PONE-D-23-27950Mume Fructus reduces interleukin-1 beta-induced cartilage degradation via MAPK downregulation in rat articular chondrocytesPLOS ONE

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Additional Editor Comments:

After careful consideration of the manuscript, we decided to recommend a major revision. It is a requirement that the authors should address a point-by-point of all the reviewers (total of three) comments before that the manuscript might be further evaluated and considered for publication.

In particular, the authors should change the conclusions in a way that these are support by the results and conduct all the additional and necessary experiments to support their observation. They should also revise their statistical analysis, confirm the number of replicates (biological and technical) being used and add statistics to western blots and/or immunofluorescence.

We are looking forward for an improved version of the manuscript with the necessary data, revised manuscript and the answers to reviewers.

Good luck.

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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: No

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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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

Reviewer #3: Yes

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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: No

Reviewer #3: Yes

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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 primary goal of the manuscript titled "Mume Fructus reduces interleukin-1 beta-induced cartilage degradation via MAPK down regulation in rat articular chondrocytes" is to clarify the biological process by which a herbal remedy works to mitigate the damaging effects of IL1β on cartilage, using this well-established in vitro model of osteoarthritis.

To support this theory, the authors have looked into the expression of key ECM-destructive enzymes, inflammatory cytokines, and components of the MAPK signaling cascade. They also looked at how MF affected macrophage inflammation brought on by LPS. The findings indicate that MF treatment reduces inflammation in LPS-treated macrophages and decreases OA signaling in chondrocytes produced by IL-1β.

They suggested that Mume Fructus (MF) could be a promising therapeutic agent for OA based on their hypothesis that MF treatment lowers OA pathogenesis by decreasing inflammation via MAPK–NF-κB signaling.

This assertion is corroborated by the western blotting technique results as well as qPCR analysis. After utilizing HPLC to examine the chemical content of MF, the researchers came to the conclusion that the 4-CQA component of MF is what inhibits OA signaling in chondrocytes.

Overall, the authors' results are well-supported by thorough research, and the manuscript itself has theoretical and structural significance. Nonetheless, the writers must address the following several issues:

1. The authors concluded that MF inhibited interleukin-1 beta-induced cartilage degradation via MAPK down regulation, citing references 17–19. They also mentioned that IL1β induces its destructive effects through activation of the MAPK signaling pathway. More proof, though, is required to support this. This theory might be validated by gene silencing certain MAPK signaling pathway components.

2. What software has been utilized for the analysis of immunofluorescence and western blot images?

3. Fig. 3 (B&C) are the same whether LPS treatment is applied or not. They are combinable.

4. Table 2 should include a description of the abbreviations for the phosphorylated forms of NF-ΚB, p65, ERK, JNK, and P38.

5. Verify the chromogram again in Fig. 7-D. There is no greater abundance of the 4-CQA peak in MF extract than in PM.

6. In Discussion, lines 346–348 "The current study revealed that…… chondrocytes," but we are unable to draw the conclusion that the primary cause of the decrease in MMP3/13-ADAMTS-5 is the accumulation of COL2A as a result of MF treatment of chondrocytes. While over-expression of COL2A1 may be a useful treatment strategy for osteoarthritis, it is unable to decrease matrix metalloproteinase enzyme expression.

7. The authors found that the NFĸB and MAPK pathways are in charge of the OA cartilage destruction after studying the IL1β induced inflammatory signaling pathway in rat chondrocytes. Along these lines, a pertinent question concerning LPS-induced inflammation in macrophages emerges and needs to be addressed. Is there a comparable signaling pathway regarding the inflammation that LPS causes in macrophages?

Reviewer #2: This manuscript explores the effects of MF and PM on normal rat chondrocytes in an inflammatory environment induced by IL1β, which simulates osteoarthritic conditions, along with testing these extracts for the inti-inflammatory properties in RAW 264.7 cells. The authors report a dose dependent reduction of MMP3, MMP13 and ADAMTS4 at gene and protein levels as well as induction of COL2A1 production. In addition, they report decrease of NO and proinflammatory chemokines like TNFα, IL6 and IL1β but only in MF-treated cells due to increased levels of 4-CQA in the PM extract.

The conclusions that the authors report are not supported by the results and a major point is repletion of the results presented in the manuscript which are not consistent. Below are some minor and major points that lead to rejection of the manuscript at this current form.

1. Metallopeptidases should be replaced with metalloproteinases throughout the manuscript.

2. Line 64: Are there any references for the use of Jaseng Woongayoungsin-hwan in herniated discs and lower limb pain?

3. Line 71: improves life functions needs to be rephrased. The same for the next sentence lines 72-74.

4. Since MF and PM are basically the same fruit, why both were tested?

5. GAPDH antibody is missing from Table 2.

6. Why concentrations of MF ranging from 0 to 100 μg/mL were used? What is the evidence that the activity is efficient in this range?

7. According to Fig1A, MF treatment seems to induce cell proliferation. How was this taken into consideration for all the subsequent experiments?

8. Why are there no statistical analysis in western blots? If the authors have not performed a statistical test, all statements for significant results need to be rephrased.

9. No information for the biological/technical replicates has been included in methods or in the legends.

10. The representative images and the quantification in Fig2C are not in agreement.

11. What is the reason for using GAPDH in some WB and ACTβ in other for normalisation?

12. Since the authors aim to show the anti-inflammatory a=effects of MF in RAW 264.7 cells, Western blots for TNFα, IL6 and IL1β must be performed as well.

13. It seems that there is a repetition of results in Fig1 and Fig5 as well as Fig2 and Fig6 with regards to MF. Furthermore, it is not clear what were the concentrations used for MF or PM in Fig6. In addition, since the experiments were repeated, why are the fold changes in all 3 gene expressions different for MF?

14. The authors wrote that “the neochlorogenic and chlorogenic acid contents were similar in MF and PM extracts, but 4-CQA was more abundant in MF than in PM as per peak area value analysis”. Where is the quantification for this? This is not evident from Fig7D and the chromatograms should be presented equally, i.e. all baselines should start at 0 mAU.

15. Finally, the authors conclude that the differences that they observe are due to the higher concentration of 4-CQA in PM. To support this, an isolation of the 3 ingredients should be performed and these should be tested separately in primary cells.

16. A major limitation is that there is no support of these findings from in vivo experiments.

Reviewer #3: Park and coworkers demonstrated a connection between Mume Fructus (MF) and its anti-inflammatory modulation in rat chondrocytes and macrophages. They observed through both RT-PCR and WB analyzes a MF-mediated down-regulation of catabolic genes (MMPs and ADAMTS) and an up-regulation of anabolic genes COL2A1 and aggrecan, which reinforce the concept of MF's anti-inflammatory properties. They also observed similar anti-inflammatory conditions in macrophages, suggesting further confirmation of the benefits of MF in inflammatory modulation. After having observed these benefits, they went into more detail and they observed the role of MF in the regulation of the MAPK-NF-kB inflammatory axis. Last but not least, they detected the molecule responsible for anti-inflammatory benefits (4-CQA) in rat chondrocytes. In my opinion, the paper is well structured and tells a story starting from a general concept and delves further and further into molecular details up to the detection of the molecule associated with the aforementioned anti-inflammatory power. However, the short chapter dedicated to MF/NSAIDs and pleiotropic effects has not been fully developed by the authors. I will attach my comments on this for more information.

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Reviewer #1: Yes: Davood Nasrabadi

Reviewer #2: No

Reviewer #3: Yes: Alessandro Marazza

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Attachments

Attachment

Submitted filename: Reviewer comments#PLOSONE.docx

Attachment

Submitted filename: 231126 PLOSE ONE Mume Fructus Review.docx

Mume Fructus reduces interleukin-1 beta-induced cartilage degradation via MAPK downregulation in rat articular chondrocytes

PONE-D-23-27950R1

Dear Dr. Ha,

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.

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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,

Adel Tekari, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Congratulations and good continuation in the field.

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 #3: All comments have been addressed

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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 #3: Yes

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

Reviewer #1: Yes

Reviewer #3: Yes

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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 #3: Yes

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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 #3: Yes

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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: The manuscript has been substantially improved.‎ The authors have addressed all questions and improved the manuscript. I have no objection to accepting the article.

Reviewer #3: The authors resolved all technical problems concerning western blot analysis (biological replicates) and they show consistency in their data. Most of my questions were not answered, but the authors decided to be more focused in their project and to avoid to answer some of my questions concerning investigation of additional inflammatory pathways. Although it's a pity to not have a wider story concerning Mume Fructus and its potential anti-inflammation benefit, the data showed in this paper is valid and, as already said before, consistent. For this reason, my last word is to accept the article for pubblication.

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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: Davood Nasrabadi

Reviewer #3: Yes: Alessandro Marazza

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