The wide spectrum anti-inflammatory activity of andrographolide in comparison to NSAIDs: A promising therapeutic compound against the cytokine storm

Mitchell Low; Harsha Suresh; Xian Zhou; Deep Jyoti Bhuyan; Muhammad A. Alsherbiny; Cheang Khoo; Gerald Münch; Chun Guang Li

doi:10.1371/journal.pone.0299965

Peer Review History

Original SubmissionFebruary 19, 2024
10 Apr 2024 Decision Letter - Asif Ali, Editor PONE-D-24-06364The wide spectrum anti-inflammatory activity of andrographolide in comparison to NSAIDs: a promising therapeutic compound against the cytokine stormPLOS ONE Dear Dr. Suresh, 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 manuscript by Low et al., titled "The Wide Spectrum Anti-inflammatory Activity of Andrographolide in Comparison to NSAIDs: A Promising Therapeutic Compound Against the Cytokine Storm," has been thoroughly reviewed. It presents significant findings on andrographolide's potential as an anti-inflammatory agent, offering insights into its utility against cytokine storms compared to NSAIDs. Both reviewers commend the manuscript's relevance to clinical, pharmaceutical, and medical biology research, predicting it will be well-received within the scientific community. However, for publication consideration, specific revision requested by reviewers are required to address issues of clarity, reproducibility, and accessibility. ============================== Please submit your revised manuscript by May 25 2024 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). 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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, Asif Ali 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 amend lines 80 – 82 where you state “synthetic approaches to drug development have not lived up to their promise” to neutral language. 3. Thank you for stating the following financial disclosure: “This study was partially supported by a Research Partnership Grant (RPG) from the Western Sydney University and LIPA Phar-maceuticals (Prof. Nikolaus Sucher), and a Western Sydney University Research Grant Scheme (RGS) grant (Prof. Nikolaus J. Sucher and Prof. Gerald Münch).” Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 4. 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. [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: The manuscript proposes addressing the gap between NSAIDs and cytokine storms by incorporating the natural product andrographolide. The study underscores the promising therapeutic potential of andrographolide as a broad-spectrum anti-inflammatory agent capable of effectively targeting cytokine storms, suggesting its viability as an alternative to NSAIDs in clinical management. Before finalizing the manuscript for publication, I would like to address a couple of points. 1. In the Materials and Methods section, please provide the catalog numbers of the reagents utilized to enhance accessibility for others. 2. Could you explain how THP-1 cell differentiation into macrophages was confirmed, including whether cell surface staining or morphological staining techniques were employed for validation? Any supporting figures should go to supplementary figures. Did you still maintain PMA after 24 differentiation or you used regular culture medium after differentiation? How was the cell viability during differentiation? Please explain this in a sentence. 3. How many cells were acquired and what flow rate was utilized for the flow cytometry analysis in the Materials and Methods section? 4. Please properly cite FlowJo v10 whether together with country of origin or company. 5. In Figure 1, please avoid using green and red colors together to prevent potential confusion for individuals with color blindness. Reviewer #2: Review Comments The manuscript "The wide spectrum anti-inflammatory activity of andrographolide in comparison to NSAIDs: a promising therapeutic compound against the cytokine storm." by Low et al. is a good research analysis of Andrographolide is a naturally derived bioactive compound. This research article provides sufficient evidence of new bioactive compounds with existing NSAIDs drugs. This manuscript is of interest to the clinical, pharmaceutical and medical biology researchers, as well as different biology researchers and I expect that the article will be well-cited. I have the following minor comments to consider. 1. Authors add details (like method, replicates, etc.) in their table’s legend. 2. Authors provide raw data of supplementary tables with details? ****** 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: Sera Averbek Reviewer #2: Yes: Pawan Kumar ******** [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.0299965.r001
Revision 1
23 May 2024 Author Response Response to Editor, Journal 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 https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf Response: The manuscript has been amended to PLOS ONE standards. 2. Please amend lines 80 – 82 where you state “synthetic approaches to drug development have not lived up to their promise” to neutral language. Response: Change made in manuscript (Ln 82-83). 3. Thank you for stating the following financial disclosure: “This study was partially supported by a Research Partnership Grant (RPG) from the Western Sydney University and LIPA Phar-maceuticals (Prof. Nikolaus Sucher), and a Western Sydney University Research Grant Scheme (RGS) grant (Prof. Nikolaus J. Sucher and Prof. Gerald Münch).” Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. Response: Funding amendment added. Statement also included in the Cover Letter. 4. 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. Response: The citation list is complete and correct, including the inclusion of new citations. Response to Reviewer #1: The manuscript proposes addressing the gap between NSAIDs and cytokine storms by incorporating the natural product andrographolide. The study underscores the promising therapeutic potential of andrographolide as a broad-spectrum anti-inflammatory agent capable of effectively targeting cytokine storms, suggesting its viability as an alternative to NSAIDs in clinical management. Before finalizing the manuscript for publication, I would like to address a couple of points. 1. In the Materials and Methods section, please provide the catalog numbers of the reagents utilized to enhance accessibility for others. Response: The authors feel that adding catalog numbers for individual reagents would be unnecessary since catalog numbers for Sigma-Aldrich and other suppliers are easily accessible upon a cursory search. Further, some of the used reagents might be discontinued by suppliers at a future date, so catalog information might become obsolete. In any case, the purity for any used analytical standards are listed in the ‘Chemicals and Reagents’ section. 2. Could you explain how THP-1 cell differentiation into macrophages was confirmed, including whether cell surface staining or morphological staining techniques were employed for validation? Any supporting figures should go to supplementary figures. Did you still maintain PMA after 24 differentiation or you used regular culture medium after differentiation? How was the cell viability during differentiation? Please explain this in a sentence. Response: There was no staining used. THP-1 cells are non-adherent, when they differentiate, they adhere, so non adherent cells were washed away with fresh PMA free medium. Confluence was near 100% so no significant cytotoxicity was observed. Further clarification regarding MTT assay S1. supporting data has been added to the ‘Protocol for MTT viability determination’ section. 3. How many cells were acquired and what flow rate was utilized for the flow cytometry analysis in the Materials and Methods section? Response: The number of cells seeded prior to flow cytometry is now clearly stated as 10,000 in the ‘Determination of multiple cytokines in LPS-stimulated THP-1 cells using a Bioplex cytokine assay’ section. Clarifications regarding the flow rate, flow cytometry parameters and S2. supporting data have also been added to the ‘The regulation of NF-kB signalling pathway using a FACS Canto II flow cytometer’ section. 4. Please properly cite FlowJo v10 whether together with country of origin or company. Response: FlowJo v10 sourcing information added to the ‘The regulation of NF-kB signalling pathway using a FACS Canto II flow cytometer’ section. 5. In Figure 1, please avoid using green and red colors together to prevent potential confusion for individuals with color blindness. Response: The authors consider the use of red and green to be unavoidable, since limited colour combinations are available for clear differentiation. Please note that all trace lines in Fig 1. have different legends for data points, unique for each therapeutic, with an inverted triangle used for Diclofenac (green) and a solid circle used for Andrographolide (red). Response to Reviewer #2: The manuscript "The wide spectrum anti-inflammatory activity of andrographolide in comparison to NSAIDs: a promising therapeutic compound against the cytokine storm." by Low et al. is a good research analysis of Andrographolide is a naturally derived bioactive compound. This research article provides sufficient evidence of new bioactive compounds with existing NSAIDs drugs. This manuscript is of interest to the clinical, pharmaceutical and medical biology researchers, as well as different biology researchers and I expect that the article will be well-cited. I have the following minor comments to consider. 1. Authors add details (like method, replicates, etc.) in their table’s legend. Response: Replicate information has now been added to each table and figure. Replicates were n=3 for all data except Table 2 (THP-1 multicytokine test) where n=2. The reasoning for using n=2 replicates in the THP-1 cytokine inhibition tests were the high cost of the testing kits at the time of purchase (~ $5-10K AUD). 2. Authors provide raw data of supplementary tables with details? Response: Information regarding supporting files S1. and S2. are now added in a separate section after the References in accordance with PLOS ONE formatting. Clarifications regarding the two supporting files have been added to the ‘Protocol for MTT viability determination’ and ‘The regulation of NF-kB signalling pathway using a FACS Canto II flow cytometer’ sections respectively. Attachments Attachment Submitted filename: Response_to_Reviewers_v1.0.docx https://doi.org/10.1371/journal.pone.0299965.r002
27 Jun 2024 Decision Letter - Asif Ali, Editor The wide spectrum anti-inflammatory activity of andrographolide in comparison to NSAIDs: a promising therapeutic compound against the cytokine storm PONE-D-24-06364R1 Dear Dr. Suresh, 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, Asif Ali 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: Accept this publication. Authors have answered all comments and there is no need of further revision. ****** 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: Sera Averbek Reviewer #2: Yes: Pawan Kumar ******** https://doi.org/10.1371/journal.pone.0299965.r003
Formally Accepted
8 Jul 2024 Acceptance Letter - Asif Ali, Editor PONE-D-24-06364R1 PLOS ONE Dear Dr. Suresh, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 Dr. Asif Ali Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0299965.r004

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