PONE-D-22-16987Ursolic acid regulates gut microbiota and corrects the imbalance of Th17/Treg cells in T1DM ratsPLOS ONE

Dear Dr. Zhang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. It was felt that while the study has merit, it lacks conclusive and mechanistic experiments.  It was also felt that the authors need to discuss the rationale for use of ursolic acid and how it compares to metformin.  Many other important concerns including the language needs to be addressed by a point by point response to all the comments of the reviewers.

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Pradeep Dudeja

Academic Editor

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: I Don't Know

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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: This manuscript by Chen et al. studies the potential of Ursolic acid to alter gut microbiota and correct Th17/Treg imbalance in Type 1 diabetes utilizing a rat model. The study is novel and supports the conclusions. There are several concerns;

Major concerns

1. The figures are difficult to follow, they are not labeled correctly and in some the statistics have not been conducted (Fig 1A,B,Fig 2B,C etc.) . Please make sure each figure is in one page with sub figures labeled accordingly. In addition, the figure legends should be very descriptive, and any abbreviation used (eg MET) should be defined in the legend as well.

2. Figure 2F is very wordy and the text is unclear even if the large image is downloaded. Please increase clarity of the figure.

3. The change in immune cells seems to be occurring in the spleen. Have the authors also looked at local tissue? Peyer’s patches, mesenteric lymph nodes and lamina propria? If not, could you speculate how gut microbiota can impact splenic Th17/Treg differentiation? What is the effect of UA?

4. All the beneficial effects of UA were similar to that of metformin an already well tolerated drug. Are there any side effects of UA? What is the benefit of using it over metformin? Please discuss

5. Please state molecular weight of proteins on the representative western blots

Minor concerns

1. The authors have mentioned that a recent study has shown there is an imbalance in Th17 and Tregs (this has been published in 2019) Is there any other recent study showing this?

2. Introduction is initiated with an abbreviation, please avoid doing so.

3. Scientific names should be in italics Page 4 line 90-91 (Salvia officinalis etc.) Please correct

Reviewer #2: In this manuscript, the authors describe their studies on the effects of ursolic acid (UA) in reversing gut microbiota dysbiosis and imbalanced Th17/Treg cells in T1DM rats. The experiments are technically well-designed and study results are appropriately described. However, rationale for choosing UA (in preference to many other phytochemicals showing hypoglycemic, antioxidant, anti-inflammatory, antitumor and neuroprotective properties) is not well addressed. Further, the studies described here are far from confirmatory to infer whether the ameliorating effects of UA on T1DM complications are direct effects of UA or mediated via correcting dysbiosis. This can be confirmed only by direct treatment with UA in vitro in cell culture experiments or in vivo in germ free animals.

There are multiple grammatical or typo errors that need attention (for example type I diabetes, not diabetic, in the Abstract). Also, the statements like “The results probably provided new research strategies for the prevention and treatment of T1DM” significantly dampens the scientific merit of the study.

Overall, the manuscript needs major revision with additional studies to confirm the claim that UA effects are via gut microbiota, not direct effects, or by downplaying the claim by revised writing of the Abstract, Results and Discussion/Conclusion sections.

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Reviewer #1: No

Reviewer #2: Yes: Alip Borthakur, Ph.D.

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Ursolic acid regulates gut microbiota and corrects the imbalance of Th17/Treg cells in T1DM rats

PONE-D-22-16987R1

Dear Dr. Zhang,

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 and correction of minor spelling mistakes as outlined by one of the reviewer.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Pradeep Dudeja

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 #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 #3: Yes

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

Reviewer #1: Yes

Reviewer #3: 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 #1: 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 #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: All comments have been addressed and is now suitable for publication.

Reviewer #3: Dear Authors

Even after professional editing, there are many mistakes in the manuscript. I have listed a couple of them.

Line 55: Spelling mistake: Druges should be drugs

Line 117: Bllank

Please check the consistency for faecal/fecal

Line 244: should be performed

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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 #3: No

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