PONE-D-24-31421Therapeutic potential of fisetin in hepatic steatosis: insights into autophagy pathway regulation and endoplasmic reticulum stress alleviation in high-fat diet-fed mice

Dear Dr. Panahi,

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Additional experiements are required in order to conclude that Fisetin has indeed a role on liver and that ER stress and autophagy are really modulated

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Reviewer comments:

The manuscript titles “Therapeutic potential of fisetin in hepatic steatosis: insights into autophagy pathway regulation and endoplasmic reticulum stress alleviation in high-fat diet-fed mice” by Sattari et al demonstrates the efficacy of Fisetin in the amelioration of NAFLD through alleviation of ER stress and enhanced autophagy. The authors show in mice model of HFD, that administration of Fisetin targeted autophagy and ER stress pathway and increased AMPH phosphorylation and helped in the management of NAFLD. Although the study is of interest in the current scenario of metabolic disease over-burden, following comments should be addressed to make the study suitable for publication.

Major Comments:

1. Can the authors explain the similar effects seen between HFD+FSN and HFD+HCQ for glucose and insulin homeostasis in Fig 2.

2. No difference in glucose homeostasis is observed for HFD+HCQ and HFD+FNS+HCQ groups in the physiological assays in Fig 2 and 3.

3. Since the study is based on role of Fisetin on autophagy in NAFLD amelioration, can the authors explain the lack of any deleterious seen for HFD+HCQ group, since HCQ is known to inhibit autophagic process completely. As reported by the authors in Fig 4, NAFLD, steatosis, ballooning and inflammation scores are either similar or lower than HFD+FSN group.

4. Authors are requested to compare the statistical significance of the data between HFD+HCQ and HFD+FNS+HCQ groups, wherever applicable.

5. There are some regularly histology experiments that are missing from the study as listed below:

a) ORO stain in liver sections

b) Sirius red stain

c) a-SMA staining

6. Quantification for fat vacuoles for Figure 4.

7. The authors requested to clarify the model in Fig 7, where they have shown Fisetin to inhibit autophagy, similar to HCQ.

8. The authors should do experiments related to autophagy and ER stress analysis in isolated hepatocytes treated Fisetin or in combination with HCQ to demonstrate the direct effect of Fisetin on authophagy in hepatocytes. This would ensure that the effect of Fisetin is directly on hepatocytes during NAFLD treatment and not through other systemic effects.

Minor Comments:

1. References should be provided for Lines 81-83, page 3.

2. Current guidelines for Glucose tolerance tests advise a fasting period of 6hrs before the experiment. The authors are requested to explain why they have selected 10hrs. The authors should also clearly mention the time the GTT and ITT experiments were done.

3. the authors are requested to mention the housekeeping gene used for qPCR normalization and indicate whether it showed any differences between the treated groups.

4. Please indicate whether totalAUC or incremental AUC was used to plot area under the curve for GTT and ITT experiments.

5. Please indicate individual data points in the graphs to indicate numer of samples and the sread of the data. Also include the exact mice numbers used for each experiment and the criteria on which any mouse was not included.

Reviewer #2: In this manuscript, Sattari and co-authors investigated fisetin's influence on ER stress, autophagy pathway regulation, and hepatic lipid accumulation, providing promising insights into its potential for mitigating liver dysfunction. While the study is well-conceived and provides valuable insights into FSN’s potential in NAFLD, several issues need to be addressed to strengthen the manuscript.

Major Points.

1. The manuscript demonstrates a reduction in ER stress markers with FSN treatment. To strengthen this argument an experiment utilizing ER stress inducers (e.g., tunicamycin) in conjunction with FSN treatment could confirm whether FSN’s effects on autophagy and lipid accumulation are indeed mediated through ER stress modulation.

2. Figure 6a: The quantitative graphics show results that differ from those observed in the western blot images. I suggest the authors replace the images.

3. Figure 6d: The transcriptional level of mTORC1 alone is not sufficient to indicate its activity. To accurately assess mTORC1 activity, it is necessary to analyze its phosphorylation state or the phosphorylation of its downstream target, S6K.

4. Figure 6g: The transcriptional level of ULK1 is not sufficient to indicate autophagic activity. The most suitable indicator for assessing autophagy activation is the phosphorylation of ULK1 at activating sites (e.g., Ser317 or Ser555). ULK1 phosphorylation at these sites is closely associated with the direct activation of the autophagy pathway, making it a key indicator of whether autophagy has been initiated.

5. Figure 5: While mRNA levels of ER stress markers provide valuable insights, they should be complemented with protein-level and functional analyses to accurately assess ER stress and its cellular impact. To comprehensively evaluate ER stress, The authors should measure protein levels of key ER stress markers (e.g., GRP78, CHOP) or their phosphorylated states (e.g., p-PERK, p-eIF2α).

6. The manuscript suggests that FSN and HCQ together are more effective but does not explain the mechanism behind this synergy. the authors should discuss synergistic effects of FSN and HCQ.

Minor Points.

1. Correct the typo in Figure 4e’s y-axis title from "scor" to "score"

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Reviewer #1: No

Reviewer #2: Yes:  Hwan-Woo Park

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Submitted filename: fistin manuscript.docx

Therapeutic potential of fisetin in hepatic steatosis: insights into autophagy pathway regulation and endoplasmic reticulum stress alleviation in high-fat diet-fed mice

PONE-D-24-31421R1

Dear Dr. Panahi,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Catherine Mounier

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

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

Reviewer #2: Yes

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The manuscript is scientifically sound, well-structured, and provides reasonable justifications for the limitations in experimental design. Given the rigorous methodology and clear data presentation, I recommend acceptance in its current form.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #2: Yes:  Hwan-Woo Park

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