PONE-D-24-12616Berberine potentiates liver inflammation and fibrosis in the PI*Z hAAT transgenic murine modelPLOS ONE

Dear Dr. Brantly,

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.

This is an interesting and important study question, along with a great point on the discordance of in vitro vs. in vivo models. You will see that the reviewers have several major points that need to be addressed; some of these can be addressed with an explicit limitations section. If you have banked samples (e.g., for liver chemistry) it would be ideal to include these analyses given the nature of the topic (safety and liver toxicity). Because of what appears might be a sex-specific response (which is also not surprising for this topic), the issue with sample size in the animal experiments is a substantial concern. You should know at this stage, that while I think this work is important, this specific point makes me uncomfortable endorsing the manuscript further if sample size cannot be addressed. Other points from me that should be examined are as follows:

•  Are the images in figure 5 consecutive sections from the same liver? Is that truly representative if so?
• There should be more said about the chosen in vivo dose either in the methods, discussion, or both. This should bein the context of other studies, what people typically would consume as a supplement, and also to the fact that BBR does not have good oral bioavailability. REF 33 somewhat gets at this as you already mentioned, and I understand that oral is the appropriate route (since that is how people consume it), but these are all factors that are important to consider for translational context.
• The biochemical/mechanistic literature on glucose metabolism is probably the richest set of detailed cell biology information available for BBR. I was surprised not to see this synthesized in the context of a liver-specific study, particularly where a major sensor of cellular metabolism is discussed.

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Additional Editor Comments:

This is an interesting and important study question, along with a great point on the discordance of in vitro vs. in vivo models. You will see that the reviewers have several major points that need to be addressed; some of these can be addressed with an explicit limitations section. If you have banked samples (e.g., for liver chemistry) it would be ideal to include these analyses given the nature of the topic (safety and liver toxicity). Because of what appears might be a sex-specific response (which is also not surprising for this topic), the issue with sample size in the animal experiments is a substantial concern. You should know at this stage, that while I think this work is important, this specific point makes me uncomfortable endorsing the manuscript further if sample size cannot be addressed. Other points from me that should be examined are as follows:

- Are the images in figure 5 consecutive sections from the same liver? Is that truly representative if so?

- There should be more said about the chosen in vivo dose either in the methods, discussion, or both. This should bein the context of other studies, what people typically would consume as a supplement, and also the fact that BBR does not have good oral bioavailability. REF 33 somewhat gets at this as you already mentioned, and I understand that oral is the appropriate route (since that is how people consume it), but these are all factors that are important to consider for translational context.

- The biochemical/mechanistic literature on BBR effects on glucose metabolism is probably the richest set of information available. I was surprised not to see this synthesized in the context of a liver-specific study.

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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: 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: Lu et al. investigated the therapeutic effects and toxicities of berberine, a commonly used nature dietary supplement, on alpha-1 antitrypsin deficiency, using transgenic Pi*Z hepatocytes and Pi*Z mice. It a very interesting approach since there is evidence that berberine has both negative and positive effects on the liver. In my point of view, the study adds new and important information to the role of berberine on diseased individuals. There are some points to be clarified:

1) A major limitation is the low number of animals used in the study (n=7 including males and females) which is aggravated by the fact that there is a high level of variation in the BBR treated group and also that the observed effects appear to be gender dependent (see fig 3). This issue could be resolved by increasing the number of animals in each group.

2) It is well known that altered mitochondrial physiology and decreased ATP availability has been associated with some properties of berberine (antitumoral, antihyperglycemic and maybe toxic effects). Do authors think that the decrease in ATP content could also be related with the toxic effects observed in this study?

3) Fig 2 – resolution was low, difficult to see the red points.

4) Fig 7 and 9 – The white arrows used are confusing with the histological image. Perhaps using black arrows would be better.

Reviewer #2: Alpha1-antitrypsin deficiency (AATD) is an inherited disorder arising due to mutations in alpha1-antitrypsin (AAT) gene leading to its polymerization and consecutive proteotoxic liver injury. Since there are no approved treatments for AATD-associated liver disease, Lu et al. tested the potential usefulness of berberine, a compound widely used in alternative medicine. This is a nice piece of work, however, a couple of issues need to be addressed prior to publication. In particular:

- The authors used RIPA buffer to measure intracellular AAT levels. Given that polymerized AAT is highly insoluble, it would be useful to check, whether RIPA buffer solubilizes as much AAT as buffers with higher SDS amounts. If not, this fact should be mentioned as a limitation.

- Line 303-the statement that reduction of intracellular AAT levels was due to enhancing of the autophagy is not supported by data and should be tuned down. For example, one could say that it might be due to increased degradation.

- It would be useful to report serum liver enzyme levels as a surrogate of hepatocellular injury.

- It would be useful to use a second method of fibrosis quantification/stellate cell activation (i.e. collagen RT-PCR, hydroxyproline assay or a comparable technique).

- In addition to PAS-D staining, it would be nice to quantify the intrahepatic AAT levels in treated/non-treated animals via immunoblotting.

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Reviewer #1: No

Reviewer #2: No

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Berberine potentiates liver inflammation and fibrosis in the PI*Z hAAT transgenic murine model

PONE-D-24-12616R1

Dear Dr. Brantly,

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.

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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Sincerely,

Nicholas A. Pullen, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

I have confirmed adequate effort has been made to address previous comments. Therefore, I am happy to recommend the manuscript for publication.

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: (No Response)

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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

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

**********

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

**********

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: (No Response)

Reviewer #2: Some comments could not be answered due to insufficient manpower, but in my opinion, the manuscript is still useful for the audience of PLoS One. The author should use buffers with higher SDS content in their future experiments (such as Laemmli 4x reducing buffer).

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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

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