Anti-Inflammatory and Anti-Airway Remodeling Activities of Jakyakgamcho-Tang in a Mouse Model of COPD

Round 1

Reviewer 1 Report

The authors for this paper investigate the potential of a traditional Chinese drug as a therapy for the treatment of the chronic airways disease COPD. This is an interesting and warranted study because even though there are current COPD treatments that are somewhat effective, they have significant side effects. Authors show here that JGT is able to reduce some key features of disease, such as airway inflammation, lung remodelling and significantly alter signalling pathways important in disease progression.

General Comments:

·         This is a valid proof-of-concept study showing the effects of JGT in the development of COPD. What would have been exciting would be if the authors could show that after the development of disease (after 8 wks smoke), JGT was able to reduce features of disease, which can last for weeks after smoking cessation.  

·         The authors didn’t indicate how the concentrations of JGT used relate to actual doses that would be given to patients. Were the doses used in this study similar to what a patient would take?

o   Another point to discuss, especially for readers who don’t know much about Chinese medicine, is how easy or practical would it be to produce enough of this drug to treat the millions of people with COPD? If it can be demonstrated that it works as well as or better than traditional western medicine, would it be an easy transition to making it available worldwide?

 

Specific comments/questions:

Introduction:

·   More detail is needed on what the common treatments of COPD are, their shortcomings and side effects etc.

o   Authors should also talk about other treatment strategies that are common in COPD, e.g smoking cessation, pulmonary rehabilitation, oxygen therapy etc and what their limitations are, to make a stronger case for why JGT would be a better option than all these.

Methods:

·         Authors don’t say how many mice were used for each experiment group.

·     Was the 1hr of smoking for the mice done all at once, or spread across a couple sessions during the day?

o   A schematic drawing of the model with when all the different treatments are, would be helpful.

·         Authors should state that Roflumilast is the positive control in the experiments.

·         The method description for BALF ELISAs says data is presented as pg/mg protein. Protein in the BALF wasn’t mentioned or measured, should it read pg/ml?

·         Was the whole lung BALFed, or just one side?

·     Was RNA isolated from the same tissue that was BALFed, or from non-balfed tissue? This will significantly alter the results/interpretation of data.

·         One very obvious omission is the lack of an emphysema measurement in the study. Authors talk about it being an important feature of disease, but don’t measure it in any way or show the effect of JGT on emphysema. This really needs to be done.

 

Results:

·         In general, more detail is needed in the description of the results. Each result section should specifically comment on each part of the figures that are shown, i.e. Fig5 part a, b, c and d should each have some description and/or explanation.

·         Figure 2 needs to have some enumeration of the infiltrated cells, whether it is a specific cell count, or a change in intensity of colour, there needs to be quantification of the staining data.

·         Figure 3: Seems to be missing neutrophil counts, neutrophils are an integral part of COPD disease, and their numbers need to be counted. Also, each graph should be labelled with the cell type to easily and quickly identify which graph is which (either a title for each graph or in the y-axis label).

·         Figure 4: The change in TNF-a between NC and COPD is from about 6pg/ml to 7pg/ml, I’m not sure that that would be significant. Which post-test did the authors use with the ANOVA analysis?

·         Figure 5: authors should add “mRNA” to each of the y-axis labels.

 

Discussion:

The authors make the assertion that JGT has anti-remodelling effects, however I don’t think that has convincingly been shown. mRNA for TGF-b, A-SMA and MMP9, while this is a start, is not enough. Authors need to show an effect on other important markers of remodelling, e.g collagen, fibronectin, smooth muscle mass etc. some of which can be done by IHC so authors already have the tissue for this.

There is also no discussion of how JGT compares to the control Roflumilast that was used. The authors don’t make a case for if or why JGT was better than Roflumilast, which I assume is the main point of the study.

Author Response

August 25, 2022

Editor in Chief

Applied Science

 

 

Dear Editor

We have revised the manuscript entitled “Anti-Inflammatory and Anti-Airway Remodeling Activities of Jakyakgamcho-Tang in a Mouse Model of COPD” according to the suggestions made by reviewers. We have edited the text to reflect all comments from our reviewers. We sincerely appreciate all of the reviewers for their time and effort in reviewing this manuscript. All of their suggestions and recommendations were constructive and critical, resulting in a significant enhancement of the manuscript. Answers to their comments are included below.

 

 

Reviewer Comments:

Reviewer 1

 

General Comments:

1. This is a valid proof-of-concept study showing the effects of JGT in the development of COPD. What would have been exciting would be if the authors could show that after the development of disease (after 8 wks smoke), JGT was able to reduce features of disease, which can last for weeks after smoking cessation.  

Answer: This is very important point. Thank you for critical question. In this study, we focused on the prevent effect of JGT in the process of COPD development. However, it is important to evaluate the effects of JGT after the development of disease as reviewer’s suggestion. We will examine the treatment effects of JGT in further study.

 

2. The authors didn’t indicate how the concentrations of JGT used relate to actual doses that would be given to patients. Were the doses used in this study similar to what a patient would take?

Answer: We converted the mouse dose (100~200 mg/kg) to human dose (8.11~16.22 mg/kg) based on the body surface area according to the previous study (Springett, G.M., et al., EBioMedicine, 2015. 2(12):1987-1995). Now, we have added the concertation of JGT for patients on page 2, line 92-94.

 

3. Another point to discuss, especially for readers who don’t know much about Chinese medicine, is how easy or practical would it be to produce enough of this drug to treat the millions of people with COPD? If it can be demonstrated that it works as well as or better than traditional western medicine, would it be an easy transition to making it available worldwide?

Answer: As your comments, we have added these information (page 10, line 254-256).

 

Specific comments/questions:

Introduction:

1. More detail is needed on what the common treatments of COPD are, their shortcomings and side effects etc. Authors should also talk about other treatment strategies that are common in COPD, e.g smoking cessation, pulmonary rehabilitation, oxygen therapy etc and what their limitations are, to make a stronger case for why JGT would be a better option than all these.

Answer: As your comment, we have added treatment and side effects of COPD drug in the introduction (page 1-2, line 43-51).

 

Methods:

1. Authors don’t say how many mice were used for each experiment group.

Answer: As your suggestion, we have added the number of mice used for each experiment (page 2, line 90).

 

2. Was the 1hr of smoking for the mice done all at once, or spread across a couple sessions during the day?

Answer: We exposed mice to cigarette smoke per group (n = 8/group). 

 

3. A schematic drawing of the model with when all the different treatments are, would be helpful.

Answer: We have added a schematic drawing of the model in Figure 1 (page 3).

 

4. Authors should state that Roflumilast is the positive control in the experiments.

Answer: Now, we have added the information about Roflumilast (page 2, line 92).

 

5. The method description for BALF ELISAs says data is presented as pg/mg protein. Protein in the BALF wasn’t mentioned or measured, should it read pg/ml?

Answer: This is ours mistake. We have now changed it (page 3, line 114).

 

6. Was the whole lung BALFed, or just one side?

Answer: We collected BALF from the whole lung through a tracheal cannula. We added this sentence in materials and methods (page 3, line 110).

 

7. Was RNA isolated from the same tissue that was BALFed, or from non-balfed tissue? This will significantly alter the results/interpretation of data.

Answer: RNA was isolated from non-balfed tissue.

 

8. One very obvious omission is the lack of an emphysema measurement in the study. Authors talk about it being an important feature of disease, but don’t measure it in any way or show the effect of JGT on emphysema. This really needs to be done.

Answer: We have added the average alveolar intercepts, reflecting the degree of emphysema in Figure 3 (page 3, line 118-124).

 

Results:

1. In general, more detail is needed in the description of the results. Each result section should specifically comment on each part of the figures that are shown, i.e. Fig5 part a, b, c and d should each have some description and/or explanation.

Answer: As your comments, we have changed the description of the results.

 

2. Figure 2 needs to have some enumeration of the infiltrated cells, whether it is a specific cell count, or a change in intensity of colour, there needs to be quantification of the staining data.

Answer: This is a critical point. Thank you for great question. We have added the histological changes, including inflammatory cell infiltration and average alveolar intercepts (page 6, line 178-181).

 

3. Figure 3: Seems to be missing neutrophil counts, neutrophils are an integral part of COPD disease, and their numbers need to be counted. Also, each graph should be labelled with the cell type to easily and quickly identify which graph is which (either a title for each graph or in the y-axis label).

Answer: This is an important point. In the present study, we also counted neutrophils and eosinophils in BALF. The numbers of neutrophils and eosinophils were higher in CS/LPS-treated group than in control group. The roflumilast and JGT-treated mice showed decreased numbers of lymphocytes, neutrophils, and eosinophils compared to the CS/LPS-exposed animals. However, these changes were not statistically significant. In addition, each graph in Figure 4 has been labelled with the cell type.

 

4. Figure 4: The change in TNF-a between NC and COPD is from about 6pg/ml to 7pg/ml, I’m not sure that that would be significant. Which post-test did the authors use with the ANOVA analysis?

Answer: The data are presented as means ± standard errors of mean (SEM). A one-way analysis of variance (ANOVA) was used to do statistical comparisons in GraphPad Prism ver-sion 6 (CA, USA). A p value of 0.05 or less was considered significant.

 

5. Figure 5: authors should add “mRNA” to each of the y-axis labels.

Answer: As your comments, each graph in Figure 6 has been added “mRNA”.

 

Discussion:

1. The authors make the assertion that JGT has anti-remodelling effects, however I don’t think that has convincingly been shown. mRNA for TGF-b, A-SMA and MMP9, while this is a start, is not enough. Authors need to show an effect on other important markers of remodelling, e.g collagen, fibronectin, smooth muscle mass etc. some of which can be done by IHC so authors already have the tissue for this.

Answer: This is a critical point. Thank you for suggestion. For analysis of collagen deposition in lung tissues, paraffin sections were stained with Sirius red and we have now added these data in Figure 7.

 

2. There is also no discussion of how JGT compares to the control Roflumilast that was used. The authors don’t make a case for if or why JGT was better than Roflumilast, which I assume is the main point of the study.

Answer: Thank you for a critical question. We have now added this information in the discussion (page 12, line 332-334).

 

We appreciate you once more for providing us with the opportunity to revise our manuscript. As previously mentioned, we believe that the reviewers’ suggestion and recommendation considerably supported in the advancement of this paper. We hope that the manuscript is now acceptable for publication in Applied Science

 

Sincerely,

Professor. Hyo-Jung Kwun, DVM, PhD

Department of Veterinary Pathology, College of Veterinary Medicine, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 305-764, Korea.

Tel.: +82-42-821-6755

Fax: +82-42-821-8903

E-mail address: hyojung@cnu.ac.kr

 

Author Response File: Author Response.docx

Reviewer 2 Report

Dear Editor,

Thank you for giving me the chance to review the manuscript titled 

"Anti-Inflammatory and Anti-Airway Remodeling Activities of 2 Jakyakgamcho-Tang in a Mouse Model of COPD".

I read with great interest this excellent work.

I appreciate the great work the authors performed in investigating this traditional Korean medicine in the treatment of severe and prevalent disease (COPD).

I would like to thank the authors for their work and I believe that this manuscript could be accepted without modifications.

Best regards,  

 

Author Response

August 25, 2022

Editor in Chief

Applied Science

 

 

Dear Editor

We have revised the manuscript entitled “Anti-Inflammatory and Anti-Airway Remodeling Activities of Jakyakgamcho-Tang in a Mouse Model of COPD” according to the suggestions made by reviewers. We have edited the text to reflect all comments from our reviewers. We sincerely appreciate all of the reviewers for their time and effort in reviewing this manuscript. All of their suggestions and recommendations were constructive and critical, resulting in a significant enhancement of the manuscript. 

We appreciate you once more for providing us with the opportunity to revise our manuscript. As previously mentioned, we believe that the reviewers’ suggestion and recommendation considerably supported in the advancement of this paper. We hope that the manuscript is now acceptable for publication in Applied Science

 

Sincerely,

Professor. Hyo-Jung Kwun, DVM, PhD

Department of Veterinary Pathology, College of Veterinary Medicine, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 305-764, Korea.

Tel.: +82-42-821-6755

Fax: +82-42-821-8903

E-mail address: hyojung@cnu.ac.kr