PONE-D-20-01855

Low dose of luteolin activates Nrf2 in the liver of mice dependent on an administration timing: luteolin activates Nrf2 at the start of the active phase but not that of inactive phase of mice

PLOS ONE

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PLOS ONE

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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: Yes

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

Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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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: This manuscript is very interesting for relationship of the luteolin-induced Nrf activation and circadian rhythms in vivo. In fact, author showed that low-dose luteolin activates Nrf2 and increase plasma luteolin levels in active phase. However, I have a few questions. Thus, I propose that to improve the manuscript and to increase potential impact figures in this field of scientific research.

Major comment

1: In Fig.1, author showed that protein levels of Nrf2 and HO-1 were induced in ZT12, but not ZT0. However, this can’t be investigated. Because Nrf2 levels in control level (luteolin 0 mg/kg) is different at ZT0 and ZT12. If author wish to compare the two groups, author should be measure the protein expressions on the same membrane.

2: In Fig.2, author indicated mRNA level of Nrf2 was regulated by circadian rhymes. This point is very interested. However, I have a question. Why did Nrf2 protein level decrease in ZT12 compared to ZT0? Author should be measure Nrf2 levels in nuclear fraction and keap1 protein level in cytoplasm.

3: For statistical analysis, it is better to do a statistical test using one-way analysis but not Dunnett’s test. Because various concentration of luteolin treated for mice in this animal experiment. This point is very important.

Reviewer #2: The authors of 'Low dose of luteolin activates Nrf2 in the liver of mice dependent on an administration timing: luteolin activates Nrf2 at the start of the active phase but not that of inactive phase of mice' present a structured manuscript, focusing on the effects of low dose of luteolin in mice liver, especially with regard to its regulation of Nrf2 transactivation. Based on the displayed results, it can indeed be suggested that luteolin activates Nrf2 to upregulated gene expressions of HO-1 & NQO1 even by the oral administration with extremely lower doses. Furthermore, it is interesting that these effects are clearly dependent on the timing of administration. Although the manuscript deserves further interest, some minor shortcomings and issues should be addressed.

MINOR

1/ Fig4: Why was NOT assessed the effects by lower dose administration like 0.1 mg/kg.

2/ Discussion section (P13line288): How were the values of 0.04-1.7 mg/kg calculated? I just wonder these should be 0.004-0.17 mg/kg, assuming human body wight is approximately 50 kg.

General

Please define all abbreviations at first use, eg BMAL1, CRY, & PER.

Title: Please improve it with correct English expressions.

Introduction:

P3line55: Please correct the spelling of Nrf2.

P3line65: …suggested that an Nrf2-dependent…

P4line78: …, our previous study…

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Reviewer #1: No

Reviewer #2: No

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Low dose of luteolin activates Nrf2 in the liver of mice at start of the active phase but not that of the inactive phase

PONE-D-20-01855R1

Dear Dr. Ashida,

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

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With kind regards,

Nobuyuki Takahashi, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments:

Authors properly addressed all of reviewers' comments.

So, I have decided to "accept" this manuscript without sending it to the reviewers again.

Reviewers' comments:

Nothing.