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Open AccessArticle
Peer-Review Record

Phytoestrogen Agathisflavone Ameliorates Neuroinflammation-Induced by LPS and IL-1β and Protects Neurons in Cocultures of Glia/Neurons

Biomolecules 2020, 10(4), 562;
Reviewer 1: Anonymous
Reviewer 2: Anonymous
Biomolecules 2020, 10(4), 562;
Received: 12 February 2020 / Revised: 27 February 2020 / Accepted: 27 February 2020 / Published: 7 April 2020
(This article belongs to the Special Issue Pharmacology of Medicinal Plants)

Round 1

Reviewer 1 Report

  This study presents the neuroprotective and anti-inflammatory effects of Agathisflavone using a
co-culture model of neurons and macrophages isolated from rat. The experimental design well construcetd
and the results are crealy demonstrated the neuroprotective and anti-inflammatory effects of Agathisflavone.
The major concern of the present study is that most of the results comes from ICC.
As is well knownm that sometimes the results of ICC are misleading,
t would be great if several biochemical parameters such as western blot of
cleaved-caspase 3 or iNOS added.

The minor points is several typo should be corrected.For example, Page 5 line 13, ER-a.

Author Response

We thank the reviewer for the comments. We carefully re-read the manuscript and corrected all typos. Please note the manuscript was proof-read by a native English speaker and last author of this paper (Prof Dr Arthur Morgan Butt).

Although we agree that western blot is a good method to analyze protein expression levels, we use immunocytochemistry (ICC) because it allows the detection of proteins of interest in specific cells. This is achieved by analysis of localization/co-localization of cleaved-caspase 3 and the other proteins of interest analyzed in specific cells (using double/triple staining). This analysis is necessary because we used a co-culture system in which glial cells (astrocytes, microglia and oligodendrocytes) and neurons are mixed together. Western blot would not allow us to recognise the response of individual class of cells to neuroinflammation. Please note that our ICC results were corroborated by qPCR analysis, which demonstrated that agathisflavone primarily acted through controlling inflammation and, thus, protecting neurons.

Reviewer 2 Report

This manuscript describes the neuron protective effect of agathisflavone (FAB) on LPS/IL1b-induced neuroinflammation in vitro in glia/neurons co-culture system. FAB polarized microglia from M1 to M2, and regulated neuroinflammatory genes expression. The authors presented evidence to support their clams; however, some issues have to be revised.

  1. The authors indicated that the inflammation inhibitory effect of FAB could be mediated through the interaction with estrogen receptors (ER). There was no enough information stated in the section of “Introduction.” The information on the relationship of ER and NFkB should be addressed. Also, the ER-associated resulted should be included in “Abstract.”
  2. Additionally, the results of immunocytochemistry of ER should be presented.
  3. In Fig.1D and Fig.2A, the distribution of beta-tubulin 3 expressed cells was quite different (e.g. control group). The authors should explain this difference in the section of “Discussion.”
  4. The format of reference should be revised.  


Author Response

  1. We thank the reviewer for this suggestion. We made the requested changes in the abstract and introduction to make our outcomes clear. Although FAB has been proven (in other models) to act via estrogen receptors, the anti-inflammatory effect of FAB on the neuroinflammatory insult induced by IL-1β was not mediated via estrogen receptors.


  1. We addressed the effect of agathisflavone on estrogen receptors and NFkB expression in the p. 23, in the discussion.


  1. We thank the reviewer for this insightful input. We added the following sentence on page 11, Figure 2 legend: “Representative images were chosen from the field that better represented astrocytes-neurons proximity.” There was no difference between the density of neurons, but the representative images per se were chosen to represent astrocytes and neurons relationship.


  1. References were re-formatted. Thank you so much for bringing this to our attention.


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