Metabolomic Investigation of Synergistic Mechanism for Fangfeng Extract Preventing LPS Induced Neuroinflammation in BV-2 Microglia Cells

Round 1

Reviewer 1 Report

Manuscript ID: applsci-1318577

Title

Metabolomic investigation of synergistic mechanism for Fangfeng extract preventing LPS induced neuroinflammation in BV-2 microglia cells

Aim of the study:

1. This study will enable us to identify potential protein targets and metabolite intermediates for FFE exerting its protective function in BV-2 cells, it also provides potential application of this drug pair in neuroinflammation related diseases treatment.
2. We attempt to conduct the untargeted metabolomics investigation to disclose the potential and molecular mechanism of FFE for neuroinflammation protection.

 

Major comments:

1. The control group is not explained in text, is it a positive or negative control? This is crucial for a fair review of the results.
2. Radix Saposhnikoviae here means the root of Saposhnikovia divaricata (SD)?
3. The test was conducted using a mixture of various ingredients or elements (concoction) or single plant? It is confusing for the reader because the authors stated that : “the detailed mechanism of the combined effects are not clear and systematic evaluation hold great promise for deciphering TCM prescription mode of action” – here the prescription would means a concoction rather than just Fangfeng extract.
4. “it also provides potential application of this drug pair in neuroinflammation related diseases treatment”-it what way this study elucidated this?

 

Typos found:

the negtive mode

might been affected

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

In the manuscript by Zhu et al., the authors performed MS based metabolomics to understand the mechanisms underlying anti-neuroinflammatory effect of Fangfeng extract on LPS-induced neuroinflammation using BV-2 microglia cells. They demonstrated that treatment with Fangfeng extract reduces the mRNA levels of proinflammatory markers like IL-6 and IL-1beta. Further, they performed metabolomics using BV-2 microglia cells after LPS treatment with or without Fangfeng extract and demonstrated that metabolites and metabolic pathways which may have potential to reduce neuroinflammation are differentially regulated after Fangfeng extract. Although, the study is interesting, there are several concerns that need to be addressed.

1) Introduction: It lacks the rationale to study metabolites regulation or use of metabolomics approach for understanding the mechanisms of Fangfeng extract. It would be good to add some literature related to the importance of studying the metabolites in the context of neuroinflammation.

2) Materials and Methods: Did authors verify the authenticity of Fangfeng as the authors procured it from a local market than from a verified vendor?

In section 2.7, please spell out the  abbreviation for "VIP".

Did authors consider both up and downregulated metabolites in their analysis? Please discuss and separate the up and downregulated metabolites for each comparison treatment comparison.

3) Results: In section 3.5 (Figure 2), it is unclear on what basis the authors described that LPS group was far away from other groups in the OPLS-DA analysis? Also, its unclear from the plots that how QF treatment groups are closer to control group? From the plots, it appears that control and LPS are on the negative side of axis, while the treatments are on positive side. Please address this and make appropriate changes to the text.

Please clarify the selection criteria for identifying the differentially expressed metabolites (DAMs) from the data. Does it include both up and downregulated DAMs? Please separate the up and downregulated DAMs and also separate DAMs based on comparison groups (Con vs. LPS; LPS vs. QF treatment groups). Please update the text accordingly and discuss what metabolites are differentially regulated in each of these groups and how that will be relevant to LPS-induced neuroinflammation and reversal of neuroinflammation with FF extract.

In fig 4, please specify what the number next to colored circles in each panel indicate?

In the discussion, it was mentioned that "dopamine, palmitic acid, corticosterone and eicosapentanoic acid metabolites were regulated by FFE", however, it was unclear from the results (Table 2) about what metabolites are regulated in LPS group when compared to control and QF group when compared to LPS.

 

 

 

 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors have taken the necessary steps to address the given comment. 

Author Response

Response to Reviewer 1 Comments

Thank you very much for your affirmation of this article.

 

 

Reviewer 2 Report

The data points in figure 4 and figure 5 are falling beyond the axes limits. Please change the range of axes in these figures.

 

Author Response

Response to Reviewer 2 Comments

The coordinate axis problem in Figure 4 and figure 5 has been modified completely according to your requirements. Thank you very much.