N-(p-Coumaroyl) Serotonin Ameliorates LPS-Induced Inflammation in BV2 Microglia via MAPK/NF-κB Inactivation and HO-1/NQO1 Upregulation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript titled “N-(p-Coumaroyl) Serotonin Ameliorates LPS-Induced Inflammation in BV2 Microglia via MAPK/NF-κB Inactivation and HO-1/NQO1 Upregulation” presents a well-structured investigation into the anti-inflammatory mechanisms of a natural phenolic amide. The study is suitable for publication, as it reveals a compound of significant interest to researchers in the natural product field. The findings contribute valuable insights toward the development of novel therapeutic anti-inflammatory agents derived from natural sources.

Minor comments:

The effect of this compound on cellular inflammatory responses and associated signaling pathways were investigated in a previous study by Seok Han Yun  and his team (Doi: 10.3892/etm.2025.13053) using ELISA, Western blotting and immunocytochemistry, what in the novelty of the current study?

Please integrate the following information into the Results section (likely in the first paragraph describing the compound) by mentioning previous reports on this naturally occurring compound, citing key references on how this compound was previously isolated from natural source/s. Also, include the chemical structure of the compound. 

Please avoid beginning sentences with abbreviations anywhere in the manuscript.

Ensure the following terms are consistently italicized throughout the manuscript such as Escherichia coli, in vitro, and in vivo.

Line 121: The sentence requires revision. Please review and modify spelling mistakes.

In all figures: Please change the label “P” to the lowercase “p” and ensure it is italicized.

STAT3 is missing from the abbreviation list. 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors investigate the potential modulatory effects of N-(p-Coumaroyl) serotonin (CS) on inflammatory responses in BV2 microglial cells treated with LPS. The study aims to explore the molecular mechanisms by which CS might mitigate inflammation, with a focus on cytokine production, nitric oxide levels, and signaling pathways, including MAPK/NF-κB and TLR4/MyD88 activation. The authors report findings from ELISA, Western blotting, nitric oxide assays, and immunocytochemistry, suggesting that CS could inhibit key pro-inflammatory mediators and downregulate the NF-κB pathway in LPS-treated BV2 cells. While the topic is promising and relevant to neuroinflammation and neurodegenerative diseases, several critical issues need to be addressed before the manuscript can be considered for publication.

Major Issues:

1. Use of Cell Lines
   The study relies solely on BV2 microglial cells to explore the effects of CS on inflammation. While BV2 cells are a common model for microglial studies, the inclusion of an additional cell line or primary cell cultures would strengthen the manuscript by demonstrating whether the observed effects are cell type-specific or generalizable. Incorporating another model could provide a broader context for the findings.

2. Western Blot Results
   The Western blot data raise significant concerns. Specifically, the β-actin bands appear as double bands in multiple figures, which is highly unusual. In my experience, such results typically suggest problems with sample integrity, often due to inefficient inhibition of proteases or phosphatases during lysate preparation. The authors should consider repeating these experiments using freshly prepared lysates and ensure that appropriate inhibitors are used to maintain protein integrity.

3. MyD88 Antibody Specificity
   The results involving MyD88 are also problematic. As a well-characterized target, MyD88 typically shows clear and specific bands. However, the lack of specificity observed here is surprising. This may be linked to issues in sample degradation, possibly due to insufficient protease inhibition. The authors should carefully reassess the conditions under which the protein lysates were prepared to ensure the reliability of their antibody results.

4. ELISA Results
   The levels of TNF-α and IL-6 reported in the ELISA assays are lower than expected for BV2 cells treated with LPS. Given the strong inflammatory response typically seen in this model, these results suggest potential issues with assay sensitivity or experimental conditions. The authors should verify the reproducibility of these results and possibly repeat the assays under optimized conditions.

5. Literature Discussion
   The manuscript would benefit from a more thorough discussion of relevant studies in the field. Notably, the study by Chavero Vargas et al. (Biology, 2025), which explores hypoxia's role in BV2 cell inflammation, could provide an important framework for understanding the inflammatory processes in BV2 cells. Addressing similar studies would enhance the manuscript and provide a more robust comparison to existing research.

6. Introduction and Discussion
   The introduction is rather brief and does not provide sufficient background on the research topic or the significance of the study. A more detailed review of the current literature and a clearer explanation of the study’s objectives would improve this section. Similarly, the discussion is underdeveloped and lacks depth. The authors should expand this section to include a more comprehensive interpretation of their results and consider the broader implications of their findings in the context of neuroinflammation and neurodegenerative diseases.

 

While the manuscript addresses an interesting and relevant topic, several issues with experimental design, data interpretation, and literature integration need to be resolved before it can be considered for publication. If the authors are able to address these concerns—particularly with regard to experimental reproducibility, data quality, and contextualizing their findings within the existing literature—I believe the manuscript has potential. However, in its current form, the manuscript cannot be accepted for revision and should be resubmitted as a new submission after appropriate revisions.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I'm sadly not satisfied with the revised MS. The authors declined to improve the MS accordingly to my suggestions (e.g. cell line, antibodies, etc.).

Author Response

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Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

Not satisfied.

Author Response

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Author Response File: Author Response.pdf