Sargassum horneri (Turner) C. Agardh Extract Regulates Neuroinflammation In Vitro and In Vivo

Round 1

Reviewer 1 Report

Reference: cimb-1981502

Sargassum horneri (Turner) C. Agardh extract regulates neuroinflammation in vitro and in vivo

Authors: Jun Hwi Cho, Dae Hyun Kim, Jong Suk Lee, Mi-Suk Seo, Mi Eun Kim and Jun Sik Lee

This is a study on the effect of an ethanolic extract obtained from an alga, S. honeri, on the inflammatory response mediated by a cell line on microglia (BV-2) and in in vivo mice challenged with LPS.

Comments

• The introduction and discussion should be greatly improved both in the flow of ideias and its interconnections, as well as language and grammar.
• Although the compounds present in the extract were isolated and identified none of them was tested alone to verify which one had the main anti-inflammatory effects.
• The in vitro toxicity of the extract has been evaluated but the same was not tested in the in vivo experiments. A fourth group of animals should be included in the in vivo protocol of experiments, a group treated only with extract. This has not been done.
• It is not clear to me in the in vivo protocol why mice were treated with LPS 1mg/kg for 24h. It is also not clear to me how the concentration of LPS and extract were chosen to perform the in vivo A reference to support the use the dose of LPS chosen should be included.
• It is not clear whether the compounds present in the extract reach the CNS.
• The model using LPS as a challenge to trigger CNS inflammatory response leads to activation of immune mechanisms that in many aspects are different from those involved in neuroinflamation that is observed in neurodegenerative diseases as Alzheimer or Parkinson.

Author Response

Reviewer I

This is a study on the effect of an ethanolic extract obtained from an alga, S. honeri, on the inflammatory response mediated by a cell line on microglia (BV-2) and in in vivo mice challenged with LPS.

Comment 1: The introduction and discussion should be greatly improved both in the flow of ideias and its interconnections, as well as language and grammar.

Response: We thank the reviewer for this helpful comment. We carefully re-read and revise, and the English proofreading was confirmed by a professional company (www.biosciencewriters.com).

Comment 2: Although the compounds present in the extract were isolated and identified none of them was tested alone to verify which one had the main anti-inflammatory effects.

Response: We thank the reviewer for this helpful comment. As for the results confirmed by the UPLC-MS/MS results, there were no commercially available products, so additional confirmation was not possible, but we intend to conduct continuous research future experiments.

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Comment 3: The in vitro toxicity of the extract has been evaluated but the same was not tested in the in vivo experiments. A fourth group of animals should be included in the in vivo protocol of experiments, a group treated only with extract. This has not been done.

Response: Thank you for your constructive comments on this work. Based on the concentration used in the cell experiment, the amount consumed by the animal was calculated and the experiment was carried out. No particular toxicity was observed during the course of the experiment. The reason that the only S. horneri-treated group was not included, because no experimental significance was observed.

Comment 4: It is not clear to me in the in vivo protocol why mice were treated with LPS 1mg/kg for 24h. It is also not clear to me how the concentration of LPS and extract were chosen to perform the in vivo experiments. A reference to support the use the dose of LPS chosen should be included.

Response: We thank the reviewer for this helpful comment. The concentration of LPS (1mg/kg for 24h) for the inflammation test was conducted with reference to the concentrations of previously reported experiments. We added a reference in Materials and Methods section. As can be seen from Figure 5, we observed that the microglia in the brain were sufficiently activated and conducted the experiment.

Comment 5: It is not clear whether the compounds present in the extract reach the CNS.

Response: We thank the reviewer for this helpful comment. We think it is possible because there are reports that materials extracted from seaweed reach the brain, therefore the effect seems to have been observed in animal experiments.

Comment 6: The model using LPS as a challenge to trigger CNS inflammatory response leads to activation of immune mechanisms that in many aspects are different from those involved in neuroinflamation that is observed in neurodegenerative diseases as Alzheimer or Parkinson.

Response: We thank the reviewer for this helpful comment. We tried to find a material that inhibits neuroinflammation and our results suggest a potential as a new treatment for Alzheimer’s or Parkinson’s disease.

Author Response File: Author Response.docx

Reviewer 2 Report

Manuscript ID cimb-1981502, Jun Hwi Cho et al.

Sargassum horneri (Turner) C. Agardh extract regulates neuroinflammation in vitro and in vivo

The main objective of the present study is to investigate the anti-neuroinflammatory effects of S. horneri extract on microglia in vitro and in vivo. In BV-2 microglia cells, the S. horneri extract significantly decreased LPS-induced NO production, diminished the expression of iNOS, COX-2, and cytokines production on LPS-stimulated microglia activation, and elicited anti-neuroinflammatory effects by inhibiting phosphorylation of p38 MAPK and NF-kB. In LPS-challenged mice brain, S. horneri inhibited astrocytes and microglia activation. Collectively, S. horneri exerted anti-neuroinflammatory effects on LPS-stimulated microglia cells activation by inhibiting neuroinflammatory factors and NF-kB signaling.

I agree that the S. horneri extract negatively regulates neuroinflammation and might potentially be useful for therapy of the CNS diseases. My questions (minor) are as follows.

1.     Based on the result of the suppressive effects of S. horneri extracts on inflammatory responses in cells and mice, the authors describe that the extracts might be beneficial for various CNS injuries such as Alzheimer’s and Parkinson’s disease. However, since young adult mice (8 weeks) were used in the current paper, this prediction might be appropriate.

2.     In Figure 6. The high-resolution UPLC-MS/MS result of ethanol extract of S. horneri isolated 4 sulfoglycolipid compounds at retention time 17.37, 18.27, 20.39, and 21.39 min. It would be better to show these sulfoglycolipid compounds have anti-inflammatory effects.

Author Response

Reviewer I

The main objective of the present study is to investigate the anti-neuroinflammatory effects of S. horneri extract on microglia in vitro and in vivo. In BV-2 microglia cells, the S. horneri extract significantly decreased LPS-induced NO production, diminished the expression of iNOS, COX-2, and cytokines production on LPS-stimulated microglia activation, and elicited anti-neuroinflammatory effects by inhibiting phosphorylation of p38 MAPK and NF-kB. In LPS-challenged mice brain, S. horneri inhibited astrocytes and microglia activation. Collectively, S. horneri exerted anti-neuroinflammatory effects on LPS-stimulated microglia cells activation by inhibiting neuroinflammatory factors and NF-kB signaling.

I agree that the S. horneri extract negatively regulates neuroinflammation and might potentially be useful for therapy of the CNS diseases. My questions (minor) are as follows.

Comment 1: Based on the result of the suppressive effects of S. horneri extracts on inflammatory responses in cells and mice, the authors describe that the extracts might be beneficial for various CNS injuries such as Alzheimer’s and Parkinson’s disease. However, since young adult mice (8 weeks) were used in the current paper, this prediction might be appropriate.

Response: We thank the reviewer for this helpful comment. In this experiment, we tried to find a material that inhibits neuroinflammation, and we think that it can offer new therapeutic potential for Alzheimer’s and Parkinson’s diseases.

Comment 2: In Figure 6. The high-resolution UPLC-MS/MS result of ethanol extract of S. horneri isolated 4 sulfoglycolipid compounds at retention time 17.37, 18.27, 20.39, and 21.39 min. It would be better to show these sulfoglycolipid compounds have anti-inflammatory effects.

Response: We thank the reviewer for this helpful comment. As for the results confirmed by the UPLC-MS/MS results, there were no commercially available products, so additional confirmation was not possible, but we intend to conduct continuous research future experiments.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

I agree that the manuscript was revised in response to my comment.

Author Response

Reviewer II

 Comment 1: I agree that the manuscript was revised in response to my comment.

Response: We thank for the comments to improve the quality of the paper.  

Author Response File: Author Response.docx