PONE-D-24-12536Anti-inflammatory effect of the combined treatment of LMT-28 and kaempferol in a collagen-induced arthritis mouse modelPLOS ONE

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PLOS ONE

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Additional Editor Comments:

Thank you for submitting your manuscript. It is very interesting. However, I think this manuscript need more discussion. Please revise your manuscript according to my and reviewers comments.

1. In abstract, authors need introduce what is Kaempferol and LMT-28. This manuscript requires detail information about Kaempferol and LMT-28. The therapeutic mechanism including molecular mechanism such as receptor is unknown. At least, authors should discuss about this point.

2. In CIA model, authors should show the histological scores and anti-IIC antibody levels. Please refer Nat Commun. 2021 Jan 4;12(1):94.

3. The inhibitory effect of Kaempferol and LMT-28 in various cells is dose-dependent?

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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: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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: Yes

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: PONE-D-24-12536

Anti-inflammatory effect of the combined treatment of LMT-28 2 and kaempferol in a collagen-induced arthritis mouse model

Comments to Authors:

This study presents an intriguing investigation into the combined effects of LMT-28 and kaempferol in addressing rheumatoid arthritis. Given the significant burden autoimmune diseases place on patients, conquering them remains a paramount objective in modern medicine.

The paper is well-written, with clear data presentation and discussion. However, some aspects could benefit from further elaboration:

1) Mechanism of Synergy: The authors should delve deeper into the anti-inflammatory mechanisms of LMT-28 and kaempferol, explaining how their combination leads to superior therapeutic effects compared to individual treatment.

2) Clarifying Figure 1: A clear link needs to be established between Figure 1A's time points and the data presented in Figure 1C.

3) Updated Reference: A recent review on the IL-6/gp130 pathway should be cited to strengthen the argument.

4) Quantifying Histological Findings: In Figure 1C, the analysis of histological features in CIA mouse joint tissue via H&E staining requires a scoring system. The results should be presented graphically alongside the images to enhance clarity.

5) Missing Methodology: The "Materials and Methods" section lacks details regarding the experimental methods used with chondrocytes C28/I2. This needs to be addressed.

Reviewer #2: This study describes the synergistic effects of LMT-28 and kaemppferol on rheumatoid arthritis (RA), particularly on differentiation, migration, and invasion of synovial cells. Overall, the study is well designed and the manuscript is written well. There are several comments:

1. Authors need to discuss more in detail what made you specifically choose kaempferol as a candidate drug to investigate the synergistic effect of LMT-28. How about other anti-inflammatory drugs?

2. In a previous study, LMT-28 was reported to be effective in suppressing the development of RA by inhibiting the activation of the IL-6 signaling pathway in a CIA mouse model. In this manuscript, however, the activation of signaling pathways did not seem to be inhibited by single-treatment of LMT-28. Authors should explain why the effect of LMT-28 on signaling pathway activation in RA-FLS was shown to be different in this study.

3. Authors described that LMT-28 or the combination therapy decreased or improved the incidence of RA. Since the treatment was administered before the onset of RA, it can be considered to be effective in preventing RA. The preventative effect is different from reducing or improving existing arthritis. Please revise these statements throughout the manuscript.

4. Gene nomenclature for all mouse- and human-derived genes, such as transcription factors and MMPs, needs to be modified according to the standard notation.

Reviewer #3: PONE-D-24-12536

Comments to Authors:

This is a well conducted study regarding the combination effect of LMT-28 and kaempferol on collagen-induced arthritis (CIA). The authors showed the suppressive effects of combination treatment with LMT-28 and kaempferol on the progression of arthritis in CIA model. In addition, the combination treatment inhibited Th17-cell differentiation both in vitro and in vivo and activated regulatory T cell proportion in vivo. Combination treatment also inhibited various functions of RA-FLS and chondrocytes in terms of their proliferation, migration, and invasion.

These results collectively indicated that the combination treatment with LMT-28 and kaempferol has a potential therapeutic merit to treat rheumatoid arthritis. In order to make more complete story, authors need to address following points.

1. In the abstract, authors need to choose either combined treatment or combination treatment for consistency.

2. Enhanced effects on signaling pathway / synergistic effects on RA development

-> Enhanced (negative or antagonistic) effects should be make more sense in the context.

3. Line 82, disease activity -> disease severity

4. Line 95, synergistic effects -> potential effects since it is before experiment . Therefore, they do not know whether they have any synergistic effects.

5. Line 98, by regulating -> by downregulating

6. Introduction-Rationale. It is not entirely clear to me why the authors decided to test the LMT-28/kaempferol combination as a potential RA treatment (in vitro/in vivo). Although often different treatments may be combined to produce a greater effect, still the authors need to expand on the underlying biology of the individual compounds and why their combination may yield synergistic effect.

7. Lines 38 – 40 and 324 – 325: the sentences are ambiguous. Can the authors please reformulate these sentences?

8. Lines 334 – 336: the sentences are ambiguous. Can the authors please reformulate these sentences?

9. Lines 301 -302: lack references.

10. In Figure 6, the efficacy of the combination treatment was measured using the chondrocyte C28/I2 cell line. I couldn't find this in the material and methods section. Please add it.

11. Line 324, enhanced effect -> enhanced negative or inhibitory effect

12. Line 336, synergistic effect ->synergistic negative or inhibitory effect

13. Line 362, co-treatment improved the invasive activity of chondrocytes -> co-treatment dampened or neutralized the invasive activity of chondrocytes

14. Line 410, enhanced effect -> enhanced negative or inhibitory effect.

15. It would be better to make all the units as mg/kg instead of mpk.

16. Figure 1A Kaem 10 mpk -> Kaempferol 10 mg/kg

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Anti-inflammatory effect of the combined treatment of LMT-28 and kaempferol in a collagen-induced arthritis mouse model

PONE-D-24-12536R1

Dear Dr. Heo,

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.

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Kind regards,

Masanori A. Murayama

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for re-submitting your manuscript. I am grad for sending you this letter. Your manuscript is remarkable for publish. Congratulations.

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

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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 #2: Yes

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

Reviewer #1: Yes

Reviewer #2: 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 #2: 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 #2: 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 author's revisions and responses to major revisions have all proven to be scientifically valid and reliable.

Reviewer #2: Authors properly made responses to reviewer's comments and revised the manuscript well, which is most likely acceptable for publication.

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Reviewer #1: No

Reviewer #2: No

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