Betanin purification from red beetroots and evaluation of its anti-oxidant and anti-inflammatory activity on LPS-activated microglial cells

Hosein Ahmadi; Zahra Nayeri; Zarrin Minuchehr; Farzaneh Sabouni; Mehdi Mohammadi

doi:10.1371/journal.pone.0233088

Peer Review History

Original SubmissionMarch 16, 2020
8 Apr 2020 Decision Letter - Gautam Sethi, Editor PONE-D-20-07544 Betanin purification from red beetroots and evaluation of its anti-oxidant and anti-inflammatory activity on LPS-activated microglial cells PLOS ONE Dear Dr mohammadi, 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. We would appreciate receiving your revised manuscript by May 23 2020 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Gautam Sethi Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 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 1. We understand that you purchased red beetroots from your local market for this study. In your Methods section, please provide additional details regarding the source of this material. Please provide the geographic coordinates and names of the purchase location. if available, as well as any further details about the purchased items (e.g., lot number, source origin, description of appearance) to ensure reproducibility of the analyses. Additional Editor Comments (if provided): The authors should check the effect of Betanin on NF-kB activation as suggested by Reviewer#2. The discussion section should be improved by discussing relevant literature such as PMID: 31315033; PMID: 25083991; PMID: 31085174; PMID: 18566231 etc. [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. 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: Mehdi and colleages have purified betanin and characterized with TLC, UV-visible, HPLC, ESI-MASS, FT-IR spectroscopy. Investigation on the inhibitory effect of betanin on activated microglia was evaluated and authirs have displayed betanin inhibited lipopolysaccharide induced microglial, generation of nitric oxide free radicals, reactive oxygen species, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-1 beta (IL-1β). Their docking results demonstrated that betanin have moderate negative binding energy against active sites of TNF-α, IL-6 and iNOS. I have one major issue which is with the resolution and presentation of Figure-6 which is docking figure. Author should make it more visible and easily understandable while adding appropriate leveling and also mention values for negative binding energy. Reviewer #2: The manuscript by Ahmadi et al. entitled “Betanin purification from red beetroots and evaluation of its anti-oxidant and anti-inflammatory activity on LPS-activated microglial cells” describes the anti-oxidant and anti-inflammatory role of natural product betamin. Although the findings in this manuscript are potentially interesting, there are still some questions need to be addressed. Here is a list of several comments: 1. Figures are not well-prepared. The resolution of all the figures is very poor. 2. For FACS staining of microglial cells, combination of CD11b and CD45 labelling will be more proper otherwise it’s very hard to distinguish microglia from macrophages. Alternatively, specific microglial marker MEM119 can be used. 3. For its anti-inflammatory activity on LPS-activated microglial cells, the author should also validate by checking phospho-NF-κB p65. As another master regulator of inflammation, does p38 signalling also involve in betamin mediated anti-inflammatory activity? ****** 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: No 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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0233088.r001
Revision 1
27 Apr 2020 Author Response Editor Comments (if provided): 1. We understand that you purchased red beetroots from your local market for this study. In your Methods section, please provide additional details regarding the source of this material. Please provide the geographic coordinates and names of the purchase location. if available, as well as any further details about the purchased items (e.g., lot number, source origin, description of appearance) to ensure reproducibility of the analyses. Related explanation was added to the Materials section The authors should check the effect of Betanin on NF-kB activation as suggested by Reviewer#2. Response: We tried to perform more experiments as you suggested but due to the Coronavirus outbreak, the laboratories are still closed, and also having access to materials for such experiments will take 2-3 months in this new situation. However, we added more explanation from the results already published in the literature that may be a proper response for this question as below (same explanation was also added to the manuscript). In LPS-activated macrophages, p38 immediately phosphorylated at a tyrosine residue and performs a crucial role in inflammation . Activating of p38 MAPKs is involved in the upregulation of inflammatory mediators, including TNF-α, iNOS, and cyclooxygenase-2 (COX-2) (1). On the other hand, activation of the p38 and NF-κB pathways could be accomplished by the TNFR1 and TNF-α (2). The obtained results of attenuating in the expression of Tnf-α and Nos2 at mRNA level, TNF-α protein, and the product of iNOS (NO•), could be attributed to either the direct effect of p38 on these factors or the negative effect of betanin on the performance of p38. The discussion section should be improved by discussing relevant literature such as PMID: 31315033; PMID: 25083991; PMID: 31085174; PMID: 18566231 etc. This section was improved by adding more discussion accordingly. Comments to the Author Reviewer #1: I have one major issue which is with the resolution and presentation of Figure-6 which is docking figure. Author should make it more visible and easily understandable while adding appropriate leveling and also mention values for negative binding energy Response: Thank you for your comment. Modification on all figures including Figure 6 was made to improve their qualities. Reviewer #2: 1. Figures are not well-prepared. The resolution of all the figures is very poor. Response: The quality of all the figures were improved according your suggestion 2. For FACS staining of microglial cells, combination of CD11b and CD45 labelling will be more proper otherwise it’s very hard to distinguish microglia from macrophages. Alternatively, specific microglial marker MEM119 can be used. Response: We use CD11b as the most useful immunohistochemical marker for microglial cells in healthy brains (3–5). Similarly, several studies have also used cd11b alone to identify microglial cells especially in in-vitro studies like the primary culture of microglial cells (6–9). However, more specific microglial marker TMEM119 could be used to distinguish microglial cells from other immune cells (10). 3. For its anti-inflammatory activity on LPS-activated microglial cells, the author should also validate by checking phospho-NF-κB p65. As another master regulator of inflammation, does p38 signalling also involve in betamin mediated anti-inflammatory activity? Response: We tried to perform more experiments as you suggested but due to the Coronavirus outbreak, the laboratories are still closed, and also having access to materials for such experiments will take 2-3 months in this new situation. However, we added more explanation from the results already published in the literature that may be a proper response for this question as below (same explanation was also added to the text). In LPS-activated macrophages, p38 immediately phosphorylated at a tyrosine residue and performs a crucial role in inflammation (11). Activating of p38 MAPKs is involved in the upregulation of inflammatory mediators, including TNF-α, iNOS, and cyclooxygenase-2 (COX-2) (1). On the other hand, activation of the p38 and NF-κB pathways could be accomplished by the TNFR1 and TNF-α (2). The obtained results of attenuating in the expression of Tnf-α and Nos2 at mRNA level, TNF-α protein, and the product of iNOS (NO•), could be attributed to either the direct effect of p38 on these factors or the negative effect of betanin on the performance of p38. 1. Yi YS, Son YJ, Ryou C, Sung GH, Kim JH, Cho JY. Functional roles of Syk in macrophage-mediated inflammatory responses. Mediators Inflamm. 2014;2014. 2. Blaser H, Dostert C, Mak TW, Brenner D. TNF and ROS Crosstalk in Inflammation. Trends Cell Biol. 2016 Apr;26(4):249–61. 3. Hickstein DD, Ozols J, Williams SA, Baenziger JU, Locksley RM, Roth GJ. Isolation and characterization of the receptor on human neutrophils that mediates cellular adherence. J Biol Chem. 1987;262(12):5576–80. 4. Sasaki A. Microglia and brain macrophages: An update. Neuropathology. 2017;37(5):452–64. 5. Jeong H-K, Ji K, Min K, Joe E-H. Brain Inflammation and Microglia: Facts and Misconceptions. Exp Neurobiol. 2013;22(2):59–67. 6. Lacagnina MJ, Fabisiak TJ, Grace PM. Mapping microglial reactivity in the brain after sciatic nerve injury. Brain Behav Immun [Internet]. 2017;66(2017):e16. Available from: https://doi.org/10.1016/j.bbi.2017.07.068 7. Morrison HW, Filosa JA. Sex differences in astrocyte and microglia responses immediately following middle cerebral artery occlusion in adult mice. Neuroscience [Internet]. 2016;339:85–99. Available from: http://dx.doi.org/10.1016/j.neuroscience.2016.09.047 8. Hutter-Schmid B, Humpel C. Primary mouse brain pericytes isolated from transgenic Alzheimer mice spontaneously differentiate into a CD11b+ microglial-like cell type in vitro. Exp Gerontol [Internet]. 2018;112(July):30–7. Available from: https://doi.org/10.1016/j.exger.2018.08.003 9. Jia X, Cong B, Wang S, Dong L, Ma C, Li Y. Secondary damage caused by CD11b+ microglia following diffuse axonal injury in rats. J Trauma Acute Care Surg. 2012;73(5):1168–74. 10. Bennett ML, Bennett FC, Liddelow SA, Ajami B, Zamanian JL, Fernhoff NB, et al. New tools for studying microglia in the mouse and human CNS. Proc Natl Acad Sci U S A. 2016;113(12):E1738–46. 11. Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, et al. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Vol. 372, Nature. 1994. p. 739–46. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0233088.r002
29 Apr 2020 Decision Letter - Gautam Sethi, Editor Betanin purification from red beetroots and evaluation of its anti-oxidant and anti-inflammatory activity on LPS-activated microglial cells PONE-D-20-07544R1 Dear Dr. mohammadi, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Gautam Sethi Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0233088.r003
Formally Accepted
1 May 2020 Acceptance Letter - Gautam Sethi, Editor PONE-D-20-07544R1 Betanin purification from red beetroots and evaluation of its anti-oxidant and anti-inflammatory activity on LPS-activated microglial cells Dear Dr. Mohammadi: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Gautam Sethi Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0233088.r004

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