Chemical Inhibition of NRF2 Transcriptional Activity Influences Colon Function and Oestrogen Receptor Expression in Mice at Different Ages

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this manuscript authors investigated whether chemical inhibition of NRF2 transcriptional activity may influence distal colon contractions in female mice. Authors found that the seven-day treatment of ML385 significantly downregulated TA altering contractility. Additionally, young females treated with ML385 exhibited an increase in goblet cells number and significantly increased ERα, but not ERβ, especially in older mice. Moreover, GPR30 basal levels were significantly higher in older mice within the epithelial layer, and ML385 treatment led to a downregulation of GPR30 in 6-month-old mice. 

Although the manuscript and topic are interesting, and the manuscript is generally well written, there are several flaws that do not allow its publication. In particular:

Line 1: Authors must specify the type of paper as showed in the journal template

Lines 39-45: Since NRF2/KEAP1 signaling plays a key role in this manuscript, the mulstifaceted role of this pathway deserves to be highlighted. In fact, this pathway plays a significant role in the onset and progression of several cancerous and non-cancerous diseases (see PMID: 39456522, PMID: 39034715, PMID: 39534872). 

2.5. Gene expression analysis: Primers sequences should be moved in a dedicate table 

Figure 3A and D: Images quality is very low and higher magnifications are needed

Figure 5: Images quality is very low, negative controls are absent. Instead of "Green-cell membranes", authors must write the probe/protein used. Higher magnifications are also necessary. Moreover, ERa and ERb are nuclear receptors but I see red everywhere. Why authors did not block tissue autofluorescence (e.g. with Sudan Black B)? 

An accurate revision of typing errors is necessary

Abbreviations must be written in full length when mentioned for the first time

Author Response

Reviewer 1

In this manuscript authors investigated whether chemical inhibition of NRF2 transcriptional activity may influence distal colon contractions in female mice. Authors found that the seven-day treatment of ML385 significantly downregulated TA altering contractility. Additionally, young females treated with ML385 exhibited an increase in goblet cells number and significantly increased ERα, but not ERβ, especially in older mice. Moreover, GPR30 basal levels were significantly higher in older mice within the epithelial layer, and ML385 treatment led to a downregulation of GPR30 in 6-month-old mice. 

Although the manuscript and topic are interesting, and the manuscript is generally well written, there are several flaws that do not allow its publication. In particular:

Line 1: Authors must specify the type of paper as showed in the journal template

Response: Thank you for the comment. The article type was added.

Lines 39-45: Since NRF2/KEAP1 signaling plays a key role in this manuscript, the mulstifaceted role of this pathway deserves to be highlighted. In fact, this pathway plays a significant role in the onset and progression of several cancerous and non-cancerous diseases (see PMID: 39456522, PMID: 39034715  , PMID: 39534872). 

Response: Thank you for bringing up this interesting review papers. We added this point to the introduction part (ref. 1, 2, 3)

2.5. Gene expression analysis: Primers sequences should be moved in a dedicate table 

Response: Thank you for the comment. We moved primers sequence to a dedicated table (table 1)

Figure 3A and D: Images quality is very low and higher magnifications are needed

Figure 5: Images quality is very low, negative controls are absent. Instead of "Green-cell membranes", authors must write the probe/protein used. Higher magnifications are also necessary. Moreover, ERa and ERb are nuclear receptors but I see red everywhere. Why authors did not block tissue autofluorescence (e.g. with Sudan Black B)? 

Response: Thank you for the comment and bringing up lack of negative control. We added negative control figures to supplementary material (Figure S1). We also repeated the staining using Sudan Black B to decrease the autofluorescence and it blocked some background (Figure 5). We believe that received results are more specific now. We are highly grateful to the Reviewer for this valuable tip.

Presented data were done at magnification 600x. We also added the name of the dye for cell membrane staining. We used DiOC6 (3,3'-Dihexyloxacarbocyanine Iodide).

An accurate revision of typing errors is necessary

Response: Thank you for the comment. The manuscript was checked for typos.

Abbreviations must be written in full length when mentioned for the first time

Response: Thank you for the comment. We added full names for abbreviations.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Review of the manuscript entitled: Chemical inhibition of NRF2 transcriptional activity influences colon function and oestrogen receptors expression in mice at different age. Currently, environmental pollution is a very serious health problem. Many xenobiotics act as estrogen receptor agonists, so it is very important to know the mechanism of action of these receptors in the digestive system. Moreover, estrogen receptors are responsible for cell proliferation, so it can expand knowledge about the causes of digestive system cancers. The manuscript is interesting and aesthetic but there are many larger and smaller errors that should be corrected.

1.     Half of the abstract is missing in the system. Why? manuscript and data in the system should be identical.

2.     The introduction is written correctly but I have the impression that the authors do not know what the dot is for. e.g. lines 63-69 are one extremely long sentence. Why? There are more such places in the manuscript. Please, use dots and punctuation marks. Shorter sentences are more understandable. Correct the entire manuscript.

3.     The methodology is described in detail, which is a big plus of the work. Unfortunately, I am concerned about the high similarity factor (52%) the longest fragments come from 10.1016/j.freeradbiomed.2022.09.014. If possible please try to modify the methodologies or provide references.

4.     In my opinion the results are described correctly. However, in fig 1 in the description of the axis a reference gene should be added.

5.     Overall the discussion is consistent. Did the authors consider other molecular pathways? It is well known that AhR and PPARγ are in crosstalk with ERs why is this not in the discussion? PPARγ also controls expressions NRF2, this should be in the discussion.

6.     Conclusions should be improved, what is the medical aspect of the research? What does it result from? Please expand the conclusions.

Author Response

Reviewer 2

Review of the manuscript entitled: Chemical inhibition of NRF2 transcriptional activity influences colon function and oestrogen receptors expression in mice at different age. Currently, environmental pollution is a very serious health problem. Many xenobiotics act as estrogen receptor agonists, so it is very important to know the mechanism of action of these receptors in the digestive system. Moreover, estrogen receptors are responsible for cell proliferation, so it can expand knowledge about the causes of digestive system cancers. The manuscript is interesting and aesthetic but there are many larger and smaller errors that should be corrected.

1. Half of the abstract is missing in the system. Why? manuscript and data in the system should be identical.

Response: Thank you for the comment. We apologise for this mistake. The abstract was uploaded twice and in the system window it was complete. Probably there were some problems during draft saving. We hope that this time the abstract uploaded to the system will be complete.

2. The introduction is written correctly but I have the impression that the authors do not know what the dot is for. e.g. lines 63-69 are one extremely long sentence. Why? There are more such places in the manuscript. Please, use dots and punctuation marks. Shorter sentences are more understandable. Correct the entire manuscript.

Response: Thank you for the comment. We shortened some sentences to make them more legible.

 

3. The methodology is described in detail, which is a big plus of the work. Unfortunately, I am concerned about the high similarity factor (52%) the longest fragments come from 10.1016/j.freeradbiomed.2022.09.014. If possible please try to modify the methodologies or provide references.

Response: Thank you for the comment. The same aspect was brought up by the Editor and we corrected methods description. We also added references to some fragments of methodology.

 

4. In my opinion the results are described correctly. However, in fig 1 in the description of the axis a reference gene should be added.

Response: Thank you for the comment. The reference gene name was added to OY axis.

5. Overall the discussion is consistent. Did the authors consider other molecular pathways? It is well known that AhR and PPARγ are in crosstalk with ERs why is this not in the discussion? PPARγ also controls expressions NRF2, this should be in the discussion.

Response: Thank you for this valuable comment. Indeed this pathway may be interesting to understand the oestrogen signalling within the colon tissue. We will consider adding this pathways in our next studies.

We agree that the activation of PPARγ can enhance the antioxidant response by upregulating NRF2 target genes. This interaction is particularly significant in the context of inflammation, metabolic diseases, and oxidative stress-related diseases. Similarly, AhR and PPARγ activation can lead to the modulation of estrogen-responsive genes, however, the interaction between these receptors is rather highlighted in the contexts of adipogenesis, breast cancer progression, and metabolic diseases, but not in the intestinal diseases. Therefore, it is not a common association in the intestinal tissue. Nevertheless, we highlighted indicated crosstalk’s in the discussion part (line 353-361).

 

6. Conclusions should be improved, what is the medical aspect of the research? What does it result from? Please expand the conclusions.

Response: Thank you for this valuable comment. We added clinical implications to the conclusion part (lines 372-377).

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Although some minor improvement, the quality of the article did not chance after revision. 

Author Response

Thank you for your valuable and constructive comments on our manuscript. We appreciate your recognition of our work and your thoughtful suggestions, which have helped us improve the quality of the manuscript. Below are our detailed responses to your comments:

Q: Line 1: Authors must specify the type of paper as showed in the journal template

Response: Thank you for the comment. The article type was added.

Q: Lines 39-45: Since NRF2/KEAP1 signaling plays a key role in this manuscript, the mulstifaceted role of this pathway deserves to be highlighted. In fact, this pathway plays a significant role in the onset and progression of several cancerous and non-cancerous diseases (see PMID: 39456522, PMID: 39034715  , PMID: 39534872). 

Response: Thank you for bringing up this interesting review papers. We added this point to the introduction part indicating some review papers highlighting the pros and cons of NRF2 activation (lines 42-49).

Q: 2.5. Gene expression analysis: Primers sequences should be moved in a dedicate table 

Response: As suggested we moved primers sequence to a dedicated table (table 1).

Q: Figure 3A and D: Images quality is very low and higher magnifications are needed

Response: Thank you for the comment. We apologise for omitting this point in our previous version. It was not done on purpose. As recommended we redone the staining for panel D and made higher magnifications of samples presented in panel A and D.

Q: Figure 5: Images quality is very low, negative controls are absent. Instead of "Green-cell membranes", authors must write the probe/protein used. Higher magnifications are also necessary. Moreover, ERa and ERb are nuclear receptors but I see red everywhere. Why authors did not block tissue autofluorescence (e.g. with Sudan Black B)? 

Response: Thank you for the comment and bringing up lack of negative control. We added negative control figures to supplementary material (Figure S1). We also repeated the staining using Sudan Black B to decrease the autofluorescence and it blocked some background. We created a new figure 5. We hope that received results are more specific now. We are highly grateful to the Reviewer for this valuable tip.

In the new Figure 5 the presented data were done at magnification 600x. We also added the name of the dye for cell membrane staining (DiOC6 (3,3'-Dihexyloxacarbocyanine Iodide)).

An accurate revision of typing errors is necessary

Response: Thank you for the comment. The manuscript was checked for typos and errors were corrected using the British English language.

Abbreviations must be written in full length when mentioned for the first time

Response: Thank you for the comment. We added full names for abbreviations which we located in the manuscript.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

The quality of the manuscript has not be improved since IHC images are blurry.