Berberine ameliorates vascular dysfunction by a global modulation of lncRNA and mRNA expression profiles in hypertensive mouse aortae

Na Tan; Yi Zhang; Yan Zhang; Li Li; Yi Zong; Wenwen Han; Limei Liu

doi:10.1371/journal.pone.0247621

Peer Review History

Original SubmissionNovember 12, 2020
6 Jan 2021 Decision Letter - Yu Huang, Editor PONE-D-20-35678 Berberine ameliorates vascular dysfunction by a global modulation of lncRNA and mRNA expression profiles in hypertensive mouse aortae PLOS ONE Dear Dr. Liu, 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. The authors provided evidence for the potential mechanisms responsible for the beneficial effect of berberine against Ang II-induced hypertension and showed that in Ang II treatment increased the levels of 578 lncRNAs and 554 mRNAs while reduces the levels of 320 lncRNAs and 377 mRNAs in mouse aortas, and these changes can be reversed by berberine treatment in Ang II-infused mice. In general, this experiments were well designed and the results were clearly presented. However, both reviewers raised a number of constructive comments and suggestions for the authors to address. They include 1. Is there any study showing the association between the 5 LncRNAs with blood pressure regulation? 2. Are the 5 LncRNAs majorly expressed in endothelial cells? 3. More information is necessary for the animal protocol (i) What is the dosage of AngII osmotic pumps implanted in the mice? (ii) What are sources and the solvents for Ang II and berberine? And (iii) Is berberine administered by oral gavage or other method? 4. The authors shall briefly describe the limitations of the current study in the Discussion. 5. There are correlation about the changes of expression profiles of lnc RNA and mRNAs with the berberine treatment in hypertension. However, it is unclear whether such changes lead to the improvement of endothelial function. Future investigations are needed to confirm that the changes lead to endothelial dysfunction in hypertension in relation to the vaso-protective effect of berberine. Please submit your revised manuscript by Feb 04 2021 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Yu Huang Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Please ensure that in your methods section you have provided details of the sources of all materials, chemicals, equipment and instrumentation used in your study, including manufacturer/supplier names where relevant. This is in line with our reproducibility criterion for publishing, see https://journals.plos.org/plosone/s/criteria-for-publication#loc-3 [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: Yes ****** 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 ****** 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: Na Tan et al performed the microarray and qRT-PCR to show the regulatory effects of berberine on the expression profiles of lnc RNA and mRNAs in hypertensive mouse aortae. The experiments provide significant and novel results about the molecular mechanisms of berberine in hypertension; and the manuscript are well written. I only have a few minor suggestions. Sugesstions: 1. More information is necessary for the animal protocol: i. What is the dosage of AngII osmotic pumps implanted in the mice? ii. What are sources and the solvents for Ang II and berberine? iii. Is berberine administered by oral gavage or other method? 2. The authors can mention the limitations of current study in the discussion part. There are correlation about the changes of expression profiles of lnc RNA and mRNAs with the berberine treatment in hypertension; nevertheless, it is not sure whether such changes lead to the improvement of endothelial function. Further investigations are needed to prove that the changes lead to the endothelial dysfunction in hypertension and vaso-protective effects of berberine. Reviewer #2: This study mainly investigated the potential mechanism underlying the therapeutic effects of berberine against Ang II-induced hypertension. By using microarray technology, the authors revealed that in Ang II group, 578 lncRNAs and 554 mRNAs were up-regulated, 320 lncRNAs and 377 mRNAs were downregulated in the aortae, while some of them were reversed by berberine treatment. By deep analysis of these differentially expressed (DE) genes, they found that these DE genes were closely associated with NO production and vascular tone regulation. Based on these findings, the authors proposed 5 potential LncRNAs that may participate in the blood pressure-lowering effect of berberine. In general, this study is well designed and written. However, I have several concerns. 1. Is there any study showing the association between the 5 LncRNAs with blood pressure regulation? 2. Are the 5 LncRNAs majorly expressed in endothelial cells? ****** 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: Wai San Cheang Reviewer #2: Yes:** Chenglin Zhang [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. https://doi.org/10.1371/journal.pone.0247621.r001
Revision 1
29 Jan 2021 Author Response The authors thank the constructive comments and suggestions of the two reviewers and we respond point-by-point as follows. We performed some new experiments to support our conclusion and also made necessary discussion. Response to Reviewers' Comments: Reviewer 1 Na Tan et al performed the microarray and qRT-PCR to show the regulatory effects of berberine on the expression profiles of lncRNA and mRNAs in hypertensive mouse aortae. The experiments provide significant and novel results about the molecular mechanisms of berberine in hypertension; and the manuscript are well written. I only have a few minor suggestions. Authors’ response: We thank this reviewer for the very positive appraisal of our findings and manuscript. 1. More information is necessary for the animal protocol: 1) What is the dosage of Ang Ⅱ osmotic pumps implanted in the mice? Authors’ response: The dosage of Ang Ⅱ is 1mg/kg/day. We revised with the detail in Materials and Methods section (please see page 4, line 106 in the revised manuscript) as “Ang II (1mg/kg/day)- or PBS-loaded osmotic pumps were implanted in mice”. 2) What are sources and the solvents for Ang Ⅱ and berberine? Authors’ response: Ang Ⅱ and berberine chloride were respectively purchased from Tocris Bioscience (Bristol, UK) and Sigma-Aldrich Chemical (St Louis, MO, USA). We revised with the detail in Materials and Methods section (please see page 4, lines 110-112 in the revised manuscript) as “Ang II and berberine chloride were respectively purchased from Tocris Bioscience (Bristol, UK) and Sigma-Aldrich Chemical (St Louis, MO, USA). Ang II was dissolved in PBS. Berberine was dissolved in drinking water”. 3) Is berberine administered by oral gavage or other method? Authors’ response: The mice received berberine administration (100 mg/kg/day) or vehicle in drinking water. We revised with the detail in Materials and Methods section (please see page 4, line 108 in the revised manuscript) as “Then the mice received berberine administration (100 mg/kg/day) or vehicle in drinking water for 2 weeks.” 2. The authors can mention the limitations of current study in the discussion part. There are correlation about the changes of expression profiles of lncRNA and mRNAs with the berberine treatment in hypertension; nevertheless, it is not sure whether such changes lead to the improvement of endothelial function. Further investigations are needed to prove that the changes lead to the endothelial dysfunction in hypertension and vaso-protective effects of berberine. Authors’ response: We thank this reviewer for the constructive suggestion. We discussed the limitations in the revised manuscript (Page 12, lines 465-477). Reviewer 2 This study mainly investigated the potential mechanism underlying the therapeutic effects of berberine against Ang II-induced hypertension. By using microarray technology, the authors revealed that in Ang II group, 578 lncRNAs and 554 mRNAs were up-regulated, 320 lncRNAs and 377 mRNAs were downregulated in the aortae, while some of them were reversed by berberine treatment. By deep analysis of these differentially expressed (DE) genes, they found that these DE genes were closely associated with NO production and vascular tone regulation. Based on these findings, the authors proposed 5 potential LncRNAs that may participate in the blood pressure-lowering effect of berberine. In general, this study is well designed and written. However, I have several concerns. Authors’ response: We thank this reviewer for the positive appraisal of our findings and manuscript. 1. Is there any study showing the association between the 5 LncRNAs with blood pressure regulation? Authors’ response: Till now, there is no evidence to state the association between the 5 LncRNAs with blood pressure regulation. But we will further explore the regulatory effects of these 5 lncRNAs on endothelial function and blood pressure in hypertension in future study. In addition, we discussed this limitation in the revised manuscript (Page 12, lines 465-477). 2. Are the 5 LncRNAs majorly expressed in endothelial cells? Authors’ response: In order to explain this point, we primarily cultured endothelial cells (ECs) and smooth muscle cells (SMCs) of the aortae from C57/6J mice. We performed qRT-PCR to examine the expressions of the 5 lncRNAs both in ECs and in SMCs. As shown in S7 Fig, the levels of lncRNA ENSMUST00000155383, AK041185, and ENSMUST00000144849 were significantly higher in ECs than those in SMCs. LncRNA uc.335+ and lncRNA NR_028422 were also clearly expressed in ECs; but the expression level of lncRNA uc.335+ was similar in ECs and SMCs and the expression level of lncRNA NR_028422 in SMCs was higher than that in ECs. Moreover, we discussed this in the revised manuscript (Page 11, lines 446-455). Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0247621.r002
10 Feb 2021 Decision Letter - Yu Huang, Editor Berberine ameliorates vascular dysfunction by a global modulation of lncRNA and mRNA expression profiles in hypertensive mouse aortae PONE-D-20-35678R1 Dear Dr. Liu 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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, Yu Huang 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 ******** 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: (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 #1: Yes:** Wai San Cheang Reviewer #2: No https://doi.org/10.1371/journal.pone.0247621.r003
Formally Accepted
15 Feb 2021 Acceptance Letter - Yu Huang, Editor PONE-D-20-35678R1 Berberine ameliorates vascular dysfunction by a global modulation of lncRNA and mRNA expression profiles in hypertensive mouse aortae Dear Dr. Liu: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Yu Huang Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0247621.r004

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