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

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: No

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The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: Luteolin Alleviates CUMS-Induced Depressive-like Behavioral Deficits in Mice

Through Blocking the JAK2/STAT3 Pathway

This article presents an interesting focus on the natural flavonoid luteolin (LUT) for the treatment of depression. The authors conducted an impressive battery of behavioral tests that supported their hypothesis, as well as some biological inflammatory tests.

I was impressed by the behavioral results, and I recommend publishing this article if the authors can address all the comments below:

Major comments:

1. Introduction: There is a lack of information about luteolin, particularly regarding the herbs that contain this flavonoid. How can luteolin influence the monoamine system, specifically the serotonin system? Are there any side effects associated with its use? Additionally, which companies offer luteolin for purchase?

2. Since luteolin is anticipated to have effects on treating depression, it would have been beneficial to include a positive control group that received an SSRI drug such as escitalopram or fluoxetine. However, since this control group was not included, it would be beneficial to explain why it was not included and to mention it as a limitation in the article.

3. Animals began receiving treatments after the first day of the chronic unpredictable mild stress (CUMS) model, indicating that they had not yet displayed any symptoms of anxiety or depression. Under these circumstances, the treatment serves as a preventive measure rather than a remedy for existing anxiety and depression. Therefore, the point of view of the article needs to be changed. In addition, the effective treatment received was at a high dose of luteolin (40 mg/kg), which may be because the high dose of luteolin in this treatment stabilizes this group in the baseline stage and does not allow it to develop the desired effects of depression and anxiety. It needs to be discussed in this article.

4. The study primarily examines the JAK2/STAT3 pathway and its relationship with inflammatory cytokines. However, it fails to clarify the link between blood and brain pathways, specifically how changes in the JAK2/STAT3 signaling in the brain affect blood cytokine levels. Additionally, the role of the hippocampus—selected as the focus area—remains unclear in terms of how it regulates cytokine levels in the blood.

Minor comments:

1. The open field test is an anxiety and motor test and not a depression test. In the method section, the authors wrote, “The number of entries into this central zone was recorded as measures of depressive-like behavioral deficits.” This is incorrect and should be corrected.

2. Although this study uses inbred mice (C57/BL), eight mice per group is a relatively low number of animals, particularly in behavioral tests; it would be helpful to clarify why this low number of animals is used.

3. At the FST and TST tests, it should have ignored the first two minutes and calculated the immobility time of the last four minutes. Maybe it was done during this article, but it is not mentioned.

4. Depression and anxiety are more commonly observed in females than in males; however, this study was conducted using male mice, which should be clarified.

5. The article focuses on proteins p-STAT3, STAT3, p-JAK2, and JAK2, but the connection between them is not so clear. It would be beneficial to explain the function of each protein and its relative expression in the results.

6. The western blot results should include all photos and not just the relevant cut of the proteins.

7. This article focuses on treatment for depression but ignores anxiety disorders. Nevertheless, the article measured anxiety-like behavior using the EPM test and the Open field test but failed to mention this during the discussion. It is essential to include anxiety in the discussion to provide a more comprehensive understanding.

Reviewer #2: The authors have shown that CUMS induces depressive-like behavior and increases p-JAK2, p-STAT3, and cytokine levels in mice. Furthermore, they’ve shown that luteolin reduces CUMS-induced effects on these parameters. My recommendation for this manuscript is to suggest major revisions for clarity and accurate representation of the statistics.

1. The statistical significance portrayed in the figures and legends does not match what is implied in the text. The figure captions state that the asterisks used on the LUT groups in the graphs show the significance compared to the control (no CUMS) group, but the data described in the text implies that the significance is in comparison to the CUMS group. Similarly, the figure captions state that the hashtag/number symbol was used to compare to the CUMS group, but that symbol is used on the CUMS group on the graph implying that the group was compared to itself. Either the statistics need to be described more clearly in the text or the figure captions just need to be updated.

2. Did the authors complete a power analysis to determine how many animals should be used in the behavioral tests? Typically, 10-12 animals would be used for these behavioral tests so eight seems low. If a power analysis was performed, that information should be included in the Materials and Methods section.

3. There is a discrepancy in the number of animals in the control group. The Materials and Methods section states n=6 but the results section states n=8.

4. Time spent in the central zone of the OFT was used as a measure of depressive-like behavioral deficits, and the results section states the OFT was used as a measure of motor abnormalities observed in depressed individuals. Citations should be included to show that the open field test has been used and validated in this way. The OFT is typically used to assess locomotor activity to ensure that a change in locomotor activity is not confounding the results of behavioral tests for depression, such as FST and TST, and not as a measure of depressive-like behavior itself.

5. The EPM is noted as being “commonly adopted to assess depressive-like behavioral deficits in rodents” and reference 7 is cited. Reference 7 does not use or state that EPM should be used as a measure of depressive-like behavior. Rather the EPM is used as a measure of anxiety. Because the results section described the EPM findings as anxiety, this may be a typo that can easily be fixed. If not, other references need to be included to show that the EPM has been used and validated in this way.

6. A positive control, e.g. fluoxetine, would be a welcome addition to this manuscript to validate the sensitivity of the paradigm and provide a comparison to established antidepressant treatments, however, it is not necessary for publication.

7. The results section mentions that “successful” CUMS mice were randomized into groups, but there is no mention of how “success” was defined. This criteria should be included in the Materials and Methods section.

8. The experimental set-up needs some clarification between the Materials and Methods section, the experimental workflow (Figure 1-1B), and the results section regarding the 2% ethanol solution (mentioned only in the results), 0.1 mL/kg of deionized water (mentioned only in the Materials and Methods), and DMSO (mentioned only in Figure 1-1B).

9. The results section should more transparently state the results of the low, medium, and high LUT groups. The results of the three LUT groups are often lumped together in the results section - stating that “LUT” had an effect – without mention of which dose(s) of LUT were significantly different in for each particular experiment.

10. The figure legends should state the type of tissue that was used for the experiments.

11. In section 2.2 of the results, it is stated in regard to the percentage of time spent in the open arms, “Although the other LUT doses showed a trend toward improvement, the differences were not statistically significant.” However, the graph shows that the LUT-M group was significant. The figure or text should be updated to reflect whichever information is correct. In addition, the p values have been stated, so mentioning a “trend” toward significance is irrelevant and unnecessary.

12. There is a typo in section 2.1 of the results section where “evtluate” is used instead of “evaluate”.

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what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

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Attachments

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Submitted filename: PONE-D-25-12815 - Review.docx

Luteolin Alleviates CUMS-Induced Depressive-like Behavioral Deficits in Mice Through Blocking the JAK2/STAT3 Pathway

PONE-D-25-12815R1

Dear Dr. Zhao,

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,

Zhiling Yu

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

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

Reviewer #1: Yes

**********

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

Reviewer #1: Yes

**********

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

The PLOS Data policy

Reviewer #1: Yes

**********

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

Reviewer #1: Yes

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Reviewer #1: (No Response)

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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 #1: No

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