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: 2 Date: 05 Sep 2016  
 
Reviewer: Andy Kuo  
 
Reviewer's report:  
 
Reviewer #2  
 
This is an interesting pap er that investigated one of the major clinical problems with 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, line56: "100 mg/ml of Urd" is that correct? On the page 8, line 162 stated "100 mg/kg 
weight of Urd".  
 
☞ This has been corrected at line 165.  
  
Reviewer 2: Thank you for the correction.  
 
 
 
2. What is the route of administration for the uridine? Reviewer was unable to locat e that 
information throughout the entire manuscript. Also Was urdine administered as a single bolus 
injection or as infusion?  
 
☞ We have inserted above information at line 164 -166.  
 
Reviewer 2: Thank you for the additional information, however, the route of administration for 
uridine is still missing. Please add this to the method section.   
 
 
 
3. In author/s previous publication "Metabolomics approach to serum biomarker for loperamid e-
induced constipation in SD rats" published in 2014 under "Induction 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.  
Reviewer 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).  
 
☞ This is good poi nt. Actually, in the initial study, we used sodium chloride as solvent to 
dissolve loperamide. However, we selected a different type solvent because this process is 
complex and tricky. Therefore, in this study, 0.5% tween 20 solution has been selected a be st 
solvent for loperamide.  
 
Reviewer 2: Thank you for the clarification. Please add the vehicle 0.5% Tween 20 in saline(?) 
to your method section.  
  
 
 
4. For the Western Blotting, the protein estimation and the amount of protein loaded into the gel 
was n ot mentioned 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  loaded.  
 
☞ According to your comments, we have inserted some paragraph for the protein estimation and 
the amount of protein loaded into the gel at line 188 -192 as follows;  
"Total proteins were extracted from the transverse colons of subset groups (No, U rd, 
Lop+Vehicle and Lop+Urd treated SD rats) and pRISMCs using Pro -Prep Protein Extraction 
Solution (Intron Biotechnology, Inc., Seongnam, Korea). Following centrifugation at 13,000 rpm 
and 4°C for 5 min, the protein concentrations were determined using a SMARTTM 
-----
"  
 
Reviewer 2: Thank you for the additional information.  
 
 
 
5. Page 13, lines 298 -299, sentence need to be re -written.  
 
☞ These have been corrected  at line 279 -280 as follows;  
"Among the two markers, only Myh11, indicating myosin -smooth muscle cells, was detected at 
high levels, while PGP9.5, indicating neuronal cells, was found at an extremely low level"  
 
Reviewer 2: Thank you for the additional i nformation.  
 
 
 
6. Scale bar should be included in the relevant figures e.g. Figure 1 histological structure.  
 
☞ This has been corrected.  
Reviewer 2: Thank you.  
 
 
 
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?  
 
☞ This is our mistake. We have corrected.  
 
Reviewer 2: Thank you.  
 
 
 
8. Figure 2, reviewer would like  to know why only number of fat droplets (involved in 
stimulation of defecation) and paneth cells (secret lysozyme) were quantified but not enterocytes 
(regulation of water and electrolytes) and goblet cells (regulation of mucus)?  
 
☞ According to your com ments, we have inserted the number of enterocytes and goblet cells 
into Figure 2. Also, the number of enterocytes have been inserted into Table 2.  
 
Reviewer 2: Thank you for the additional information.  
 
 
 
9. The numerical values on the Y -axis of Figure 2 B are incorrect, likely masked by the adjacent 
text.  
 
☞ This has been corrected.  
 
Reviewer 2: Thank you.  
 
 
 
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".  
 
☞ They have  been corrected at line 404 -405.  
 
Reviewer 2: Thank you.  
 
 
 
11. Page 21, line 456, sentence need to be re -written.  
 
☞ They have been corrected at 413 -415 as follows;  
"Although the above studies suggested that Urd had potential for use as a laxative com pound, 
none provided scientific evidence of its therapeutic effects on chronic constipation."  
 
Reviewer 2: There are discrepancies between the paragraph provided above and that in the 
revised manuscript. Reviewer believes that the paragraph in the revised  manuscript should be 
change to: "Although the above studies suggested that Urd can be potentially used as a laxative 
compound, there are however, no direct scientific evidence on the therapeutic effect of Urd for 
chronic constipation."  
 
 
 
12. The choice of assessing downstream signalling pathways e.g. IP3 (presumably as a 
measurement for the gastrointestinal smooth muscle contraction) and the relevant physiological 
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.  
 
☞ Accordin g to your comments, we have inserted a paragraph for IP3 and the relevant 
physiological implications on the constipation into Discussion part at 446 -455 as follows;  
"IP3 can also be considered one of the factors related to bowel movements because it induc es 
increases in intracellular Ca2+ levels through a release from the sarcoplasmic reticulum (33). 
Smooth muscle tone in the intestine is controlled by phosphorylation of the regulatory light chain 
(MLC) of myosin II. Ca2+/CaM -dependent myosin light chain k inase and myosin phosphatase 
regulated this process to control the velocity and force of actomyosin cross -bridging (34). 
Therefore, we measured the IP3 level in pRISMC to investigate the effects of Urd response to 
Lop treatment. The results of the present study suggest that Urd may play an important role in 
pRISMCs collected from the small intestine of infant rats treated with Lop and that its effects are 
tightly correlated with those of laxatives that improve chronic constipation."  
 
Reviewer 2: There are discrepancies between the paragraphs provided above and that in the 
revised manuscript. Reviewer believes that the paragraphs provided above should be use instead 
the one currently in the revised manuscript.  
 
 
 
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.  
 
☞ According to your comments, we have inserted a paragraph for the role of loperamide on the 
balance of metabolites at 483 -490 as follows;  
"Alterations in endogenous metabolites were also examined in the serum of Lop -induced 
constipation rats. Among 35 endogenous metabolites, four amino acids (alanine, glutamate, 
glutamine and glycine) and six endogenous metabolites related to the glycolysis pathway 
(acetate, glucose, glycerol, lactate, succinate and taurine) were dram atically decreased after Lop 
treatment [52]. These results provide a possibility that Urd can rescue the Lop -induced 
constipation by interfering with the balance of metabolites because Urd plays a role in the 
glycolysis pathway of galactose [53]. However, additional studies are needed to determine if this 
is the case."  
 
Reviewer 2: There are discrepancies between the paragraphs provided above and that in the 
revised manuscript. Reviewer believes that the paragraphs provided above should be use instead 
of the one currently in the revised manuscript.  
 
 
 
 
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: 2 Date: 05 Sep 2016  
 
Reviewer: Andy Kuo  
 
Reviewer's report:  
 
Reviewer #2  
 
This is an interesting pap er that investigated one of the major clinical problems with 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, line56: "100 mg/ml of Urd" is that correct? On the page 8, line 162 stated "100 mg/kg 
weight of Urd".  
 
☞ This has been corrected at line 165.  
  
Reviewer 2: Thank you for the correction.  
 
 
 
2. What is the route of administration for the uridine? Reviewer was unable to locat e that 
information throughout the entire manuscript. Also Was urdine administered as a single bolus 
injection or as infusion?  
 
☞ We have inserted above information at line 164 -166.  
 
Reviewer 2: Thank you for the additional information, however, the route of administration for 
uridine is still missing. Please add this to the method section.   
 
 
 
3. In author/s previous publication "Metabolomics approach to serum biomarker for loperamid e-
induced constipation in SD rats" published in 2014 under "Induction 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.  
Reviewer 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).  
 
☞ This is good poi nt. Actually, in the initial study, we used sodium chloride as solvent to 
dissolve loperamide. However, we selected a different type solvent because this process is 
complex and tricky. Therefore, in this study, 0.5% tween 20 solution has been selected a be st 
solvent for loperamide.  
 
Reviewer 2: Thank you for the clarification. Please add the vehicle 0.5% Tween 20 in saline(?) 
to your method section.  
  
 
 
4. For the Western Blotting, the protein estimation and the amount of protein loaded into the gel 
was n ot mentioned 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  loaded.  
 
☞ According to your comments, we have inserted some paragraph for the protein estimation and 
the amount of protein loaded into the gel at line 188 -192 as follows;  
"Total proteins were extracted from the transverse colons of subset groups (No, U rd, 
Lop+Vehicle and Lop+Urd treated SD rats) and pRISMCs using Pro -Prep Protein Extraction 
Solution (Intron Biotechnology, Inc., Seongnam, Korea). Following centrifugation at 13,000 rpm 
and 4°C for 5 min, the protein concentrations were determined using a SMARTTM 
-----
"  
 
Reviewer 2: Thank you for the additional information.  
 
 
 
5. Page 13, lines 298 -299, sentence need to be re -written.  
 
☞ These have been corrected  at line 279 -280 as follows;  
"Among the two markers, only Myh11, indicating myosin -smooth muscle cells, was detected at 
high levels, while PGP9.5, indicating neuronal cells, was found at an extremely low level"  
 
Reviewer 2: Thank you for the additional i nformation.  
 
 
 
6. Scale bar should be included in the relevant figures e.g. Figure 1 histological structure.  
 
☞ This has been corrected.  
Reviewer 2: Thank you.  
 
 
 
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?  
 
☞ This is our mistake. We have corrected.  
 
Reviewer 2: Thank you.  
 
 
 
8. Figure 2, reviewer would like  to know why only number of fat droplets (involved in 
stimulation of defecation) and paneth cells (secret lysozyme) were quantified but not enterocytes 
(regulation of water and electrolytes) and goblet cells (regulation of mucus)?  
 
☞ According to your com ments, we have inserted the number of enterocytes and goblet cells 
into Figure 2. Also, the number of enterocytes have been inserted into Table 2.  
 
Reviewer 2: Thank you for the additional information.  
 
 
 
9. The numerical values on the Y -axis of Figure 2 B are incorrect, likely masked by the adjacent 
text.  
 
☞ This has been corrected.  
 
Reviewer 2: Thank you.  
 
 
 
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".  
 
☞ They have  been corrected at line 404 -405.  
 
Reviewer 2: Thank you.  
 
 
 
11. Page 21, line 456, sentence need to be re -written.  
 
☞ They have been corrected at 413 -415 as follows;  
"Although the above studies suggested that Urd had potential for use as a laxative com pound, 
none provided scientific evidence of its therapeutic effects on chronic constipation."  
 
Reviewer 2: There are discrepancies between the paragraph provided above and that in the 
revised manuscript. Reviewer believes that the paragraph in the revised  manuscript should be 
change to: "Although the above studies suggested that Urd can be potentially used as a laxative 
compound, there are however, no direct scientific evidence on the therapeutic effect of Urd for 
chronic constipation."  
 
 
 
12. The choice of assessing downstream signalling pathways e.g. IP3 (presumably as a 
measurement for the gastrointestinal smooth muscle contraction) and the relevant physiological 
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.  
 
☞ Accordin g to your comments, we have inserted a paragraph for IP3 and the relevant 
physiological implications on the constipation into Discussion part at 446 -455 as follows;  
"IP3 can also be considered one of the factors related to bowel movements because it induc es 
increases in intracellular Ca2+ levels through a release from the sarcoplasmic reticulum (33). 
Smooth muscle tone in the intestine is controlled by phosphorylation of the regulatory light chain 
(MLC) of myosin II. Ca2+/CaM -dependent myosin light chain k inase and myosin phosphatase 
regulated this process to control the velocity and force of actomyosin cross -bridging (34). 
Therefore, we measured the IP3 level in pRISMC to investigate the effects of Urd response to 
Lop treatment. The results of the present study suggest that Urd may play an important role in 
pRISMCs collected from the small intestine of infant rats treated with Lop and that its effects are 
tightly correlated with those of laxatives that improve chronic constipation."  
 
Reviewer 2: There are discrepancies between the paragraphs provided above and that in the 
revised manuscript. Reviewer believes that the paragraphs provided above should be use instead 
the one currently in the revised manuscript.  
 
 
 
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.  
 
☞ According to your comments, we have inserted a paragraph for the role of loperamide on the 
balance of metabolites at 483 -490 as follows;  
"Alterations in endogenous metabolites were also examined in the serum of Lop -induced 
constipation rats. Among 35 endogenous metabolites, four amino acids (alanine, glutamate, 
glutamine and glycine) and six endogenous metabolites related to the glycolysis pathway 
(acetate, glucose, glycerol, lactate, succinate and taurine) were dram atically decreased after Lop 
treatment [52]. These results provide a possibility that Urd can rescue the Lop -induced 
constipation by interfering with the balance of metabolites because Urd plays a role in the 
glycolysis pathway of galactose [53]. However, additional studies are needed to determine if this 
is the case."  
 
Reviewer 2: There are discrepancies between the paragraphs provided above and that in the 
revised manuscript. Reviewer believes that the paragraphs provided above should be use instead 
of the one currently in the revised manuscript.  
 
 
 
 
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: 2 Date: 05 Sep 2016  
 
Reviewer: Andy Kuo  
 
Reviewer's report:  
 
Reviewer #2  
 
This is an interesting pap er that investigated one of the major clinical problems with 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, line56: "100 mg/ml of Urd" is that correct? On the page 8, line 162 stated "100 mg/kg 
weight of Urd".  
 
☞ This has been corrected at line 165.  
  
Reviewer 2: Thank you for the correction.  
 
 
 
2. What is the route of administration for the uridine? Reviewer was unable to locat e that 
information throughout the entire manuscript. Also Was urdine administered as a single bolus 
injection or as infusion?  
 
☞ We have inserted above information at line 164 -166.  
 
Reviewer 2: Thank you for the additional information, however, the route of administration for 
uridine is still missing. Please add this to the method section.   
 
 
 
3. In author/s previous publication "Metabolomics approach to serum biomarker for loperamid e-
induced constipation in SD rats" published in 2014 under "Induction 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.  
Reviewer 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).  
 
☞ This is good poi nt. Actually, in the initial study, we used sodium chloride as solvent to 
dissolve loperamide. However, we selected a different type solvent because this process is 
complex and tricky. Therefore, in this study, 0.5% tween 20 solution has been selected a be st 
solvent for loperamide.  
 
Reviewer 2: Thank you for the clarification. Please add the vehicle 0.5% Tween 20 in saline(?) 
to your method section.  
  
 
 
4. For the Western Blotting, the protein estimation and the amount of protein loaded into the gel 
was n ot mentioned 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  loaded.  
 
☞ According to your comments, we have inserted some paragraph for the protein estimation and 
the amount of protein loaded into the gel at line 188 -192 as follows;  
"Total proteins were extracted from the transverse colons of subset groups (No, U rd, 
Lop+Vehicle and Lop+Urd treated SD rats) and pRISMCs using Pro -Prep Protein Extraction 
Solution (Intron Biotechnology, Inc., Seongnam, Korea). Following centrifugation at 13,000 rpm 
and 4°C for 5 min, the protein concentrations were determined using a SMARTTM 
-----
"  
 
Reviewer 2: Thank you for the additional information.  
 
 
 
5. Page 13, lines 298 -299, sentence need to be re -written.  
 
☞ These have been corrected  at line 279 -280 as follows;  
"Among the two markers, only Myh11, indicating myosin -smooth muscle cells, was detected at 
high levels, while PGP9.5, indicating neuronal cells, was found at an extremely low level"  
 
Reviewer 2: Thank you for the additional i nformation.  
 
 
 
6. Scale bar should be included in the relevant figures e.g. Figure 1 histological structure.  
 
☞ This has been corrected.  
Reviewer 2: Thank you.  
 
 
 
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?  
 
☞ This is our mistake. We have corrected.  
 
Reviewer 2: Thank you.  
 
 
 
8. Figure 2, reviewer would like  to know why only number of fat droplets (involved in 
stimulation of defecation) and paneth cells (secret lysozyme) were quantified but not enterocytes 
(regulation of water and electrolytes) and goblet cells (regulation of mucus)?  
 
☞ According to your com ments, we have inserted the number of enterocytes and goblet cells 
into Figure 2. Also, the number of enterocytes have been inserted into Table 2.  
 
Reviewer 2: Thank you for the additional information.  
 
 
 
9. The numerical values on the Y -axis of Figure 2 B are incorrect, likely masked by the adjacent 
text.  
 
☞ This has been corrected.  
 
Reviewer 2: Thank you.  
 
 
 
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".  
 
☞ They have  been corrected at line 404 -405.  
 
Reviewer 2: Thank you.  
 
 
 
11. Page 21, line 456, sentence need to be re -written.  
 
☞ They have been corrected at 413 -415 as follows;  
"Although the above studies suggested that Urd had potential for use as a laxative com pound, 
none provided scientific evidence of its therapeutic effects on chronic constipation."  
 
Reviewer 2: There are discrepancies between the paragraph provided above and that in the 
revised manuscript. Reviewer believes that the paragraph in the revised  manuscript should be 
change to: "Although the above studies suggested that Urd can be potentially used as a laxative 
compound, there are however, no direct scientific evidence on the therapeutic effect of Urd for 
chronic constipation."  
 
 
 
12. The choice of assessing downstream signalling pathways e.g. IP3 (presumably as a 
measurement for the gastrointestinal smooth muscle contraction) and the relevant physiological 
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.  
 
☞ Accordin g to your comments, we have inserted a paragraph for IP3 and the relevant 
physiological implications on the constipation into Discussion part at 446 -455 as follows;  
"IP3 can also be considered one of the factors related to bowel movements because it induc es 
increases in intracellular Ca2+ levels through a release from the sarcoplasmic reticulum (33). 
Smooth muscle tone in the intestine is controlled by phosphorylation of the regulatory light chain 
(MLC) of myosin II. Ca2+/CaM -dependent myosin light chain k inase and myosin phosphatase 
regulated this process to control the velocity and force of actomyosin cross -bridging (34). 
Therefore, we measured the IP3 level in pRISMC to investigate the effects of Urd response to 
Lop treatment. The results of the present study suggest that Urd may play an important role in 
pRISMCs collected from the small intestine of infant rats treated with Lop and that its effects are 
tightly correlated with those of laxatives that improve chronic constipation."  
 
Reviewer 2: There are discrepancies between the paragraphs provided above and that in the 
revised manuscript. Reviewer believes that the paragraphs provided above should be use instead 
the one currently in the revised manuscript.  
 
 
 
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.  
 
☞ According to your comments, we have inserted a paragraph for the role of loperamide on the 
balance of metabolites at 483 -490 as follows;  
"Alterations in endogenous metabolites were also examined in the serum of Lop -induced 
constipation rats. Among 35 endogenous metabolites, four amino acids (alanine, glutamate, 
glutamine and glycine) and six endogenous metabolites related to the glycolysis pathway 
(acetate, glucose, glycerol, lactate, succinate and taurine) were dram atically decreased after Lop 
treatment [52]. These results provide a possibility that Urd can rescue the Lop -induced 
constipation by interfering with the balance of metabolites because Urd plays a role in the 
glycolysis pathway of galactose [53]. However, additional studies are needed to determine if this 
is the case."  
 
Reviewer 2: There are discrepancies between the paragraphs provided above and that in the 
revised manuscript. Reviewer believes that the paragraphs provided above should be use instead 
of the one currently in the revised manuscript.  
 
 
 
 
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