Apigetrin Abrogates Lipopolysaccharide-Induced Inflammation in L6 Skeletal Muscle Cells through NF-κB/MAPK Signaling Pathways

Round 1

Reviewer 1 Report

This study aimed to identify apigetrin through NF-κB and MAPK signaling pathways and evaluate the biological activity in the presence of different concentrations apigetrin and the presence of LPS. Major issues are necessary to be addressed before reconsideration for publication. Please see the appended points:

v Line 17: l6 cells of what? Please declare. It would better to use L16

v Results section in line 78-124: The authors should state in the results section whether it is significant or not (please indicate the P-value).

v Line 153-154: Please explain these findings in light of previous bibliographies.

v line 175: Please address the language correction.

v line 183: The authors should indicate (with references) why they selected these concentrations of apigetrin (0, 1, 2, 5, 10, and 15 μM) with or without LPS (2 μg/mL).

v line 217-218: Please edit this sentence well

v line 220: Please concise the conclusion section.

v Material and methods section should be reordered before the results section?

v Figures were not enclosed in this manuscript. Please upload the figures in the next round of the review.

Author Response

Comments and Suggestions for Authors

This study aimed to identify apigetrin through NF-κB and MAPK signaling pathways and evaluate the biological activity in the presence of different concentrations apigetrin and the presence of LPS. Major issues are necessary to be addressed before reconsideration for publication. Please see the appended points:

v Line 17: l6 cells of what? Please declare. It would better to use L16

Answer:

Thank you for your comment.

We have revised the mentioned word.

v Results section in line 78-124: The authors should state in the results section whether it is significant or not (please indicate the P-value).

Answer:

Thank you for your comment.

The P-value is indicated in each figure legend. In addition, we have added it with figures in this submission file.

v Line 153-154: Please explain these findings in light of previous bibliographies.

Answer:

Thank you for your comment.

We have revised the mentioned sentence.

v line 175: Please address the language correction.

Answer:

Thank you for your comment.

We have revised the mentioned sentence.

v line 183: The authors should indicate (with references) why they selected these concentrations of apigetrin (0, 1, 2, 5, 10, and 15 μM) with or without LPS (2 μg/mL).

Answer:

Thank you for your comment.

We have indicated the selected concentrations and added references.

1. Apigetrin treatment attenuates LPS-induced acute otitis media though suppressing inflammation and oxidative stress

It has been reported that apigetrin inhibits P65 expression at 40 and 80 uM concentrations in human middle ear epithelial cells (HMEECs).

2. Apigetrin induces extrinsic apoptosis, autophagy and G2/M phase cell cycle arrest through PI3K/AKT/mTOR pathway in AGS human gastric cancer cell

In addition, cell cycle inhibition and PI3K pathway inhibitory effects were also reported in the AGS gastric cancer cell line at 25, 50, and 100 uM concentrations of apigetrin.

Additionally, based on our MTT assay results, we selected concentrations of apigetrin with cell viability of 80% or more as a low-toxic concentration.

v line 217-218: Please edit this sentence well

Answer:

Thank you for your comment.

We have revised the mentioned sentence.

v line 220: Please concise the conclusion section.

Answer:

Thank you for your comment.

We have revised the conclusion section.

v Material and methods section should be reordered before the results section?

Answer:

Thank you for your comment.

We have moved the material and methods section to before the results section.

v Figures were not enclosed in this manuscript. Please upload the figures in the next round of the review.

Answer:

Thank you for your comment.

We did not include the figures in the manuscript but submitted them as separate files. Therefore, it seems that something has happened that is not included in the pdf. We apologize for any inconvenience this review may have caused you. We have included those figures again in this file.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript titled “Apigetrin abrogates Lipopolysaccharide-induced inflammation in L6 skeletal muscle cells through NF-κB/MAPK signaling pathways” (manuscript ref. number 1736699) describes the cellular effects of the flavone apigetrin in rat skeletal muscle cells, with focus on inflammation. The work presented elucidates the anti-inflammatory role of this flavone on muscle cells.

The manuscript contains minor points to be addressed by the authors:

Introduction

Apigetrin is not correctly defined in the first line of introduction. It is apigenin 7-O-beta-D-glucoside or 4’,5-dihydryxyflavone 7-O-glucoside. Moreover, the description of substituents is not correct, at position 7 it contains a glucoside residue.

Line 65: the sentence is not complete, it ends in “p65-mediated transactivation of pro-inflammatory.”.

Line 74: replace with “apigetrin concentrations” or “concentrations of apigetrin”.

Materials and Methods

Line 175: the sentence is incomplete, cells were grown or cultured in complete DMEM

Results

Line 83: the sentence is confusing, inhibition of cell viability was about 20%, not 80%

Line 118: apigetrin is misspelled

Line 366: ligand (singular word) is more appropriate

Discussion:

Lines 134-135: iNOS and cyclooxygenase-2 are not cytokines. The discussion is missing some anti-inflammatory effects of the aglycone apigenin in different types of skeletal muscle cells, e.g. doi: 10.1007/s12272-016-0756-2, doi: 10.3177/jnsv.61.188; doi: 10.1002/mnfr.201700218

Author Response

The manuscript titled “Apigetrin abrogates Lipopolysaccharide-induced inflammation in L6 skeletal muscle cells through NF-κB/MAPK signaling pathways” (manuscript ref. number 1736699) describes the cellular effects of the flavone apigetrin in rat skeletal muscle cells, with focus on inflammation. The work presented elucidates the anti-inflammatory role of this flavone on muscle cells.

The manuscript contains minor points to be addressed by the authors:

Introduction

Apigetrin is not correctly defined in the first line of introduction. It is apigenin 7-O-beta-D-glucoside or 4’,5-dihydryxyflavone 7-O-glucoside. Moreover, the description of substituents is not correct, at position 7 it contains a glucoside residue.

Answer:

Thank you for your comment.

We have revised the mentioned sentence.

Line 65: the sentence is not complete, it ends in “p65-mediated transactivation of pro-inflammatory.”.

Answer:

Thank you for your comment.

We have revised the mentioned sentence.

Line 74: replace with “apigetrin concentrations” or “concentrations of apigetrin”.

Answer:

Thank you for your comment.

We have revised the mentioned sentence.

Materials and Methods

Line 175: the sentence is incomplete, cells were grown or cultured in complete DMEM

Answer:

Thank you for your comment.

We have revised the mentioned sentence.

Results

Line 83: the sentence is confusing, inhibition of cell viability was about 20%, not 80%

Answer:

Thank you for your comment.

We have revised the mentioned sentence.

Line 118: apigetrin is misspelled

Answer:

Thank you for your comment.

We have revised the mentioned word.

Line 366: ligand (singular word) is more appropriate

Answer:

Thank you for your comment.

We have revised the mentioned word.

Discussion:

Lines 134-135: iNOS and cyclooxygenase-2 are not cytokines. The discussion is missing some anti-inflammatory effects of the aglycone apigenin in different types of skeletal muscle cells, e.g. doi: 10.1007/s12272-016-0756-2, doi: 10.3177/jnsv.61.188; doi: 10.1002/mnfr.201700218

Answer:

Thank you for your comment.

We have revised the mentioned sentence. In addition, we have added the suggested explanation of apigenin along with references.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

1-Thank you so much for addressing my comments in the round 1 (according to your letter). However, I hardly followed your modifications because you did not highlight changes of the submitted manuscript and indicate the modifications in the submitted manuscripts that were raised by the comments of the reviewers. Please highlight changes of the submitted manuscript and indicate (lines or pages) the modifications in the submitted manuscript.

2-The figures were not uploaded. Again, please resolve this issue to finish the review.

Author Response

Thank you for the comments. We have provided the point-by-point response in a separate file.

Author Response File: Author Response.docx

Round 3

Reviewer 1 Report

Thank you so much for addressing the comments in the previous revision. Please address these important points:

1-Some figures contain different charts. Please identify these charts. For example, Fig. 5A, Fig. 5B, Fig. 5C, Fig. 5C. The authors should identify these panels in the figure legends.

2-The same for all figures

3-Please do not forget to cite these figures in the text accordingly.

Author Response

Thank you for the comments. We have provided the point-by-point response in a separate file.

Author Response File: Author Response.docx