2-Bromo-5-Hydroxy-4-Methoxybenzaldehyde Exhibits Anti-Inflammatory Effects Through the Inactivation of ERK, JNK, and NF-kB Pathways in RAW 264.7 Cells
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors“2-Bromo-5-Hydroisxy-4-Methoxybenzaldehyde Exhibits Antiinflammatory Effects Through the Inactivation of ERK, JNK, and NF-kB Pathways in RAW264.7 Cells’’ is a good one for ferroptosis. English is not good, needs to be more clarity.
The compound ‘‘2-Bromo-5-hydroxy-4-methoxybenzaldehyde’’ is algal origin or not, clarify it.
If not then, I think it will better to publish in another journal.
Result should be more clearly stated. Moreover, it will be better to do Insilco approach, I mean molecular docking and MD simulation to prove with clarity.
Figure 1. is not clear
The discussion also needs to be improved in some of the patches.
Conclusion should be revised.
I recommended it for major revision.
Author Response
"Please see the attachment."
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsDear Authors,
I have reviewed the manuscript “2-Bromo-5-Hydroxy-4-Methoxybenzaldehyde Exhibits Anti-inflammatory Effects Through the Inactivation of ERK, JNK, and NF-kB Pathways in RAW264.7 Cells by Kim et al. In this research, authors investigated the biological/pharmacological effects of 2-Bromo-5- hydroxy-4-methoxybenzaldehyde on RAW264.7 cells. The work has significant novelty but need revision as below comments/suggestion
1) Mention the usefulness of this research finding in the abstract
2) I agree with the authors that seaweed-derived phenolic or brominated compounds have been reported for their inflammatory properties. However, I am unsure if the compound '2-Bromo-5-Hydroxy-4-Methoxybenzaldehyde' investigated in this research has been reported from marine algae/seaweeds If yes, provide the citation, and if not, then how is this manuscript related to phycology.
3) In the whole manuscript, sentences like we examined, we found, we added, and we observed have been used. Especially in the results and discussion section, such sentences have been used very frequently. I would suggest to not using first-person plural personal pronouns as much.
4) Line 43 provides reference to validate your sentence
5) Lines 57 and 59 italicize seaweed species names.
6) Correct Fig.1A as Fig.1 in the text
7) It is important to add the future perspective of this research in the conclusion section
Minor corrections
Line no. errors/corrections
18 anti-inflammatory
20 were investigated
23 demonstrate
29 various diseases
35 get activated
42 ‘however’ in place of ‘yet’
72 this study investigated
90 The cells were
96 the culture supernatants
248 it was found
278 this study confirmed
279 these results indicate
288 these findings suggest
288-289 anti-inflammatory
Comments on the Quality of English LanguageThe suggestions are added in Comments and Suggestions for Authors section
Author Response
"Please see the attachment."
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsGeneral Evaluation: The content outlines the very basics of the 2B5H4M anti-inflammatory activity, but it falls short of the scientific rigor and formatting requirements of a higher-level work. The current draft needs significant modification before it can be reconsidered.
- Abstract: This section needs editing (state explicitly the gap, approach, main findings, and importance).
- Introduction: The first paragraph of the manuscript does not indicate how the scientific gap that 2B5H4M is distinct from the structurally similar brominated vanillin derivatives. It would be helpful to add a paragraph that describes the uniqueness of this substance and the rationale for its comparison with other substances. Recent studies involving RAW264.7 and phenolic anti-inflammatory drugs (2020-2024) are not referenced.
- Materials and methods: The Methods section is incomplete. Add the necessary information. Why did the authors use only an unpaired Student’s t-test for multiple-group comparisons? There are more than two groups. Add a statistical analysis subsection with complete details.
- Results: This part is not completely backed up by the data, is very descriptive, and does not provide a thorough analysis at all. Mistakes: The low quality of Western blots and the absence of a comparable context.
- Discussion: The work's limits and biological importance are not discussed, and there is no comparison with other research on marine phenolics or vanillin derivatives. The section needs to be completely reorganized.
- The discussion section has been completely rewritten.
- Include a subheading that explains the biological relevance and potential pharmacological use of 2B5H4M.
- Insert a paragraph to compare. Without this comparison, the novelty of the work is unclear.
- Add a Limitations paragraph in the discussion
- There are no suggestions for future research
- Conclusions: If possible, strengthen this section or carefully rephrase the conclusions.
- References: Add new studies (2020–2024). Reformatting and a major update are needed.
Final Remark: The manuscript needs a significant and fundamental change overall.
Author Response
"Please see the attachment."
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for Authorsns
Author Response
Comment 1. “2-Bromo-5-Hydroisxy-4-Methoxybenzaldehyde Exhibits Antiinflammatory Effects Through the Inactivation of ERK, JNK, and NF-kB Pathways in RAW264.7 Cells’’ is a good one for ferroptosis. English is not good, needs to be more clarity.
Response:
The entire manuscript has been carefully revised for grammar, clarity, and readability. Ambiguous sentences were rewritten to improve overall English quality.
Comment 2. The origin of 2-Bromo-5-hydroxy-4-methoxybenzaldehyde is unclear. Is it algal or not?
Response:
We appreciate this important comment. In the Introduction, we now clearly state that 2B5H4M is not an algal-derived natural product but a synthetic vanillin derivative. We also explain that its brominated phenolic scaffold closely resembles bromophenols isolated from marine algae, and therefore 2B5H4M is used in this study as a marine-inspired reference compound to explore inflammation-related mechanisms that are relevant to marine phenolic structures. These clarifications have been added to the paragraph describing vanillin derivatives and brominated phenolic scaffolds in the Introduction (Lines 55-77).
Comment 3. Result should be more clearly stated. Moreover, it will be better to do Insilco approach, I mean molecular docking and MD simulation to prove with clarity.
Response:
The Results section has been revised to describe the findings more clearly and objectively, with a focus on the effects of 2B5H4M on NO/PGEâ‚‚ production, cytokine expression, MAPK phosphorylation, and NF-κB activation. In addition, in the Discussion, we now explicitly mention that future studies will include in silico approaches such as molecular docking and molecular dynamics simulations to identify potential upstream molecular targets of 2B5H4M and to further support the mechanistic interpretation of our findings (Lines 231-256).
Comment 4. Figure 1 is not clear.
Response:
Figure 1 has been redesigned with improved resolution, clearer labels, and enhanced readability (Line 92).
Comment 5. The discussion also needs to be improved in some of the patches.
Response:
The Discussion section has been substantially reorganized and expanded. We now Relate our findings more directly to the roles of NF-κB and MAPK signaling in macrophage-mediated inflammation, Compare the effects of 2B5H4M with previously reported effects of marine phenolics and vanillin derivatives, and Explain how our data support the concept of 2B5H4M as a marine-inspired brominated phenolic scaffold with anti-inflammatory potential. In addition, a separate paragraph has been added to discuss the biological relevance and potential pharmacological implications of 2B5H4M (Lines 257-284).
Comment 6. Conclusion should be revised.
Response:
The Conclusion section has been rewritten.
It now more clearly summarizes the key findings (inhibition of NO/PGEâ‚‚, cytokines, MAPK phosphorylation, and NF-κB activation in LPS-stimulated RAW264.7 cells) and emphasizes the potential of 2B5H4M as a marine-inspired anti-inflammatory lead compound, while acknowledging the need for further in vivo and mechanistic studies (Lines 297-305).
We sincerely appreciate the reviewer’s thoughtful and constructive feedback, which has helped us to improve the clarity and scientific quality of the manuscript. All revisions made in response to the reviewer’s comments are highlighted in red in the revised manuscript, and we hope that the current version satisfactorily addresses all concerns.
Reviewer 3 Report
Comments and Suggestions for AuthorsDear Authors,
Thank you for submitting the revised version of your manuscript. I appreciate the revisions made. However, after evaluating the second-round submission, it is clear that several essential issues raised during the first review cycle remain insufficiently addressed. Consequently, the manuscript cannot be accepted in its current form and requires another revision.
- Although the authors state that the reference list has been fully reorganized and updated, the revised manuscript does not reflect this. Only one new reference (from 2021) appears to have been added, and there are no substantive citations from 2022–2025. A major update of the literature was previously requested, and this requirement has not been fulfilled. The authors should include multiple relevant and recent studies (2022–2025) and cite them meaningfully within the Introduction, Results, and especially the Discussion.
- The Discussion section has been expanded, but it still does not critically compare the findings with recent studies and does not include the updated literature that was previously requested. The manuscript needs a clearer comparison with recent work to demonstrate what is new and why the study is important.
I invite the authors to revise the manuscript comprehensively, addressing all outstanding concerns. If these issues are resolved with careful revision, the manuscript will progress toward suitability for publication.
Author Response
“The reference list has not been updated with recent literature (2022–2025). The manuscript should include multiple relevant studies and cite them meaningfully within the Introduction, Results, and Discussion.”
Response:
We thank the reviewer for highlighting this important point. In response, we conducted a comprehensive update of the literature and have now incorporated recent and relevant studies (published between 2023 and 2025) addressing marine-derived phenolic compounds and phenolic aldehydes associated with anti-inflammatory activity.
Marine-derived phenolic anti-inflammatory compounds (Ghallab et al., 2024; Silva et al., 2024; Goya et al., 2025), Phenolic aldehydes and vanillin derivatives linked to macrophage-mediated inflammation (Wang et al., 2024; Choudhary et al., 2023; Lamichhane et al., 2023). These references have been integrated meaningfully into the revised manuscript within the Introduction and Discussion, highlighting updated mechanisms associated with NF-κB and MAPK suppression and demonstrating the clinical relevance of marine-inspired phenolic scaffolds. The reference list has been reorganized to reflect these additions accurately. (line 55-57, 63-77, 270-273)
Comment 2:
“The Discussion does not critically compare the findings with recent studies and needs a clearer comparison with recent work to demonstrate novelty and importance.”
Response:
We appreciate this important recommendation. The Discussion section has been substantially revised to:
Compare 2B5H4M findings with recent work on marine phenolics and vanillin derivatives Distinguish non-brominated phenolic aldehydes vs. brominated scaffolds Clarify what remains unknown and how this work fills a knowledge gap Emphasize why 2B5H4M represents a unique contribution. A new paragraph has been added summarizing how our findings align with, extend, or diverge from recent studies, and delineating why this compound is a relevant marine-inspired candidate despite being synthetic. (line 270-284)
These changes strengthen the argument for the novelty, biological significance, and mechanistic relevance of 2B5H4M in the context of recent phenolic-based anti-inflammatory research.
Closing Statement
We are grateful for the reviewer’s valuable and insightful comments, which have greatly contributed to improving the clarity and quality of this manuscript.
We have made comprehensive revisions in response, and we sincerely hope that the revised version meets the journal’s standards for publication.
All revisions made in response to the reviewer’s comments are highlighted in red in the revised manuscript for ease of reference.
