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MDPIMDPI
by
  • Chih-Yuan Ko1,2,3,
  • Chung-Hsin Wu4 and
  • Szu-Chuan Shen5,*
  • et al.

Reviewer 1: Anonymous Reviewer 2: Anonymous Reviewer 3: Anonymous

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study by Ko et al showed the effect of Ficus formosana extract (FFE) on peripheral neuropathy in ovarietomized diabetic mice. Here are some of my major and minor comments.


Major comments:

 

1.     In abstract:  The results seem to be general. And the conclusion mainly focused on the oxidative stress response of FFE but I think the study did not completely justify that. As searched this study was also submitted to a library (https://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0021-NTNU40372)- The abstract submitted there is more detailed and conclusions are more precise. With this, I think it is also recommended to input in the manuscript or acknowledge that this study abstract was submitted in an online library.

2.     Published studies on Ficus sp. have shown its antidiabetic effect as you have cited in the introduction. What made the authors choose this specific species of Ficus family? And why? Maybe including these reasons and citations can improve the introduction of the manuscript.

3.     Add limitation of the study.

4.     One of my main concerns is that the conclusion and discussion of the paper needs to be more improved. The paper focused on oxidative response but only one marker of oxidative stress was performed. This could be confirmed more with other oxidant markers and/or antioxidant markers. The potential summary said it inhibits ROS but there was no data on that on your paper.

5.     Another major comment is that the low and high dose of FFE is not discussed. What are the differences between these two dosages? As your results show it seems that there is a trend that IL-1 and IFN-g decreased more in LFFE than HFFE and some patterns are also observed in other markers. What could be the possible explanation for these trends.  

 

 

Minor comments

6.     IENFD  abbreviation in Line102 is not explained  

7.     In DM induction, why 50mg/kg of STZ, I recommend inserting citation or pretest results if done. I

8.     What is the basis of choosing the low-dose and high-dose of FFE?

9.     I suggest including Study design of the study as Figure 1 to make the study easier to understand.

10.  I am not very familiar with von Frey filament test and thermal hyperalgesia test but in these kinds of test to avoid biases when observing the behavior responses, did an expert or are these tests done as a blind test?

11.  Figure 2 needs to be changed as the photos are not clear. How was the immunohistochemistry data quantified? Please mention in the method part. Magnification was only mentioned in Figure 2B how about figure 2C and 2D? It seems like the picture was also taken from different parts or side- if you could find the same spot in each group for more reliable interpretation of results.

12.  Figure 3C, ALA group did not have standard deviation- Make sure that the indicated N=6 for each group in all assays are indicated.

 

 

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Minimal editing needed. 

Author Response

Reviewer 1 Comments:

This study by Ko et al explored the potential effect of Ficus formosa extract (FFE) on peripheral neuropathy in ovariectomized diabetic mice and found out that FFE can protect sciatic nerve through lowering oxidative stress and suppressing inflammatory markers. Here are some of my major and minor comments.

 

  1. In abstract: The results seem to be general. And the conclusion mainly focused on the oxidative stress response of FFE but I think the study did not completely justify that. As searched this study was also submitted to a library (https://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0021-NTNU40372)- The abstract submitted there is more detailed and conclusions are more precise. With this, I think it is also recommended to input in the manuscript or acknowledge that this study abstract was submitted in an online library.

Reply:

Agree. This manuscript offers a clear and precise presentation of the results and conclusions, allowing readers an effortless understanding. This approach distinguishes it from a typical master's thesis. The author of this master's thesis, Li-Wen Chen, is also one of authors of this manuscript. The works Miss Chen did of this study is addressed in the section of “Author contribution”.

 

  1. Published studies on Ficus sp. have shown its antidiabetic effect as you have cited in the introduction. What made the authors choose this specific species of Ficus family? And why? Maybe including these reasons and citations can improve the introduction of the manuscript.

Reply:

Thank you very much for the precise suggestion. Although the antidiabetic effect of Ficus sp. has been studied well, however, the research regarding Ficus sp. to DPN is limited. In addition, the relevant descriptions regarding the antidiabetic effect of Ficus sp. were addressed in the Discussion section accordingly (lines 217-234).

 

  1. Add limitation of the study.

Reply:

Accept. The relevant descriptions were addressed in the Discussion section (lines 235-243).

 

  1. One of my main concerns is that the conclusion and discussion of the paper needs to be more improved. The paper focused on oxidative response but only one marker of oxidative stress was performed. This could be confirmed more with other oxidant markers and/or antioxidant markers. The potential summary said it inhibits ROS but there was no data on that on your paper.

Reply:

Agree. Some of the relevant descriptions regarding the anti-oxidative stress/ROS of FFE to sciatic nerves in diabetic mice were addressed in the section of Discussion (lines 177-185).

 

  1. Another major comment is that the low and high dose of FFE is not discussed. What are the differences between these two dosages? As your results show it seems that there is a trend that IL-1 and IFN-g decreased more in LFFE than HFFE and some patterns are also observed in other markers. What could be the possible explanation for these trends.

Reply:

Thanks for the great suggestion. The Figure 3 shows the effect of FFE on mRNA expression of ovariectomized diabetic mice. The LFFE and HFFE are compared to DM group individual. In addition, the inflammatory markers of LFFE and HFFE includes IL1-beta, IL 6 and COX-2 shows no statistical significant difference except for the IFN-γ.

 

  1. IENFD abbreviation in Line102 is not explained.

Reply:

We apologize for the oversight. IENFD is the abbreviation for intraepidermal nerve fiber density. The abbreviation IENFD has been clarified in the revised manuscript (line 102).

 

  1. In DM induction, why 50mg/kg of STZ, I recommend inserting citation or pretest results if done.

Reply:

Accept. The relevant reference was cited in the Materials and Methods section (line 269).

 

  1. What is the basis of choosing the low-dose and high-dose of FFE?

Reply:

Thanks for the great suggestion. The dosage of FFE in this study referred to the experiences of our previous studies, for example the dosages of guava or Ruellia tuberosa L. extracts in DM rats are ranged from 0-400mg/kg body weight. Therefore, the dosage of FFE we used in the present study is 0, 20 and 200mg/kg body weight in DM (DPN) mice.

References:

(1) Shen SC, Cheng FC, Wu NJ. 2008. Effect of guava (Psidium guajava Linn.) leaf soluble solids on glucose metabolism in type 2 diabetic rats. Phytotherapy Research. 22:1458-1464.

(2) Huang DW, Lo YM, Chang WC, Lin CY, Chen JA, Wu JSB, Huang WC, Shen SC. 2020. Alleviative effect of Ruellia tuberosa L. on NAFLD and hepatic lipid accumulation via modulating hepatic de novo lipogenesis in high-fat diet plus strep-tozotocin-induced diabetic rats. Food Science & Nutrition. 8(10): 5710-5716.

 

  1. I suggest including Study design of the study as Figure 1 to make the study easier to understand.

Reply:

Accept. The experimental design of this study was added in the supplementary Figure of revised manuscript.

 

  1. I am not very familiar with von Frey filament test and thermal hyperalgesia test but in these kinds of test to avoid biases when observing the behavior responses, did an expert or are these tests done as a blind test?

Reply:

Thank you for your inquiry. The von Frey filament test and the thermal hyperalgesia test are commonly used methods to evaluate pain behaviors in rodents, especially for assessing mechanical allodynia and thermal nociception respectively. To ensure the integrity and reliability of our data and to avoid any potential biases during behavioral observation, it is indeed crucial that these tests are conducted in a blinded manner. In this study, all behavioral tests were performed by trained experts who were blinded to the treatment groups, ensuring an unbiased interpretation of the results.

 

  1. Figure 2 needs to be changed as the photos are not clear. How was the immunohistochemistry data quantified? Please mention in the method part. Magnification was only mentioned in Figure 2B how about figure 2C and 2D? It seems like the picture was also taken from different parts or side- if you could find the same spot in each group for more reliable interpretation of results.

Reply:

Agree. The old photos in Figure 2 have been substituted by the high resolution photos in the revised manuscript. The measurements of immunohistochemistry data were addressed in the Materials and Methods section (lines 311-314). The magnification of figure 2B, 2C and 2D were added in the figure legend (lines 123-124).

 

  1. Figure 3C, ALA group did not have standard deviation- Make sure that the indicated N=6 for each group in all assays are indicated.

Reply:

Accept. Figure 3C has been corrected in the revised manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is an interesting paper, I suggest that could be publish after read the second version.

Author Response

Reviewer 2 Comments:

  1. This is an interesting paper, I suggest that could be publish after read the second version.

Reply:

Thank you for your positive feedback and for recognizing the value of our work. We are delighted to hear that you find our paper interesting. We have taken all your comments and suggestions into consideration and will ensure that the revised version addresses any concerns. We are committed to providing a comprehensive and improved second version for your review.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors of this manuscript investigated the alleviative effect of Ficus formosana extract on peripheral neuropathy in ovariectomized diabetic mice. There are a few points of clarification that I would like to address to the authors:

1.      There is no chemical characterization of the tested extracts, as well as which active compound dominates and is responsible for alleviative effect on peripheral neuropathy. The authors should at least address this topic in the discussion section based on the available literature.

2.      Considering that the effect of the genus Ficus in diabetic mice has been extensively investigated in numerous previous studies, the authors should compare obtained results for Ficus formosana with other Ficus species in the discussion section and highlight differences.

3.      Page 7, Line 211, the reference for extract preparation is missing and why the authors opted for that way of obtaining the extract.

4.      Page 7, Line 211, the voucher specimen number of the plant material under which the drug was deposited in the Herbarium is missing.

5.      Page 7, Line 224, did the authors, according to specific reference, carry out the animal experimental  procedure?

Author Response

Reviewer 3 Comments:

The authors of this manuscript investigated the alleviative effect of Ficus formosana extract on peripheral neuropathy in ovariectomized diabetic mice. There are a few points of clarification that I would like to address to the authors:

 

  1. There is no chemical characterization of the tested extracts, as well as which active compound dominates and is responsible for alleviative effect on peripheral neuropathy. The authors should at least address this topic in the discussion section based on the available literature.

Reply:

Thank you so much for the precious suggestion. We suggest that the mitigation of DPN in ovariectomized diabetic mice by FFE could be attributed to its rich content of polyphenols and flavonoids. Presently, efforts are underway in our laboratory to isolate and identify the active constituents within the FFE using techniques such as column chromatography and high-performance liquid chromatography. The relevant descriptions were addressed in the Discussion section (lines 217-221). Moreover, the following preliminary data showed that one of the possible active compounds were identified including umbelliferone from FFE. However, more evidences are needed to prove it.

 

  1. Considering that the effect of the genus Ficus in diabetic mice has been extensively investigated in numerous previous studies, the authors should compare obtained results for Ficus formosana with other Ficus species in the discussion section and highlight differences.

Reply:

Accept. The relevant descriptions were addressed in the Discussion section (lines 222-234).

 

  1. Page 7, Line 211, the reference for extract preparation is missing and why the authors opted for that way of obtaining the extract.

Reply:

Thank you very much for the precise suggestion. The relevant reference was cited in the Materials and Methods section (line 248) and also added in the reference list (lines 409-411) to elucidate the reason and procedure to prepare the extract.

 

  1. Page 7, Line 211, the voucher specimen number of the plant material under which the drug was deposited in the Herbarium is missing.

Reply:

Agree. A voucher specimen (TaiBNET204841) has been deposited in the herbarium of Biodiversity Research Center, Academia Sinica, Taiwan. The relevant descriptions were addressed in the Materials and Methods section (lines 248-250).

 

  1. Page 7, Line 224, did the authors, according to specific reference, carry out the animal experimental procedure?

Reply:

Yes, we did. The relevant procedure was described in the Materials and Methods section and reference was also cited (lines 266-272, 455-456).

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The revised manuscript has been improved a lot. Minor comments of adding the abbreviations and its meaning in the Sup. Figure 1 for better and easier understanding. 

Author Response

  1. The revised manuscript has been improved a lot. Minor comments of adding the abbreviations and its meaning in the Sup. Figure 1 for better and easier understanding.

Reply:

Accept. The relevant abbreviations were addressed in the legend of Supplementary figure accordingly (lines 379-382).

Reviewer 3 Report

Comments and Suggestions for Authors

The authors improved the manuscript according to suggestions.

Author Response

Thank you very much for the great suggestions to improve the quality  of this manuscript.