Reviewer ’s report  
 
Title: Uridine stimulate laxative effect in the loperamide -induced constipation of SD rats 
through  the regulation of mAChRs signaling pathway and mucin secretion  
 
Version: 0 Date: 10 Jul 2016  
 
Reviewer: Andy Kuo  
 
Reviewer's report:  
 
This is an intere sting paper that investigated one of the major clinical problems wrt opioid 
induced constipation using uridine in the loperamide -induced constipation rat model.  The 
Authors have presented novel findings with some of the underlying mechanisms of uridine in  the 
alleviation of loperamide -induced constipation. There are however, several issues that the 
authors need to address. Specifically, the methods, summary of the findings, discussion and the 
flow of the manuscript can be improved.   
 
 
 
 
1. Page 3, line 56: "100 mg/ml of Urd" is that correct? On the page 8, line 162 stated "100 
mg/kg weight of Urd".  
 
 
 
2. What is the route of administration for the uridine? Reviewer was unable to locate that 
information throughout the entire manuscript. Also Was urdin e administered as a single 
bolus injection or as infusion?  
 
 
 
3. In author/s previous publication "Metabolomics approach to serum biomarker for 
loperamide -induced constipation in SD rats" published in 2014 under "Inducti on of 
constipation" stated "….. injection of loperamide (1.5 mg/kg weight) in 0.9% sodium 
chloride twice a day for 3 days" (however, 4 mg/kg was mentioned in the discussion 
section?). This  is different to what stated in this manuscript whereby 4 mg/kg of 
loperamide was given.     
 
Reviewe r curious why does the dose changed (or how was the dose selected, as this is 
higher than references cited in the paper published in 2014).  
 
 
4. For the Western Blotting, the protein estimation and the amount of protein loaded into the 
gel was not men tioned in this manuscript. Also, on the Figures 4 and 6, the actin control 
clearly showed that the proteins were overloaded. This raised concerns where the 
differences shown are due to the treatment and/or due to the different amount of proteins 
been loade d.  
 
 
5. Page 13, lines 298 -299, sentence need to be re -written.  
 
 
6. Scale bar should be included in the relevant figures e.g. Figure 1 histological structure.  
 
 
7. Reviewer is unable to locate S1 as per referenced on the page 16, line 345. Or this is a 
typographical error which meant to be refereed to Table 2?  
 
 
8. Figure 2, reviewer would like to know why only number of fat droplets (involved in 
stimul ation of defecation) and paneth cells (secret lysozyme) were quantified but not 
enterocytes (regulation of water and electrolytes) and goblet cells (regulation of mucus)?   
 
 
9. The numerical values on the Y -axis of Figure 2B are incorrect, likely masked by the 
adjacent text.  
 
 
 
10. Page 20, lines 446 -447, what are the high/low affinity receptors that authors referring to? 
Based on the two cited references, reviewer thinks that "nucleoside carrier/s or nucleoside 
transporter/s" should be used instead of "receptor/s".  
 
 
11. Page 21, line 456, sentence need to be re -written.   
 
 
12. The choice of assessing downstream signalling pathways e.g. IP3 (presumably as a 
measurement for the gastrointestinal smooth muscle contraction) and the relevant 
physiologica l implications on the constipation did not specifically mentioned, discussed 
and summarised. It is likely that due to the word limitation the authors decided to leave 
this information out of the manuscript. It is however, in reviewer's opinion a summary 
table will greatly aid the readers to fully realise the implication of this study.  
 
 
13. Based on authors' previous papers, it seems plausible that uridine rescued the 
loperamide( -)induced constipation by interfering with the balance of metabolites such as  
alanine, glycerol, choline and phosphocholine. It will be ideal if authors can write a small 
paragraph to discuss this.  
 
 
Are the methods appropriate and well described?  
If no t, please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the co nclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistica l review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, fu nding, or salary from an  
organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially fro m the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization th at 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
If you can answer no to all of the above,  write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will b e included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses. I agree for my report to be made available under an Open Access Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as  confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Reviewer ’s report  
 
Title: Uridine stimulate laxative effect in the loperamide -induced constipation of SD rats 
through  the regulation of mAChRs signaling pathway and mucin secretion  
 
Version: 0 Date: 10 Jul 2016  
 
Reviewer: Andy Kuo  
 
Reviewer's report:  
 
This is an intere sting paper that investigated one of the major clinical problems wrt opioid 
induced constipation using uridine in the loperamide -induced constipation rat model.  The 
Authors have presented novel findings with some of the underlying mechanisms of uridine in  the 
alleviation of loperamide -induced constipation. There are however, several issues that the 
authors need to address. Specifically, the methods, summary of the findings, discussion and the 
flow of the manuscript can be improved.   
 
 
 
 
1. Page 3, line 56: "100 mg/ml of Urd" is that correct? On the page 8, line 162 stated "100 
mg/kg weight of Urd".  
 
 
 
2. What is the route of administration for the uridine? Reviewer was unable to locate that 
information throughout the entire manuscript. Also Was urdin e administered as a single 
bolus injection or as infusion?  
 
 
 
3. In author/s previous publication "Metabolomics approach to serum biomarker for 
loperamide -induced constipation in SD rats" published in 2014 under "Inducti on of 
constipation" stated "….. injection of loperamide (1.5 mg/kg weight) in 0.9% sodium 
chloride twice a day for 3 days" (however, 4 mg/kg was mentioned in the discussion 
section?). This  is different to what stated in this manuscript whereby 4 mg/kg of 
loperamide was given.     
 
Reviewe r curious why does the dose changed (or how was the dose selected, as this is 
higher than references cited in the paper published in 2014).  
 
 
4. For the Western Blotting, the protein estimation and the amount of protein loaded into the 
gel was not men tioned in this manuscript. Also, on the Figures 4 and 6, the actin control 
clearly showed that the proteins were overloaded. This raised concerns where the 
differences shown are due to the treatment and/or due to the different amount of proteins 
been loade d.  
 
 
5. Page 13, lines 298 -299, sentence need to be re -written.  
 
 
6. Scale bar should be included in the relevant figures e.g. Figure 1 histological structure.  
 
 
7. Reviewer is unable to locate S1 as per referenced on the page 16, line 345. Or this is a 
typographical error which meant to be refereed to Table 2?  
 
 
8. Figure 2, reviewer would like to know why only number of fat droplets (involved in 
stimul ation of defecation) and paneth cells (secret lysozyme) were quantified but not 
enterocytes (regulation of water and electrolytes) and goblet cells (regulation of mucus)?   
 
 
9. The numerical values on the Y -axis of Figure 2B are incorrect, likely masked by the 
adjacent text.  
 
 
 
10. Page 20, lines 446 -447, what are the high/low affinity receptors that authors referring to? 
Based on the two cited references, reviewer thinks that "nucleoside carrier/s or nucleoside 
transporter/s" should be used instead of "receptor/s".  
 
 
11. Page 21, line 456, sentence need to be re -written.   
 
 
12. The choice of assessing downstream signalling pathways e.g. IP3 (presumably as a 
measurement for the gastrointestinal smooth muscle contraction) and the relevant 
physiologica l implications on the constipation did not specifically mentioned, discussed 
and summarised. It is likely that due to the word limitation the authors decided to leave 
this information out of the manuscript. It is however, in reviewer's opinion a summary 
table will greatly aid the readers to fully realise the implication of this study.  
 
 
13. Based on authors' previous papers, it seems plausible that uridine rescued the 
loperamide( -)induced constipation by interfering with the balance of metabolites such as  
alanine, glycerol, choline and phosphocholine. It will be ideal if authors can write a small 
paragraph to discuss this.  
 
 
Are the methods appropriate and well described?  
If no t, please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the co nclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistica l review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, fu nding, or salary from an  
organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially fro m the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization th at 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
If you can answer no to all of the above,  write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will b e included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses. I agree for my report to be made available under an Open Access Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as  confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Please review this paper.
user
Reviewer ’s report  
 
Title: Uridine stimulate laxative effect in the loperamide -induced constipation of SD rats 
through  the regulation of mAChRs signaling pathway and mucin secretion  
 
Version: 0 Date: 10 Jul 2016  
 
Reviewer: Andy Kuo  
 
Reviewer's report:  
 
This is an intere sting paper that investigated one of the major clinical problems wrt opioid 
induced constipation using uridine in the loperamide -induced constipation rat model.  The 
Authors have presented novel findings with some of the underlying mechanisms of uridine in  the 
alleviation of loperamide -induced constipation. There are however, several issues that the 
authors need to address. Specifically, the methods, summary of the findings, discussion and the 
flow of the manuscript can be improved.   
 
 
 
 
1. Page 3, line 56: "100 mg/ml of Urd" is that correct? On the page 8, line 162 stated "100 
mg/kg weight of Urd".  
 
 
 
2. What is the route of administration for the uridine? Reviewer was unable to locate that 
information throughout the entire manuscript. Also Was urdin e administered as a single 
bolus injection or as infusion?  
 
 
 
3. In author/s previous publication "Metabolomics approach to serum biomarker for 
loperamide -induced constipation in SD rats" published in 2014 under "Inducti on of 
constipation" stated "….. injection of loperamide (1.5 mg/kg weight) in 0.9% sodium 
chloride twice a day for 3 days" (however, 4 mg/kg was mentioned in the discussion 
section?). This  is different to what stated in this manuscript whereby 4 mg/kg of 
loperamide was given.     
 
Reviewe r curious why does the dose changed (or how was the dose selected, as this is 
higher than references cited in the paper published in 2014).  
 
 
4. For the Western Blotting, the protein estimation and the amount of protein loaded into the 
gel was not men tioned in this manuscript. Also, on the Figures 4 and 6, the actin control 
clearly showed that the proteins were overloaded. This raised concerns where the 
differences shown are due to the treatment and/or due to the different amount of proteins 
been loade d.  
 
 
5. Page 13, lines 298 -299, sentence need to be re -written.  
 
 
6. Scale bar should be included in the relevant figures e.g. Figure 1 histological structure.  
 
 
7. Reviewer is unable to locate S1 as per referenced on the page 16, line 345. Or this is a 
typographical error which meant to be refereed to Table 2?  
 
 
8. Figure 2, reviewer would like to know why only number of fat droplets (involved in 
stimul ation of defecation) and paneth cells (secret lysozyme) were quantified but not 
enterocytes (regulation of water and electrolytes) and goblet cells (regulation of mucus)?   
 
 
9. The numerical values on the Y -axis of Figure 2B are incorrect, likely masked by the 
adjacent text.  
 
 
 
10. Page 20, lines 446 -447, what are the high/low affinity receptors that authors referring to? 
Based on the two cited references, reviewer thinks that "nucleoside carrier/s or nucleoside 
transporter/s" should be used instead of "receptor/s".  
 
 
11. Page 21, line 456, sentence need to be re -written.   
 
 
12. The choice of assessing downstream signalling pathways e.g. IP3 (presumably as a 
measurement for the gastrointestinal smooth muscle contraction) and the relevant 
physiologica l implications on the constipation did not specifically mentioned, discussed 
and summarised. It is likely that due to the word limitation the authors decided to leave 
this information out of the manuscript. It is however, in reviewer's opinion a summary 
table will greatly aid the readers to fully realise the implication of this study.  
 
 
13. Based on authors' previous papers, it seems plausible that uridine rescued the 
loperamide( -)induced constipation by interfering with the balance of metabolites such as  
alanine, glycerol, choline and phosphocholine. It will be ideal if authors can write a small 
paragraph to discuss this.  
 
 
Are the methods appropriate and well described?  
If no t, please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the co nclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistica l review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, fu nding, or salary from an  
organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially fro m the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization th at 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
If you can answer no to all of the above,  write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will b e included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses. I agree for my report to be made available under an Open Access Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as  confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant