Epigallocatechin-3-Gallate-Loaded Liposomes Favor Anti-Inflammation of Microglia Cells and Promote Neuroprotection

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General comments

In this manuscript, authors report interesting findings related to the anti-inflammatory efficacy of Epigallocatechin-3-gallate Loaded Liposomes in BV2 cells and LPS induced inflammatory model in rats. While the manuscript reports novel findings, few concerns remain to be addressed.

We thank the Reviewer for his/her comments. These comments add substantial value to the document. To consider the point made by the reviewer, we have modified the manuscript. We hope meeting his/her agreement through the revised version.

Specific Comments

1

Authors' claim that the ECGC is neuroprotective. For example in line 166-167 “Therefore, pretreatment of EGCG has an inhibitory effect on neuroinflammation and neuroprotection.” While EGCG seems to have prevented the change in  the morphological appearance (denoting activation of the cells), the conclusion that EGCG has neuroinflammatory and neurprotective effect based this is exagerated. Authors should correct the sentence. This is also applicable to the data from in vivo studies. Authors' have not provided any data to prove the neuroprotection like NeuN staining or such. At best the dataset provided proves the anti-inflammatory effect of ECGG liposomes.

To address the comments made by the reviewer, we rewrote the sentence as follow:

Therefore, pretreatment of EGCG has an inhibitory effect on neuroinflammation, protecting the cells from damage. and neuroprotection.

Indeed, we did not perform some experiments like NeuN staining to prove the neuroprotective effect. We addressed this point as a limitation of our study. However, the anti-neuroinflammation results necessary to a neuroprotective effect (Lee DS and Jeong GS, Br J Pharmacol. 2016 Oct; 173(19): 2894–2909). BV2 activation releases pro-inflammatory factors, which are neurotoxic and lead to cell damage. By preventing BV2 activation, EGCG provides a neuroprotective effect.

2

Authors should consider re-writing and provide more details on result/method sections. Why was this rat model chosen specifically?

Please clarify the methods section of the in vivo studies. When were the rats dosed with LPS and EGCG and when was the behavioral tests conducted? Perhaps adding an experimental timeline would help the readers follow through the flow of the paper better.

The methods section has been expanded to provide additional technical details. The experimental timeline of animal study has been considered (figure 6).

3

English language editing is required. A lot of phrases/sentences do not read very well.

We thank the Reviewer for his comment to improve the quality of this manuscript. The manuscript will be submitted to English language editing.

4

Line 103 – Change “Anti-inflammation” to anti-inflammatory”

The word has been modified: anti-inflammation

5

Line 104 – “was effectively” should be “effectively”.

The wording has been modified: it should be effectively

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Comments

Answers

General comments

The manuscript entitled “Epigallocatechin-3-gallate Loaded Liposomes favors Anti-inflammation of Microglia Cells and promotes neuroprotection” is very interesting research. It may be linked with novel treatment of neurodegenerative diseases such as PD.

We are grateful to the Reviewer for making these comments.

Specific Comments

1

In statistical analysis, I think all results of ANOVA should be reanalyzed by using post hoc.

For example, in Figure 5, the author compared among control, LPS and LPS+EGCG.

The author did not describe post hoc analysis.

We thank the Reviewer for his interest in statistical analysis. Using ANOVA, we performed “Dunnett's multiple comparisons test,”  which is also a post-hoc analysis.

However, more details has been described in figure 5 legend.

2

The author explained the neuroprotection from neuroinflammation by reducing production of TNFα, but not neurotropic factors. However, they investigated only BDNF as a neurotropic factor. Microglia is associated with neurotropic factors not only BDNF, but FGF2, IGF2 and so on. Reference: Abe N et al. Cells 2020. PMID: 32967118.

So, their discussion of neuroprotective mechanisms of EGCG may be little exaggerated.

Indeed, we did not analyze some neutrophic factors such us FGF2, IGF2, and so on to prove the neuroprotective mechanism. We addressed this point as a limitation of our study. However, the anti-neuroinflammation results necessary to a neuroprotective effect (Lee DS and Jeong GS, Br J Pharmacol. 2016 Oct; 173(19): 2894–2909). BV2 activation releases pro-inflammatory factors, which are neurotoxic and lead to cell damage. By preventing BV2 activation, EGCG provides a neuroprotective effect. The reference (Abe N et al. Cells 2020. PMID: 32967118.) has also been considered.

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Therefore, they investigated the expressions of such neurotropic factors or they should describe limitation of this study. If EGCG improve also these neurotropic factors, EGCG would be a more useful candidate of treatment of neurodegenerative diseases.

We thank the Reviewer for this excellent comment that gives us the hope to continue the investigation of EGCG, leading to a clinical trial in Parkinson's disease investigation.