Notoginsenoside R1 attenuates brain injury in rats with traumatic brain injury: Possible mediation of apoptosis via ERK1/2 signaling pathway

Xiaoxian Pei; Ling Zhang; Dan Liu; Yajuan Wu; Xiaowei Li; Ying Cao; Xiangdong Du

doi:10.1371/journal.pone.0295903

Peer Review History

Original SubmissionApril 27, 2023
5 Jun 2023 Decision Letter - Kazuhiko Kibayashi, Editor PONE-D-23-11939Notoginsenoside R1 attenuates brain injury in rats with traumatic brain injury: possible mediation of apoptosis via ERK1/2 signaling pathwayPLOS ONE Dear Dr. pei, 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 important basic study that will lead to the treatment of traumatic brain injury. It should be revised to take into account the opinions of the reviewers and the handling editor. Please submit your revised manuscript by Jul 20 2023 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. 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The period must be preceded by a space, the period must be followed by an open space, and the beginning of the sentence must be capitalized. 2. Insufficient references. Formatting is not standardized. Authors' first names and last names are reversed in the bibliography, making it impossible to search the bibliography. For example, reference 23 is not searchable because the first and last names are reversed. It is necessary to check all references one by one. 3. The point of administration of the NGR1 needs to be indicated in the text. The reason for administering the NGR1 before and after the creation of the injury needs to be stated. Since therapeutic drugs are not administered before brain injury in humans, the results of this study may not be applicable to humans. [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: Partly ******** 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. 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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The manuscript PONE-D-23-11939 is well written by Pei et al. Their report about neuroprotective effect of Notoginsenoside R1 (NGR1) on traumatic brain injury is interesting and novel. However , the protective effect of NGR1 on blood brain-barrier (BBB)still needs more tests. I strongly suggest that the authors do immunofluorescence test ,for example, measuring the ratio of Evans blue / albumin:DOI: 10.1186/s12987-022-00356-6 Minor comments : Authors also should justify whether NGR1 has the ability to cross the blood-brain barrier (BBB) or not? If yes, the authors can compare its molecular weight with small molecule substances which passes through BBB DOI: 10.1016/j.taap.2011.06.011 It is suggested that authors add the importance of using effective substances derived from plants that are both affordable and effective in the area of brain trauma, DOI: 10.1038/s41598-023-31891-3 and DOI: 10.1038/srep31866 Reviewer #2: I read the paper titled 'Notoginsenoside R1 attenuates brain injury in rats with traumatic brain injury: possible mediation of apoptosis via ERK1/2 signaling pathway' with great interest. This study showed that otoginsenoside R1 (NGR1) as an extracted phytoestrogen has potent neuroprotective, anti-inflammatory, and anti-apoptotic properties with Bcl-2, Bax, caspase 3, and ERK1/2-related molecules by western blotting and pro-inflammatory cytokines by real-time quantitative PCR. They also showed morphological changes by Nissl staining wand Fluoro-Jade B(FJB) and terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling(TUNEL). In figure -2 and 3 we demonstrated that high dose of NGR1 results are worse than medium dose. So authors need to explain why higher dose did not work. Best regards. ****** 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 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. https://doi.org/10.1371/journal.pone.0295903.r001
Revision 1
22 Oct 2023 Author Response Dear reviewer #1: 1.Response to comment: The protective effect of NGR1 on blood brain-barrier (BBB)still needs more tests. Response: We understand that the immunofluorescence may better reveal the protective effect of NGR1 on blood brain-barrier (BBB). However, in the present study, we mainly focused on NGR1 can improve neuronal apoptosis in brain injury,and we think that western blotting of albumin may not be optimal, but should be sufficient to draw a conclusion that the protective effect of NGR1 on blood brain-barrier (BBB). Unfortunately, results are unavailable at this point. We agree with the reviewer, and have toned down our conclusions. 2.Response to comment: Authors also should justify whether NGR1 has the ability to cross the blood-brain barrier (BBB) or not? Response: NGR1 belongs to saponin monomers from traditional Chinese medicine, molecular weight < 1000 KDa, we have experienced small molecules of water-soluble substances, perhaps directly through the intercellular space propagating through the blood-brain barrier. At the same time, we looked in the literature, and experiments have shown that NGR1 could actually penetrate the blood-brain barrier when given intraperitoneally. (Doi:10.3109/10715762.2014.911853). 3.Response to comment: It is suggested that authors add the importance of using effective substances derived from plants that are both affordable and effective in the area of brain trauma. Response: It is really true as Reviewer suggested that we had update the literature on “the importance of using effective substances derived from plants” in the introduction part of the paper and enriched the introduction of the paper. At last, thank you for your arduous work and instructive advice. Dear reviewer #2: Response to comment: Authors need to explain why higher dose did not work? Response: We found that the proper dose of NGR1 was closely linked to the neuroprotective effect after a traumatic brain injury, and the intervention dose of NGR1 which was too high and too low could not have the corresponding protective effect on the brain, which was in accordance with previous literature reports, suggesting that the relation between NGR1 dose and neuroprotective effect was a U-shaped curve. Studies by Liu et al. (DOI: 10.1016/j.phymed.2021.153660) and Zhu et al. (DOI: 10.3389/fphar.2021.615998) suggest that by establishing a rat acute ischemic attack model and a medium cerebral artery occlusion/reperfusion model, both demonstrated an optimum concentration of NGR1, and zhu et al. (DOI: 10.1016/j.biopha.2021.111693) also found that higher doses of NGR1 had no stronger neuroprotective effects. Therefore, it is assumed that the higher dose we used, 80mg/kg, could have been higher than the maximum therapeutic dose of the medication and may have no protective effect on the traumatic brain injury. Special thanks to you for your good comments and thank you sincerely for your suggestions. Attachments Attachment Submitted filename: Response to Reviewer.docx https://doi.org/10.1371/journal.pone.0295903.r002
16 Nov 2023 Decision Letter - Kazuhiko Kibayashi, Editor PONE-D-23-11939R1Notoginsenoside R1 attenuates brain injury in rats with traumatic brain injury: possible mediation of apoptosis via ERK1/2 signaling pathwayPLOS ONE Dear Dr. pei, 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. I have reviewed the revised manuscript. The terminology needs to be corrected again. I will check again after the revision. Please refer to the following Additional Editor Comments for revision. Please submit your revised manuscript by Dec 31 2023 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, Kazuhiko Kibayashi 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: Dear Authors: Please consider revising the following statement: 38 Traumatic brain injury (TBI) is a severe trauma caused by violent injury to the head. TBIs occur primarily as a result of falls and traffic accidents, with a small number resulting from violence. 75 effects on TBI. NGR1 belongs to saponin monomers from traditional Chinese medicine, molecular weight < 1000 KDa, we have experienced small molecules of water-soluble substances, perhaps directly through the intercellular space propagating through the blood-brain barrier [13]. The composition of the English text is unclear. 97 controlled cortical impingement (CCI) Please check the English description of CCI. CCI is usually described as controlled cortical impact. 229 Treatment of NGR1(M) significantly reversed the changes. Please describe the results accurately. The reverse is unknown. 255 protein espression Please list the name of the protein. 374 This is the first time we have demonstrated a neuroprotective effect of NGR1 in traumatic brain injury. This statement is meant to be your personal first study. It is important to know if this is the first study that includes other researchers. [Note: HTML markup is below. Please do not edit.] [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.0295903.r003
Revision 2
24 Nov 2023 Author Response Dear Editors and Reviewers: Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled “Notoginsenoside R1 attenuates brain injury in rats with traumatic brain injury: possible mediation of apoptosis via ERK1/2 signaling pathway” (ID: PONE-D-23-11939). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion are marked in red in the paper. The main corrections in the paper and the responds to the reviewer’s comments are as flowing: Responds to the reviewer’s comments: Dear reviewer #1: 1.Response to comment: The protective effect of NGR1 on blood brain-barrier (BBB)still needs more tests. Response: We acknowledge that immunofluorescence techniques could more effectively elucidate the protective effect of NGR1 on the blood-brain barrier (BBB). Nevertheless, our current research primarily concentrated on how NGR1 ameliorates neuronal apoptosis in brain injury. While we recognize that the use of western blotting for albumin may not be the most optimal method, we believe it suffices to support a preliminary conclusion regarding the protective effect of NGR1 on the BBB. Regrettably, definitive results in this regard are not yet available. In agreement with the reviewer's insights, we have accordingly moderated the assertions in our conclusions. 2.Response to comment: Authors also should justify whether NGR1 has the ability to cross the blood-brain barrier (BBB) or not? Response: Owing to the diminutive molecular size and hydrophilic properties of phytoestrogens, compounds like NGR1, a distinct saponin present in the root of Panax notoginseng, are presumed to efficiently permeate the blood-brain barrier (Doi:10.1016/j.taap.2011.06.011). Specifically, there is a proposition of NGR1's potential modulatory role within the mammalian Central Nervous System (CNS), as suggested in recent studies (Doi:10.3109/10715762.2014.911853). 3.Response to comment: It is suggested that authors add the importance of using effective substances derived from plants that are both affordable and effective in the area of brain trauma. Response: Indeed, as the Reviewer astutely recommended, we have updated our manuscript to incorporate an extensive review of literature emphasizing 'the importance of using effective substances derived from plants' in the introduction. This enhancement significantly enriches the context and framework of our paper, providing a more comprehensive background for our study. At last, thank you for your arduous work and instructive advice. Dear reviewer #2: Response to comment: Authors need to explain why higher dose did not work? Response: Our research indicates that the efficacy of NGR1 in providing neuroprotection following a traumatic brain injury is intricately associated with its dosage. Notably, both excessively high and low doses of NGR1 failed to offer the anticipated protective effects on the brain. This observation aligns with existing literature, suggesting a U-shaped curve relationship between NGR1 dosage and its neuroprotective impact. Studies by Liu et al. (DOI: 10.1016/j.phymed.2021.153660) and Zhu et al. (DOI: 10.3389/fphar.2021.615998), employing rat models of acute ischemic attack and middle cerebral artery occlusion/reperfusion, respectively, identified an optimal concentration range for NGR1. Furthermore, Zhu et al. (DOI: 10.1016/j.biopha.2021.111693) reported that higher doses of NGR1 did not correspond to increased neuroprotective effects. Consequently, we hypothesize that the higher dose used in our study, 80mg/kg, might have surpassed the maximal therapeutic threshold, potentially rendering it ineffective in mitigating traumatic brain injury. Special thanks to you for your good comments and thank you sincerely for your suggestions. We tried our best to improve the manuscript and made some changes in the manuscript. These changes will not influence the content and framework of the paper. And here we did not list the changes but marked in red in revised paper. We appreciate for Editors/Reviewers’ warm work earnestly, and hope that the correction will meet with approval. Once again, thank you very much for your comments and suggestions. Attachments Attachment Submitted filename: Response to Reviewer.docx https://doi.org/10.1371/journal.pone.0295903.r004
4 Dec 2023 Decision Letter - Kazuhiko Kibayashi, Editor Notoginsenoside R1 attenuates brain injury in rats with traumatic brain injury: possible mediation of apoptosis via ERK1/2 signaling pathway PONE-D-23-11939R2 Dear Dr. pei, 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, Kazuhiko Kibayashi Academic Editor PLOS ONE Additional Editor Comments (optional): Dear authors: I have confirmed that the terminology has been properly corrected in the revised manuscript. In general, there is no effective treatment for traumatic brain injury, and basic research on treatment methods plays an important role in patient care. I believe that this basic research on the treatment of traumatic brain injury will bring important knowledge to the field of traumatic brain injury research. I look forward to your continued translational research leading to the treatment of traumatic brain injury. Kazuhiko Kibayashi Reviewers' comments: https://doi.org/10.1371/journal.pone.0295903.r005
Formally Accepted
7 Dec 2023 Acceptance Letter - Kazuhiko Kibayashi, Editor PONE-D-23-11939R2 Notoginsenoside R1 attenuates brain injury in rats with traumatic brain injury: possible mediation of apoptosis via ERK1/2 signaling pathway Dear Dr. pei: 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 customercare@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 Professor Kazuhiko Kibayashi Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0295903.r006

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