Protective Role of Astaxanthin in Regulating Lipopolysaccharide-Induced Inflammation and Apoptosis in Human Neutrophils
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsIn the present work where Sang Hyun and co-workers investigate the anti-inflammatory effect of astaxanthin and its mechanism in human neutrophils. As Astaxanthin is already FDA approved and there are already existing literature closely related to the significance of current work. In my view, the current work lacks both novelty and significance and does not meet the standards for publication in this journal.
Author Response
Comments 1:
In the present work where Sang Hyun and co-workers investigate the anti-inflammatory effect of astaxanthin and its mechanism in human neutrophils. As Astaxanthin is already FDA approved and there are already existing literatures closely related to the significance of current work. In my view, the current work lacks both novelty and significance and does not meet the standards for publication in this journal.
Response 1:
Dear Reviewer,
Thank you for your valuable feedback on our manuscript. We understand your concerns regarding the novelty and significance of our study. While we recognize that our research may not present groundbreaking new findings, we believe it adds valuable insights to the existing body of literature on astaxanthin's anti-inflammatory effects, particularly in human neutrophils. We would like to address these points in detail.
While astaxanthin is indeed FDA-approved for various uses such as a dietary supplement and a food coloring additive, our research focuses specifically on its effects in human neutrophils under lipopolysaccharide (LPS) stimulation. Understanding the effects of astaxanthin in different cell types is crucial because each cell type can have distinct inflammatory pathways and responses. This cell-specific research helps in developing tailored therapeutic strategies and understanding the comprehensive role of astaxanthin in the immune system.
We appreciate the opportunity to address these points and hope that you will consider these points and reevaluate the suitability of our manuscript for publication in this journal.
Thank you once again for your constructive comments.
Sincerely,
Sang Hyun and co-authors
Reviewer 2 Report
Comments and Suggestions for AuthorsThe relevance of sepsis and the function of neutrophils in inflammatory reactions are two topics covered in the introduction. More background information on astaxanthin, such as its origins, earlier research on its anti-inflammatory qualities, and its known mechanisms of action, would enhance the introduction, nevertheless. This would improve the contextualization of the study's goals.
Although the procedure for isolating neutrophils is thoroughly explained, the justification for selecting particular astaxanthin concentrations (10, 50, and 100 µM) is not given. A succinct description of the methodology used to determine these concentrations would be beneficial.
It is recommended that the authors include a brief explanation for their decision to use a 4-hour ELISA incubation period.
The findings indicate that astaxanthin significantly lowers levels of TNF-α and IL-1β in relation to cytokine production.
In terms of cytokine production, the data indicate that astaxanthin administration significantly lowers levels of TNF-α and IL-1β, whereas IL-6 has no discernible effect.
It would be beneficial for the writers to include more details on the possible pathways by which astaxanthin stimulates apoptosis and modifies ERK1/2 MAPK.
Astaxanthin has several potential therapeutic uses; however, the study should also address the difficulties and constraints in using these results in real-world clinical settings.
The main conclusions are succinctly and efficiently summarized in the conclusion. It should also draw attention to the shortcomings of the study and the necessity of additional investigation to completely grasp astaxanthin's medicinal potential.
Author Response
Dear Reviewer,
Thank you for your thorough and constructive feedback on our manuscript. We appreciate your time and effort in providing these valuable comments. We have addressed each of your points in detail below:
Comment 1:
The relevance of sepsis and the function of neutrophils in inflammatory reactions are two topics covered in the introduction. More background information on astaxanthin, such as its origins, earlier research on its anti-inflammatory qualities, and its known mechanisms of action, would enhance the introduction, nevertheless. This would improve the contextualization of the study's goals.
Response 1:
We acknowledge the need for more comprehensive background information on astaxanthin. We have now included additional details regarding its origins, previous research on its anti-inflammatory properties, and known mechanisms of action. This change can be found in page 2, line 54~76.
Comment 2:
Although the procedure for isolating neutrophils is thoroughly explained, the justification for selecting particular astaxanthin concentrations (10, 50, and 100 µM) is not given. A succinct description of the methodology used to determine these concentrations would be beneficial.
Response 2:
We have now added a sentence in the Materials and Methods that explains these concentrations were chosen based on prior studies. Specifically, these concentrations were selected to cover a spectrum from low to high doses, allowing us to observe dose-dependent effects. This change can be found in page 3, line 101.
Comment 3:
It is recommended that the authors include a brief explanation for their decision to use a 4-hour ELISA incubation period.
Response 3:
We appreciate your suggestion to explain the 4-hour ELISA incubation period. The 4-hour incubation period was chosen based on preliminary optimization experiments and literature references indicating that this duration allowed for optimal antigen-antibody binding and signal detection in our specific assay setup. This period ensures sufficient time for the cytokines to bind to the capture antibodies, providing reliable and reproducible results. We have now included this justification in the Methods section. This change can be found in page 2, line 96.
Comment 4:
The findings indicate that astaxanthin significantly lowers levels of TNF-α and IL-1β in relation to cytokine production. In terms of cytokine production, the data indicate that astaxanthin administration significantly lowers levels of TNF-α and IL-1β, whereas IL-6 has no discernible effect. It would be beneficial for the writers to include more details on the possible pathways by which astaxanthin stimulates apoptosis and modifies ERK1/2 MAPK.
Response 4:
To address your request for more details on the pathways through which astaxanthin stimulates apoptosis and modifies ERK1/2 MAPK, we have expanded the Discussion section. This change can be found in page 8, line 238-247.
Comment 5:
Astaxanthin has several potential therapeutic uses; however, the study should also address the difficulties and constraints in using these results in real-world clinical settings.
The main conclusions are succinctly and efficiently summarized in the conclusion. It should also draw attention to the shortcomings of the study and the necessity of additional investigation to completely grasp astaxanthin's medicinal potential.
Response 5:
We acknowledge the importance of addressing the challenges and constraints in translating our findings into clinical settings.
Just before the conclusion paragraph, we have included a section that highlights the limitations of our study, such as the in vitro nature of our experiments and the need for in vivo studies.
While our study showed that pretreatment with astaxanthin is highly effective, it is often impractical to administer a preventive treatment for inflammatory diseases in clinical settings. Typically, treatment is initiated after the onset of inflammation. Although there are scenarios where inflammation can be anticipated, such cases are limited. Therefore, further research should explore the efficacy of astaxanthin when administered at the onset or after the onset of inflammation.
To address these points, we have added a paragraph in the Discussion section outlining these challenges and constraints. This change can be found in page 8, line 252.
We hope these revisions address your concerns and improve the overall quality of our manuscript. Thank you once again for your insightful comments.
Sincerely,
Sang Hyun and co-authors
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript entitled, ‘Protective role of astaxanthin in regulating lipopolysaccharide-induced inflammation and apoptosis in human neutrophils’ aims to investigate the role of astaxanthin on the production of inflammatory cytokines, activation of intracellular signaling pathways, and apoptosis in lipopolysaccharide (LPS)-stimulated human neutrophils. The concept behind the study is sound yet the manuscript lacks the appropriate execution of the experimentation and detailed presentation. Therefore, the current form of the manuscript can not be recommended for publication in the Journal of Current Issues in Molecular Biology. Following are some critical comments…
1. The #Introdution section is vaguely written and fails to describe the bottlenecks in the study and practical application of this work.
2. The authors should declare the source of their tested material (astaxanthin in this case) included with the basic biochemical details.
3. The authors have not presented any in-vitro antioxidant/anti inflammatory experiments in the manuscript.
4. This manuscript has no study regarding the toxicity-related study of the tested samples on non-immune cells.
5. Why have the authors chosen only neutrophils as their target cell type? On what basis the selection was done? Why not other immune cells?
6. The results of flow cytometry experiments (#Figure 4, at least the presentation) are very confusing and should be presented with proper labeling (better to use colored images).
7. As mentioned in #line_239-240, “…our research highlights astaxanthin’s efficacy in inhibiting the ERK1/2 239 MAPK pathway associated with neutrophil activation.” However, the authors should present detailed experimental findings to support this statement.
8. The limitations of this study must be highlighted in the #Conclusion section.
Comments on the Quality of English LanguageMinor editing are required
Author Response
Comment:
The manuscript entitled, ‘Protective role of astaxanthin in regulating lipopolysaccharide-induced inflammation and apoptosis in human neutrophils’ aims to investigate the role of astaxanthin on the production of inflammatory cytokines, activation of intracellular signaling pathways, and apoptosis in lipopolysaccharide (LPS)-stimulated human neutrophils. The concept behind the study is sound yet the manuscript lacks the appropriate execution of the experimentation and detailed presentation. Therefore, the current form of the manuscript can not be recommended for publication in the Journal of Current Issues in Molecular Biology. Following are some critical comments…
Response:
Dear Reviewer,
Thank you for your thorough and constructive feedback on our manuscript. We have addressed each of your points in detail below:
Comment 1:
The #Introdution section is vaguely written and fails to describe the bottlenecks in the study and practical application of this work.
Response 1:
We acknowledge that the Introduction section was not sufficiently detailed. We have revised this section to better describe the bottlenecks in the study and the practical applications of our work. This includes a more comprehensive background on the challenges of using astaxanthin in clinical settings and its potential therapeutic benefits. This change can be found in page 2, line 54~76
Comment 2:
The authors should declare the source of their tested material (astaxanthin in this case) included with the basic biochemical details.
Response 2:
We have added a declaration of the source of our tested material (astaxanthin) along with its basic biochemical details. This change can be found in page 2, line 54 and 94.
Comment 3:
The authors have not presented any in-vitro antioxidant/anti inflammatory experiments in the manuscript.
Response 3:
While we did not conduct antioxidant experiments ourselves, we have now included a detailed discussion of previous in-vitro studies that have demonstrated the anti-inflammatory properties of astaxanthin. This change can be found in page 8, line 228.
Comment 4:
This manuscript has no study regarding the toxicity-related study of the tested samples on non-immune cells.
Response 4:
Although we did not conduct toxicity studies on non-immune cells ourselves, we recognize the importance of this aspect. Existing literature has extensively documented the safety profile of astaxanthin, particularly noting its FDA approval for use as a dietary supplement and food additive. Several studies have demonstrated the low toxicity of astaxanthin in various non-immune cell types, supporting its safety for broader applications.
We have added this point in the limitation of the study. This change can be found in page 8, line 252.
Comment 5:
Why have the authors chosen only neutrophils as their target cell type? On what basis the selection was done? Why not other immune cells?
Response 5:
Neutrophils were chosen as our target cell type due to their critical role in the innate immune response and their involvement in acute inflammatory conditions such as sepsis. While we recognize the value of studying other immune cells, it is practically challenging to conduct experiments on all cell types within the scope of a single study. Additionally, existing research has already explored the effects of astaxanthin on other immune cells, such as macrophages and lymphocytes. Therefore, focusing on neutrophils allowed us to provide a detailed and specific analysis of astaxanthin's effects in this important cell type. We have added this justification in the Introduction section.
Comment 6:
The results of flow cytometry experiments (#Figure 4, at least the presentation) are very confusing and should be presented with proper labeling (better to use colored images).
Response 6:
We apologize for the confusion. We have uploaded revised figure.
Comment 7:
As mentioned in #line_239-240, “…our research highlights astaxanthin’s efficacy in inhibiting the ERK1/2 239 MAPK pathway associated with neutrophil activation.” However, the authors should present detailed experimental findings to support this statement.
Response 7:
As the reviewer pointed out, we have changed the conclusion section. This change can be found in page 9, line 262.
Comment 8:
The limitations of this study must be highlighted in the #Conclusion section.
Response 8:
We agree that highlighting the limitations of our study is important. We have added a section in the Conclusion that discusses the limitations, such as the in-vitro nature of our experiments and the need for in-vivo validation, as well as potential challenges in translating these findings to clinical applications. This change can be found in page 8, line 252.
We hope these revisions address your concerns and improve the overall quality of our manuscript. Thank you once again for your insightful comments.
Sincerely,
Sang Hyun and co-authors
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsI consider the manuscript proper to be accepted in present form, since the authors improved it following my recommendations.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors have made satisfactory revisions to the manuscript. It can be published in the Journal of current issues in molecular biology.
