PONE-D-24-40651Therapeutic Potential of a Choline-Zinc-Vitamin E Nutraceutical Complex in Ameliorating Thioacetamide-Induced Nonalcoholic Fatty Liver Pathology in ZebrafishPLOS ONE

Dear Dr. zhou,

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.

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We look forward to receiving your revised manuscript.

Kind regards,

Fakhar Islam, PhD

Academic Editor

PLOS ONE

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Additional Editor Comments:

Reviewer-1

I am writing to submit my review of the manuscript entitled " Therapeutic Potential of a Choline-Zinc-Vitamin E Nutraceutical Complex in Ameliorating Thioacetamide-Induced Nonalcoholic Fatty Liver Pathology in Zebrafish" for your consideration. Overall, I find the manuscript's findings intriguing and the information provided useful for researchers and academia. The article has the potential to make a significant contribution to the related discipline.

However, I have some concerns regarding the clarity, detail, and precision of different sections, which I outline below:

I recommend that the authors address these concerns and provide a revised version of the manuscript for further consideration

· L92- Reduce the redundancy and make it more precise. Nonalcoholic fatty liver disease (NAFLD) has been troubling almost one-quarter of global population for several years and its prevalence continues to rise.

· L 98- Please rephrase it. To address NAFLD, operational management approaches encompass lifestyle modifications, weight reduction, and the potential utilization of various biomolecules, including caffeine and polyphenols, which have shown promise in ameliorating the condition.

· L 102- Make it more clear. However, there is no accurate evidence on their treatment's optimal dose and duration.

· L 105- These lines need to be improved. Medical interventions like T2DM drugs (Glucagon-like peptide-1 receptor agonists and Liraglutide), lipid-lowering drugs (Statins and Ezetimibe) and antihypertensive drugs are reported to have a certain effect in ameliorating NAFLD while they tend to cause a series of side effects such as diarrhea, vomiting, constipation and loss of appetite.

· L 109 This line should be precised and easily readable-The neurotransmitter acetylcholine is composed of choline, which is a constituent of mitochondrial membranes and cell membranes.

· L 122 The concet of these lines is not clear- As a lipid-soluble antioxidant, vitamin E prevents the spread of free radicals.

· L 136-138 These lines should be more clear - A new nutraceutical complex of choline bitartrate, zinc citrate, and dl-α-Tocopheryl acetate is developed to help improve the NAFLD.

· L 172 This statement is not clear it should by specify - Oil Red O (lot number: SHBN4926, Sigma, USA).

· L 211-214 Break it into two sentences for more clarification. Interfering with intracellular nucleus RNA transfer, affecting protein synthesis and enzyme activity, increasing DNA synthesis and mitosis in the nucleus of hepatocytes, promoting the development of cirrhosis.

· L 370-371 Reconsider it. The protective effects of the nutraceutical complex at concentrations of 250 and 500 μg/mL are superior to those of choline bitartrate at concentrations of 162 and 316 μg/mL when the content of choline bitartrate is equivalent (p < 0.001).

· L 386-387 Break the sentence for better comparison -the liver cells in the model control group showed loose struc ture with numerous lipid vacuoles; the positive control group at a concentration of 50.0 μg/mL showed compact liver cell structure similar to the normal control group, with a significant reduction in lipid vacuoles compared to the model control group, indicating a significant hepatoprotective effect of the positive control group.

· L 449-352 Avoid too long sentences - The concentration and purity of total RNA were determined using a UV-Vis spectrophotometer. 2.00 μg of total RNA from zebrafish samples was taken and 20.0 μL of cDNA was synthesized according to the instructions of the cDNA First Strand Synthesis Kit, and the expression of β-actin and acsl1a genes was detected by q-PCR.

· L 427- 428 Minor grammatically mistake - The KEGG Pathway enrichment bubble diagram is presented in Figure S4.

· L 443-445 You haven’t mentioned the units if it is measured by spectrophotometer- The ratio of A260/A280 was between 1.8-2.2, 444 indicating that the extracted the quality of fish total RNA is good and can be used in 445 subsequent q-PCR experiments.

· The introduction & discussion section could be improved by providing more context and background from following references,

· doi: https://doi.org/10.1016/j.phymed.2024.155353

· doi: https://doi.org/10.1016/j.phymed.2022.154090

· doi: https://doi.org/10.1016/j.pacs.2023.100569

· doi: 10.1620/tjem.2022.J083

· https://doi.org/10.7150/thno.42539

Italic all the scientific names,

· Remove grammatical mistakes

· Need to rewrite the conclusion

§ Recheck Legends description is as per figure number and discussion-

§ I urge the authors to improve the English language for better flow of literature.

§ Please check reference style throughout MS

Reviewer-2

• Mention full forms of all abbreviations at its first mention.

• Why Choline-Zinc-Vitamin E together? Justify.

• Mention the limitation and future prospects in conclusion.

• Did not found any table at your submission.

• Figure no and Result figure has not matched properly.

[Note: HTML markup is below. Please do not edit.]

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

**********

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

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: No

**********

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

**********

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: I am writing to submit my review of the manuscript entitled " Therapeutic Potential of a Choline-Zinc-Vitamin E Nutraceutical Complex in Ameliorating Thioacetamide-Induced Nonalcoholic Fatty Liver Pathology in Zebrafish" for your consideration. Overall, I find the manuscript's findings intriguing and the information provided useful for researchers and academia. The article has the potential to make a significant contribution to the related discipline.

However, I have some concerns regarding the clarity, detail, and precision of different sections, which I outline below:

I recommend that the authors address these concerns and provide a revised version of the manuscript for further consideration

· L92- Reduce the redundancy and make it more precise. Nonalcoholic fatty liver disease (NAFLD) has been troubling almost one-quarter of global population for several years and its prevalence continues to rise.

· L 98- Please rephrase it. To address NAFLD, operational management approaches encompass lifestyle modifications, weight reduction, and the potential utilization of various biomolecules, including caffeine and polyphenols, which have shown promise in ameliorating the condition.

· L 102- Make it more clear. However, there is no accurate evidence on their treatment's optimal dose and duration.

· L 105- These lines need to be improved. Medical interventions like T2DM drugs (Glucagon-like peptide-1 receptor agonists and Liraglutide), lipid-lowering drugs (Statins and Ezetimibe) and antihypertensive drugs are reported to have a certain effect in ameliorating NAFLD while they tend to cause a series of side effects such as diarrhea, vomiting, constipation and loss of appetite.

· L 109 This line should be precised and easily readable-The neurotransmitter acetylcholine is composed of choline, which is a constituent of mitochondrial membranes and cell membranes.

· L 122 The concet of these lines is not clear- As a lipid-soluble antioxidant, vitamin E prevents the spread of free radicals.

· L 136-138 These lines should be more clear - A new nutraceutical complex of choline bitartrate, zinc citrate, and dl-α-Tocopheryl acetate is developed to help improve the NAFLD.

· L 172 This statement is not clear it should by specify - Oil Red O (lot number: SHBN4926, Sigma, USA).

· L 211-214 Break it into two sentences for more clarification. Interfering with intracellular nucleus RNA transfer, affecting protein synthesis and enzyme activity, increasing DNA synthesis and mitosis in the nucleus of hepatocytes, promoting the development of cirrhosis.

· L 370-371 Reconsider it. The protective effects of the nutraceutical complex at concentrations of 250 and 500 μg/mL are superior to those of choline bitartrate at concentrations of 162 and 316 μg/mL when the content of choline bitartrate is equivalent (p < 0.001).

· L 386-387 Break the sentence for better comparison -the liver cells in the model control group showed loose struc ture with numerous lipid vacuoles; the positive control group at a concentration of 50.0 μg/mL showed compact liver cell structure similar to the normal control group, with a significant reduction in lipid vacuoles compared to the model control group, indicating a significant hepatoprotective effect of the positive control group.

· L 449-352 Avoid too long sentences - The concentration and purity of total RNA were determined using a UV-Vis spectrophotometer. 2.00 μg of total RNA from zebrafish samples was taken and 20.0 μL of cDNA was synthesized according to the instructions of the cDNA First Strand Synthesis Kit, and the expression of β-actin and acsl1a genes was detected by q-PCR.

· L 427- 428 Minor grammatically mistake - The KEGG Pathway enrichment bubble diagram is presented in Figure S4.

· L 443-445 You haven’t mentioned the units if it is measured by spectrophotometer- The ratio of A260/A280 was between 1.8-2.2, 444 indicating that the extracted the quality of fish total RNA is good and can be used in 445 subsequent q-PCR experiments.

· The introduction & discussion section could be improved by providing more context and background from following references,

· doi: https://doi.org/10.1016/j.phymed.2024.155353

· doi: https://doi.org/10.1016/j.phymed.2022.154090

· doi: https://doi.org/10.1016/j.pacs.2023.100569

· doi: 10.1620/tjem.2022.J083

· https://doi.org/10.7150/thno.42539

Italic all the scientific names,

· Remove grammatical mistakes

· Need to rewrite the conclusion

§ Recheck Legends description is as per figure number and discussion-

§ I urge the authors to improve the English language for better flow of literature.

§ Please check reference style throughout MS

Reviewer #2: • Mention full forms of all abbreviations at its first mention.

• Why Choline-Zinc-Vitamin E together? Justify.

• Mention the limitation and future prospects in conclusion.

• Did not found any table at your submission.

• Figure no and Result figure has not matched properly.

**********

6. 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: Yes:  Dr. Muhammad Afzaal

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

<div>PONE-D-24-40651R1Therapeutic potential of a choline-zinc-vitamin e nutraceutical complex in ameliorating thioacetamide-induced nonalcoholic fatty liver pathology in zebrafishPLOS ONE

Dear Dr. zhou,

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.

Please submit your revised manuscript by Mar 08 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Fakhar Islam, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

• In “Hematoxylin and Eosin Staining, and Oil Red O Staining” paragraph, kindly mention the full form of H&E.

• In line 213, kindly mention the sample name.

• In result S1, S2, S3, S4 table and Figure means which table and figure? In supporting information no figure and table found. Kindly check.

• In Table 1 , which substrate’s concentration? Please mention.

[Note: HTML markup is below. Please do not edit.]

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

**********

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: No

**********

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: Dear Editor, The authors have responded to all the raised queries, therefore, the Manuscript can be accepted

Reviewer #2: • In “Hematoxylin and Eosin Staining, and Oil Red O Staining” paragraph, kindly mention the full form of H&E.

• In line 213, kindly mention the sample name.

• In result S1, S2, S3, S4 table and Figure means which table and figure? In supporting information no figure and table found. Kindly check.

• In Table 1 , which substrate’s concentration? Please mention.

**********

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: Yes:  Dr. Muhammad Afzaal

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Attachments

Attachment

Submitted filename: Comment PLOS 1.docx

Therapeutic potential of a choline-zinc-vitamin e nutraceutical complex in ameliorating thioacetamide-induced nonalcoholic fatty liver pathology in zebrafish

PONE-D-24-40651R2

Dear Dr. zhou,

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.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

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,

Fahrul Nurkolis

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Accept in the present form!

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 #2: All comments have been addressed

**********

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 #2: Yes

**********

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

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 #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 #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 #2: (No Response)

**********

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 #2: Yes:  Sunanda Biswas

**********