. We apologize for the incorrect image previously displayed in Figure 3(f). The figure has now been replaced with the correct one, and the description of mitochondria is consistent with the image.

 

Comment 24: Figure 4. Please supplement the information in the figure legend regarding the statistical test used (if ANOVA, then which post-hoc test), analogous to the comments for the previous figures. Please also include information regarding the number of donors from whom the cells used in this test were derived.

Response 24: We appreciate the reviewer’s detailed suggestion. One-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test, which was marked in the legend of Figure 4.

 

Comment 25: Figure 5. Migration: As above: please specify which statistical tests were used and from how many donors the cells were derived.

Growth: the presented data do not include standard deviations. Does this mean that only a single experiment was performed? If so, please clarify this and comment on it appropriately. In such a case, these data should be interpreted with great caution. If more data were included in the group, please indicate the standard deviations and specify the number of biological replicates “n”. The Y-axis label should also be corrected. These values appear rather to indicate the number of cells per well (according to the description in the Methods) rather than cell density per ml.

Response 25: We thank the reviewer for the constructive comments. For the migration assay in Figure 5, we have added the statistical method in the figure 5 legend. For the cell growth data, we have supplemented the standard deviations and clearly stated the number of biological replicates (n). The label of the Y-axis has been revised to cell number per well to match the experimental description.

 

Discussion

Comments 26: Lines 421–442: this part of the Discussion is highly speculative. Expressions such as “preliminary study,” “we speculate,” phloretin “may promote,” and “the Nrf2-GPX4 axis is a highly likely mechanism” appear here. This represents a significant discrepancy with the title, which contains a strong statement. In light of such inconsistencies, the title should be moderated.

Response 26: We sincerely thank the reviewer for the rigorous comment. We agree that the original discussion was overly speculative and inconsistent with the title. We have revised the discussion to remove speculative statements and use more rigorous wording, and have also moderated the title accordingly. (revised Lines 766–786)

All modifications have been marked in the revised manuscript. We again express our sincere gratitude to the reviewer for the careful review and constructive suggestions, which have greatly improved the quality of our manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Comments and suggestions for the authors are indicated in the attached document.

Comments for author File: Comments.pdf

Author Response

Comments 1: The abstract presents an interesting and well-structured study. However, the last sentence raises a very broad implication:“lay a foundation for improving ruminant breeding efficiency and production performance.” Given that the study is at the cellular and molecular level, this conclusion could be perceived as an over-extrapolation. It would be more prudent to state that the results of this study provide a basis or preliminary evidence for future applications in animal production, possibly for meat goat production.

Response 1: We sincerely appreciate the reviewer’s thorough evaluation and constructive comments. We fully agree with the reviewer’s concern. The over-extrapolated concluding sentence has been revised to a more prudent statement that aligns with the cellular and molecular level of our study (Lines 32-26).

 

Comments 2: It is recommended to review the proposed keywords, as several of them are already included in the manuscript title. To enhance the article's visibility and indexing performance in scientific databases, it is advisable to select alternative or complementary keywords that expand the thematic or methodological scope of the study. This will facilitate more effective retrieval of the article in literature searches.  These keywords are repeated in the title and will not give the opportunity to expand the search for information. It is suggested not repeat simple or compound words that are already included in the title. It is suggested to write other keywords.

Response 2: We appreciate the reviewer’s valuable suggestion on keyword optimization. After a thorough re-evaluation of the manuscript, we confirm that the core keywords are highly consistent with the study’s central theme and cannot be replaced without compromising accuracy. To address the repetition with the title and expand the indexing scope, we have made minor non-substantive adjustments to the expression of core keywords and supplemented one complementary keyword (Lipid Peroxidation)—a core mechanism characteristic of ferroptosis that runs through the entire study and is not included in the title. The revised keywords are presented manuscript for your kind review.

 

Comments 3: Page 2, lines 44-88; Page 3, lines 89-125. Check that this section presents literature from the last ten years. Is it important to explain why the study is being conducted specifically with the Leizhou goat breed and not with another goat breed? Finally, it is recommended to provide a clear and explicit statement of the hypothesis or research question guiding the study, to offer the reader better guidance from the outset.

Response 3: We sincerely appreciate the suggestion and have carefully verified all literature cited in the Introduction section.

• The vast majority of the references are high-quality studies published in the recent 5 years in the field of ferroptosis, mesenchymal stem cell biology and ruminant breeding, which fully reflects the timeliness of the research background.
• In addition, Leizhou goats were used as experimental materials in this study because they are routine existing research materials in our laboratory and not a specific breed specially screened for this study, so no additional explanation of this material selection has been added in the Introduction. Meanwhile, as a local meat-type goat breed, the adipose-derived mesenchymal stem cells of Leizhou goats share the same core biological characteristics and regulatory mechanisms with other meat-type goat breeds. The research results based on this breed are representative and can be extended to other ruminant breeds, which does not affect the scientificity and universality of the conclusions of this study. Therefore, this material selection background has not been specifically addressed in the Introduction.
• We have added clear and explicit research hypotheses at the end of the Introduction section(Lines 146-149).

 

Comments 4: The information in this section must be consistent with the objective of the study. Page 4, lines 141-146

2.2. Cell Culture and Identification 

2.2.1. Cell Isolation and Culture

Can the amount of adipose tissue collected from each male goat be indicated, or the total amount of adipose tissue collected from the 9 male goats used in the experiment? Page 4, lines 237-242

2.9. Statistical Analysis

Can you indicate if any data normality tests were performed to consider the use of parametric statistics?

Response 4: We sincerely appreciate the reviewer’s suggestion.

• We have carefully verified this section. Its content is fully consistent with the study objective: the isolation, culture and identification of AD-MSCs serve to confirm the cell identity, laying a fundamental experimental foundation for subsequent research on phloretin's effect on AD-MSC ferroptosis.
• In this study, AD-MSCs were isolated from a single 9-day-old male Leizhou goat without pooling cells from multiple donors. This design eliminated genetic background variations among individuals and reduced experimental errors, which was suitable for our focus on phloretin's regulatory mechanism on RSL3-induced ferroptosis in goat ADSCs. Moreover, all experiments included three biological and three technical replicates, ensuring the reliability and reproducibility of the results (Lines 181-187).
• We have added the description of normality and homogeneity of variance tests as the prerequisite for parametric analysis in the 2.9 section, with the revised content clearly stated.

 

Comments 5: Page 8, lines 286-293. Will it be possible to change the color of the SRL3 treatment bars in the graphs of the following figures: Figure 2 (a) and (c), Figure 3 (a), (b), (c), (d), (e) Figure 4 (in all graphs) Figure 5 (a) and (c) Since pink is not easily visible or distinguishable from the other colors, could a contrasting color such as purple or blue be

used?

Response 5: Thank you for the suggestion. We have replaced the pink color of the RSL3 treatment bars with a more distinct blue in all the specified figures, and the revised images have been updated in the manuscript.

 

Comments 6: Page 15, lines 492-510. It will be possible to present some argument prior to the conclusions section, indicating whether the application of these biotechnologies for the purpose of improving animal production does not increase the cost of the production

process or whether the quality of meat can be improved in species that are not as commercially important, such as Leizhou goats, for example.

Response 6: Thank you for the valuable suggestion. We have added relevant arguments in the first paragraph of the discussion section, addressing the potential effects of these biotechnologies on production costs and meat quality improvement in non‑major commercial breeds such as Leizhou goats.

 

Comments 7: Page 15, lines 520-528: The conclusion should be consistent with the stated objective. The objective of this study was to provide novel molecular targets and theoretical insights for genetic breeding and healthy rearing of goats and other ruminants, as well as a scientific basis for optimizing stem cell culture systems, developing green and safe feed additives/regulatory products, and ultimately enhancing animal reproductive performance and production efficiency. Rather, the study demonstrates that experimentally induced ferroptosis (using RSL3) causes specific cellular alterations in ADSCs. This point should be clarified to improve the conceptual clarity of the conclusion. It will be possible to indicate that the generation of this knowledge serves as a technology transfer. Since the study was conducted at the in vitro cellular level, this extrapolation may be considered somewhat broad. As previously stated, it would be more prudent to indicate that the findings provide a theoretical basis or preliminary evidence that could guide future applied research in animal production

or reproductive biotechnology.

Response 7: We sincerely appreciate your rigorous and constructive comments, which have significantly improved the manuscript. We have revised the conclusion section accordingly: We clarified that this study was based on an RSL3-induced ferroptosis model in goat AD-MSCs in vitro. We avoided over-interpretation and confirmed that the results only provide in vitro evidence. We emphasized the potential value of phloretin as a green agent for AD-MSC transplantation. We added the limitations and future perspectives, indicating that further in vivo studies are still needed.

All modifications have been marked in the revised manuscript. We again express our sincere gratitude to the reviewer for the careful review and constructive suggestions, which have greatly improved the quality of our manuscript.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript has been substantially improved. 

I have only three small comments:

1. in abstract please indicate the cells originated from a single donor (it is important). The phrase "we established (...) (AD-MSCs) from Leizhou goats" should be changed to "from Leizhou goat". The readers should be informed clearly in this matter.
2. line 187 (and potentially in other sites in the manuscript) - not double antibodies but double ANTIBIOTICS. 
3. Figure 1 should have clarified marking. Now it is confusing: panels are marked with (a), (b) etc, and individual images are again marked with a, b, etc. additionally, panel 3 (c) should described in the legend more in detail. 
   
   

Figure issue: 

The only instance that may have been flagged by the software concerns Figure 1C (fluorescence microscopy). In this case, the images presented in adjacent panels appear to represent the same field of view shown in different channels (blue, green, and merged images). This likely explains why the algorithm detected similarity. However, this is a standard and appropriate way of presenting fluorescence microscopy data.

I would recommend that the authors correct the figure legend, as it is currently not clearly stated that these panels correspond to the same field of view shown across different channels.

with kind regards

Comments on the Quality of English Language

I am not a native speaker. Some sentences seems to be too convoluted and not very clear. Some expression sound not very scientific (I pointed it in the main review part). But generally in my opinion language requires minor corrections.

Author Response

Comments 1: in abstract please indicate the cells originated from a single donor (it is important). The phrase "we established (...) (AD-MSCs) from Leizhou goats" should be changed to "from Leizhou goat". The readers should be informed clearly in this matter.

Response 1: We greatly appreciate this important comment. According to the suggestion, we have revised the abstract to clearly state that the AD-MSCs used in this study were isolated from a single Leizhou goat. The expression “from Leizhou goats” has been updated to “from Leizhou goat” throughout the manuscript to ensure accuracy. The corrections have been marked in red in the corresponding parts of the manuscript.

Comments 2: line 187 (and potentially in other sites in the manuscript) - not double antibodies but double ANTIBIOTICS.

Response 2: We apologize for this typographical error. We have carefully checked the whole manuscript and corrected “double antibodies” to double antibiotics at line 187 and all other relevant positions.

Comments 3: Figure 1 should have clarified marking. Now it is confusing: panels are marked with (a), (b) etc, and individual images are again marked with a, b, etc. additionally, panel 3 (c) should described in the legend more in detail. 

Figure issue: The only instance that may have been flagged by the software concerns Figure 1C (fluorescence microscopy). In this case, the images presented in adjacent panels appear to represent the same field of view shown in different channels (blue, green, and merged images). This likely explains why the algorithm detected similarity. However, this is a standard and appropriate way of presenting fluorescence microscopy data.

I would recommend that the authors correct the figure legend, as it is currently not clearly stated that these panels correspond to the same field of view shown across different channels

Response 3: We thank the reviewer for this constructive comment. We have standardized the labeling of all figure: panels are marked with (A), (B), etc. The corrections have been marked in red in the corresponding parts of the manuscript.

Meanwhile, we have supplemented a more detailed description of panel 3(C) in the figure legend to improve clarity.

Comments 4: Some sentences seems to be too convoluted and not very clear. Some expression sound not very scientific (I pointed it in the main review part). But generally in my opinion language requires minor corrections.

Response 4: We sincerely appreciate the reviewer for the positive comments and constructive suggestions. Regarding the language revisions, we have carefully checked the entire manuscript and revised the inappropriate expressions, with all corrections marked
in the manuscript.

All modifications have been marked in the revised manuscript. We again express our sincere gratitude to the reviewer for the careful review and constructive suggestions, which have greatly improved the quality of our manuscript.

 

Author Response File: Author Response.docx