PONE-D-24-41839“Betaone” barley water extract suppresses ovariectomy-induced osteoporosis in vivo and RANKL-induced osteoclast differentiation in vitroPLOS 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: Partly

Reviewer #2: Yes

Reviewer #3: Partly

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: 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: No

Reviewer #2: Yes

Reviewer #3: No

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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

Reviewer #3: 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: 93-96: The yield of B1W was approximately 10.8%. Please provide the extraction yield report for B1W.

99-112: Please include ultra-performance liquid chromatography data for TH and FA, not just graphs.

117-120: Were the rats 5 weeks old at the time of modeling? Female rats at this age are not yet sexually mature, which may not be the optimal time for ovariectomy.

125-135: The experimental animals were sacrificed 6 weeks post-surgery, and serum was extracted for bioanalysis. What method was used to collect the blood? Is it venous blood?

139-147: BMMSCs were extracted from 5-week-old male rats, which may be somewhat older, potentially affecting the activity of rat bone marrow cells. How were BMMs pretreated with B1W? What passage number were the subsequent BMMSCs used for cell experiments?

139-147: A 96-well plate was seeded with 10^4 cells and cultured for four days. Is there sufficient space for growth?

157-160: In the cytotoxicity experiments, was 10 ng/ml RANKL used to induce osteoclasts for four days?

182：In Table 1, what does "sense" and "anti-sense" refer to? The formatting appears incorrect.

207: Table 2 is not formatted correctly.

273-277: The timing and dosage of RANKL induction were not annotated, nor are they indicated in the corresponding Fig. 5. What is the difference between bone pit formation and TRAP staining? The results of tissue section staining for osteoporosis should be supplemented for verification.

WB Fig. 5: The gray values are not labeled, and there appears to be a significant difference in the Day 2 group. Is there any duplication in the data?

Fig. 3: TRAP-stained positive cells should appear red, but the coloration in the article appears biased. Additionally, no scale bars are marked in panels A and D.

Reviewer #2: 123：The referenced study primarily investigates the effects of 17α estradiol, prompting consideration for replacing literature that examines 17β estradiol. This substitution would enhance the experimental rationale surrounding the use of 17β estradiol.

232、233：It is possible that the designation of "Fig. 2a" instead of "Fig. 1a" in reference to the picture could be a clerical error; please take note of this correction.

274：Based on the experimental results from WB, the statement that the expression level of NFATc1 in the control group increases over time is not entirely accurate.

Reviewer #3: The study by Yongjin Lee et al. explored effects of B1W on ovariectomy (OVX)-induced bone loss in vivo and osteoclastogenesis in vitro. The paper is well-written and presents promising results. However, there are a couple of concerns to make this study more convincing:

Please add associated biomechanical testing data to double confirm B1W-induced anti-osteoporosis.

Please supplement relevant data on effects of B1W on osteogenesis and bone formation.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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“Betaone” barley water extract suppresses ovariectomy-induced osteoporosis in vivo and RANKL-induced osteoclast differentiation in vitro

PONE-D-24-41839R1

Dear Dr. Son,

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,

Liangliang Xu

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 #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: 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 #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: N/A

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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 #1: Yes

Reviewer #2: Yes

Reviewer #3: 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 #1: Yes

Reviewer #2: Yes

Reviewer #3: 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 #1: The questions have been well answered and can support the conclusions. Articles can be accepted for publication.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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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 #1: No

Reviewer #2: No

Reviewer #3: No

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