Reviewer’ s report
Title: ROLE OF SEX HORMONES AND THEIR RECEPTORS ON GASTRIC NRF2 AND
NEURONAL NITRIC OXIDE SYNTHASE FUNCTION IN AN EXPERIMENTAL HYPER-GLYCEMIA MODEL
Version: 1Date: 31 Jan 2020
Reviewer: Brian A Perrino
Reviewer's report:
This study adds interesting and important information to be considered when trying to examine
and understand the contributions of hyperglycemia to the gastric dysmotility associated withdiabetic gastroparesis.Since the underlying mechanisms of gastric dysmotility are poorly understood, the approach ofthis study to work with experimental hyperglycemic conditions will help to establish translationalrelevance.The present study follows up on previous studies showing that loss of Interstitial Cells of Cajal(ICC), neuronal nitric oxide synthase (nNOS) function, and elevated oxidative stress are hallmarksof diabetic gastroparesis, and that results from their previous in-vivo studies have demonstratedthat nNOS-mediated gastric motility is greater in healthy female compared to male rodents.Furthermore, diabetes induction impaired nNOSα activity in female, but not in male in stomach
and duodenum. Other studies have shown that supplementation of 17 β-estradiol (E2) increases
nNOS expression and nitrergic-mediated gastric motility in ovariectomized rats. E2 has multiplebeneficial actions that include neuroprotection, maintaining glucose homeostasis, and activation ofNO synthesis in vascular smooth muscle. Also, Nuclear factor (erythroid-derived 2)-like 2 (Nrf2),
an antioxidant-responsive transcription factor, has emerged as a potent target for alleviating
clinical manifestations of diabetes mellitus (DM), in addition to its regulation of gastric nitrergicfunction.Taken together, the objective of this study is to investigate whether selective activation of gastricestrogen receptors improves gastric Nrf2-nNOS expression in an in-vitro experimentalhyperglycemic model.
Overall, the results are clear and support the conclusions of each experiment; namely that in NG
conditions, ER antagonist, but not PR antagonist inhibit NO relaxation, and ER antagonist, but notPR antagonist reduce nNOSa, Nrf2, DHFR, and ERa/b mRNA levels. Also, nNOSa, Nrf2, GCH-1, and ERa , protein expression were reduced; while ERb expression was upregulated. In HGmedium, ER agonists restored the impaired NO relaxation from HG medium, and restored the HG-induced reduction in mRNA levels and protein expression of nNOS, DHFR, Nrf2, ERa and ERb.NO enzymatic activity was assayed by nitrite production to show that enzyme activity is correlatedwith protein expression. Mannitol was used as the osmotic control for high glucose to eliminatethe osmotic variable as having an effect.
Major concern:
Previous findings showing co-expression of ERa and ERb in enteric neurons indicates that
estrogenic effects could be mediated through neurogenic reflexes. So this study utilized EFS underNANC conditions to stimulate neural release of NO and evoke nitrergic relaxations of the gastricmuscle strips. However, it might strengthen the conclusions that estrogenic effects are mediatedthrough neurogenic reflexes by examining an exogenous NO donor such as SNP. For example,neural NO relaxation was inhibited by HG; and restored by ER agonists. However, in the presenceof TTx, SNP will deliver NO to post-junctional cells; since this does not rely upon nNOS,relaxation may not be inhibited, and ER agonists may not have effects; but if relaxation evoked bySNP is inhibited by HG would suggest some myogenic component is disrupted by HG, and not all
the effects of HG on nitrergic relaxation are mediated by disruptions to nNOS.
Minor concerns:
The results show that HG reduced Era but not ERb; and that (Fig 6G) ERβ expression was
significantly upregulated in hyperglycemia, but restored by PPT.
Could potential explanations for these observations be addressed in the Discussion?On p 9 in the Methods it is stated that Electric field stimulation (EFS)-induced NANC relaxation
was studied in circular gastric neuromuscular strips [5,6}However, Reference 5 is about identify patient perceptions regarding the impact of gastroparesison managing their diabetes. It does not contain methods on carrying out in vitro smooth muscletension experiments.
Reference 6 is a review, that was published in 1999, not 2018, and also does not contain methods
on carrying out in vitro smooth muscle tension experiments.Please provide the correct references.
p10 Organ Bath Studies
Please indicate whether the mucosa and submucose removed from the muscle strips?And also, please indicate which anatomical region of the stomach these strips are from.
p10
Comparisons between the groups will be performed by measuring the area under thecurve (AUC) of the EFS-induced relaxation (AUCR) for 1 min and the baseline for 1 min(AUCB), according to the formula: (AUCR-AUCB)/weight of the tissue (mg) = AUC/mg tissue.
"will be" is a typop11 Were the mucosa and submucosa removed prior to western blot analysis? If not, how do you
know where ERa, ERb, DHFR, Nrf2, nNOS, and HO-1 are expressed?
p11
Please provide information on the instrumentation and software used to collect and analyze the
images.
p12- 13
Non-selective blockade of gastric ERs with ICI 182,780 reduces gastric (Fig 2A) nNOS, (Fig2B) Nrf2, (Fig 2C) GCH-1, (Fig 2D) DHFR and (Fig 2E-F) estrogen receptor (a/b) mRNAexpression. No measurable effect of RU-486, the progesteronereceptor antagonist, was observed when compared to DMEM (NG) control group. In addition,nochanges were observed with GCH-1 with either group.
Fig2C and the next sentence demonstrate that GCH-1 mRNA levels are not changed.
Are the methods appropriate and well described?
If not, please specify what is required in your comments to the authors.
YesDoes the work include the necessary controls?
If not, please specify which controls are required in your comments to the authors.
YesAre the conclusions 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 statistical review is recommended, please specify what aspects require furtherassessment in your comments to the editors.
I am able to assess the statisticsQuality of written English
Please indicate the quality of language in the manuscript:
AcceptableDeclaration 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, funding, 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 from 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 that 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 be included
on my report to the authors and, if the manuscript is accepted for publication, my named reportincluding 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 CommonsCC-BY license (http://creativecommons.org/licenses/by/4.0/). I understand that any commentswhich I do not wish to be included in my named report can be included as confidential commentsto the editors, which will not be published.
I agree to the open peer review policy of the journal
Reviewer’ s report
Title: ROLE OF SEX HORMONES AND THEIR RECEPTORS ON GASTRIC NRF2 AND
NEURONAL NITRIC OXIDE SYNTHASE FUNCTION IN AN EXPERIMENTAL HYPER-GLYCEMIA MODEL
Version: 1Date: 31 Jan 2020
Reviewer: Brian A Perrino
Reviewer's report:
This study adds interesting and important information to be considered when trying to examine
and understand the contributions of hyperglycemia to the gastric dysmotility associated withdiabetic gastroparesis.Since the underlying mechanisms of gastric dysmotility are poorly understood, the approach ofthis study to work with experimental hyperglycemic conditions will help to establish translationalrelevance.The present study follows up on previous studies showing that loss of Interstitial Cells of Cajal(ICC), neuronal nitric oxide synthase (nNOS) function, and elevated oxidative stress are hallmarksof diabetic gastroparesis, and that results from their previous in-vivo studies have demonstratedthat nNOS-mediated gastric motility is greater in healthy female compared to male rodents.Furthermore, diabetes induction impaired nNOSα activity in female, but not in male in stomach
and duodenum. Other studies have shown that supplementation of 17 β-estradiol (E2) increases
nNOS expression and nitrergic-mediated gastric motility in ovariectomized rats. E2 has multiplebeneficial actions that include neuroprotection, maintaining glucose homeostasis, and activation ofNO synthesis in vascular smooth muscle. Also, Nuclear factor (erythroid-derived 2)-like 2 (Nrf2),
an antioxidant-responsive transcription factor, has emerged as a potent target for alleviating
clinical manifestations of diabetes mellitus (DM), in addition to its regulation of gastric nitrergicfunction.Taken together, the objective of this study is to investigate whether selective activation of gastricestrogen receptors improves gastric Nrf2-nNOS expression in an in-vitro experimentalhyperglycemic model.
Overall, the results are clear and support the conclusions of each experiment; namely that in NG
conditions, ER antagonist, but not PR antagonist inhibit NO relaxation, and ER antagonist, but notPR antagonist reduce nNOSa, Nrf2, DHFR, and ERa/b mRNA levels. Also, nNOSa, Nrf2, GCH-1, and ERa , protein expression were reduced; while ERb expression was upregulated. In HGmedium, ER agonists restored the impaired NO relaxation from HG medium, and restored the HG-induced reduction in mRNA levels and protein expression of nNOS, DHFR, Nrf2, ERa and ERb.NO enzymatic activity was assayed by nitrite production to show that enzyme activity is correlatedwith protein expression. Mannitol was used as the osmotic control for high glucose to eliminatethe osmotic variable as having an effect.
Major concern:
Previous findings showing co-expression of ERa and ERb in enteric neurons indicates that
estrogenic effects could be mediated through neurogenic reflexes. So this study utilized EFS underNANC conditions to stimulate neural release of NO and evoke nitrergic relaxations of the gastricmuscle strips. However, it might strengthen the conclusions that estrogenic effects are mediatedthrough neurogenic reflexes by examining an exogenous NO donor such as SNP. For example,neural NO relaxation was inhibited by HG; and restored by ER agonists. However, in the presenceof TTx, SNP will deliver NO to post-junctional cells; since this does not rely upon nNOS,relaxation may not be inhibited, and ER agonists may not have effects; but if relaxation evoked bySNP is inhibited by HG would suggest some myogenic component is disrupted by HG, and not all
the effects of HG on nitrergic relaxation are mediated by disruptions to nNOS.
Minor concerns:
The results show that HG reduced Era but not ERb; and that (Fig 6G) ERβ expression was
significantly upregulated in hyperglycemia, but restored by PPT.
Could potential explanations for these observations be addressed in the Discussion?On p 9 in the Methods it is stated that Electric field stimulation (EFS)-induced NANC relaxation
was studied in circular gastric neuromuscular strips [5,6}However, Reference 5 is about identify patient perceptions regarding the impact of gastroparesison managing their diabetes. It does not contain methods on carrying out in vitro smooth muscletension experiments.
Reference 6 is a review, that was published in 1999, not 2018, and also does not contain methods
on carrying out in vitro smooth muscle tension experiments.Please provide the correct references.
p10 Organ Bath Studies
Please indicate whether the mucosa and submucose removed from the muscle strips?And also, please indicate which anatomical region of the stomach these strips are from.
p10
Comparisons between the groups will be performed by measuring the area under thecurve (AUC) of the EFS-induced relaxation (AUCR) for 1 min and the baseline for 1 min(AUCB), according to the formula: (AUCR-AUCB)/weight of the tissue (mg) = AUC/mg tissue.
"will be" is a typop11 Were the mucosa and submucosa removed prior to western blot analysis? If not, how do you
know where ERa, ERb, DHFR, Nrf2, nNOS, and HO-1 are expressed?
p11
Please provide information on the instrumentation and software used to collect and analyze the
images.
p12- 13
Non-selective blockade of gastric ERs with ICI 182,780 reduces gastric (Fig 2A) nNOS, (Fig2B) Nrf2, (Fig 2C) GCH-1, (Fig 2D) DHFR and (Fig 2E-F) estrogen receptor (a/b) mRNAexpression. No measurable effect of RU-486, the progesteronereceptor antagonist, was observed when compared to DMEM (NG) control group. In addition,nochanges were observed with GCH-1 with either group.
Fig2C and the next sentence demonstrate that GCH-1 mRNA levels are not changed.
Are the methods appropriate and well described?
If not, please specify what is required in your comments to the authors.
YesDoes the work include the necessary controls?
If not, please specify which controls are required in your comments to the authors.
YesAre the conclusions 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 statistical review is recommended, please specify what aspects require furtherassessment in your comments to the editors.
I am able to assess the statisticsQuality of written English
Please indicate the quality of language in the manuscript:
AcceptableDeclaration 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, funding, 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 from 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 that 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 be included
on my report to the authors and, if the manuscript is accepted for publication, my named reportincluding 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 CommonsCC-BY license (http://creativecommons.org/licenses/by/4.0/). I understand that any commentswhich I do not wish to be included in my named report can be included as confidential commentsto 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: ROLE OF SEX HORMONES AND THEIR RECEPTORS ON GASTRIC NRF2 AND
NEURONAL NITRIC OXIDE SYNTHASE FUNCTION IN AN EXPERIMENTAL HYPER-GLYCEMIA MODEL
Version: 1Date: 31 Jan 2020
Reviewer: Brian A Perrino
Reviewer's report:
This study adds interesting and important information to be considered when trying to examine
and understand the contributions of hyperglycemia to the gastric dysmotility associated withdiabetic gastroparesis.Since the underlying mechanisms of gastric dysmotility are poorly understood, the approach ofthis study to work with experimental hyperglycemic conditions will help to establish translationalrelevance.The present study follows up on previous studies showing that loss of Interstitial Cells of Cajal(ICC), neuronal nitric oxide synthase (nNOS) function, and elevated oxidative stress are hallmarksof diabetic gastroparesis, and that results from their previous in-vivo studies have demonstratedthat nNOS-mediated gastric motility is greater in healthy female compared to male rodents.Furthermore, diabetes induction impaired nNOSα activity in female, but not in male in stomach
and duodenum. Other studies have shown that supplementation of 17 β-estradiol (E2) increases
nNOS expression and nitrergic-mediated gastric motility in ovariectomized rats. E2 has multiplebeneficial actions that include neuroprotection, maintaining glucose homeostasis, and activation ofNO synthesis in vascular smooth muscle. Also, Nuclear factor (erythroid-derived 2)-like 2 (Nrf2),
an antioxidant-responsive transcription factor, has emerged as a potent target for alleviating
clinical manifestations of diabetes mellitus (DM), in addition to its regulation of gastric nitrergicfunction.Taken together, the objective of this study is to investigate whether selective activation of gastricestrogen receptors improves gastric Nrf2-nNOS expression in an in-vitro experimentalhyperglycemic model.
Overall, the results are clear and support the conclusions of each experiment; namely that in NG
conditions, ER antagonist, but not PR antagonist inhibit NO relaxation, and ER antagonist, but notPR antagonist reduce nNOSa, Nrf2, DHFR, and ERa/b mRNA levels. Also, nNOSa, Nrf2, GCH-1, and ERa , protein expression were reduced; while ERb expression was upregulated. In HGmedium, ER agonists restored the impaired NO relaxation from HG medium, and restored the HG-induced reduction in mRNA levels and protein expression of nNOS, DHFR, Nrf2, ERa and ERb.NO enzymatic activity was assayed by nitrite production to show that enzyme activity is correlatedwith protein expression. Mannitol was used as the osmotic control for high glucose to eliminatethe osmotic variable as having an effect.
Major concern:
Previous findings showing co-expression of ERa and ERb in enteric neurons indicates that
estrogenic effects could be mediated through neurogenic reflexes. So this study utilized EFS underNANC conditions to stimulate neural release of NO and evoke nitrergic relaxations of the gastricmuscle strips. However, it might strengthen the conclusions that estrogenic effects are mediatedthrough neurogenic reflexes by examining an exogenous NO donor such as SNP. For example,neural NO relaxation was inhibited by HG; and restored by ER agonists. However, in the presenceof TTx, SNP will deliver NO to post-junctional cells; since this does not rely upon nNOS,relaxation may not be inhibited, and ER agonists may not have effects; but if relaxation evoked bySNP is inhibited by HG would suggest some myogenic component is disrupted by HG, and not all
the effects of HG on nitrergic relaxation are mediated by disruptions to nNOS.
Minor concerns:
The results show that HG reduced Era but not ERb; and that (Fig 6G) ERβ expression was
significantly upregulated in hyperglycemia, but restored by PPT.
Could potential explanations for these observations be addressed in the Discussion?On p 9 in the Methods it is stated that Electric field stimulation (EFS)-induced NANC relaxation
was studied in circular gastric neuromuscular strips [5,6}However, Reference 5 is about identify patient perceptions regarding the impact of gastroparesison managing their diabetes. It does not contain methods on carrying out in vitro smooth muscletension experiments.
Reference 6 is a review, that was published in 1999, not 2018, and also does not contain methods
on carrying out in vitro smooth muscle tension experiments.Please provide the correct references.
p10 Organ Bath Studies
Please indicate whether the mucosa and submucose removed from the muscle strips?And also, please indicate which anatomical region of the stomach these strips are from.
p10
Comparisons between the groups will be performed by measuring the area under thecurve (AUC) of the EFS-induced relaxation (AUCR) for 1 min and the baseline for 1 min(AUCB), according to the formula: (AUCR-AUCB)/weight of the tissue (mg) = AUC/mg tissue.
"will be" is a typop11 Were the mucosa and submucosa removed prior to western blot analysis? If not, how do you
know where ERa, ERb, DHFR, Nrf2, nNOS, and HO-1 are expressed?
p11
Please provide information on the instrumentation and software used to collect and analyze the
images.
p12- 13
Non-selective blockade of gastric ERs with ICI 182,780 reduces gastric (Fig 2A) nNOS, (Fig2B) Nrf2, (Fig 2C) GCH-1, (Fig 2D) DHFR and (Fig 2E-F) estrogen receptor (a/b) mRNAexpression. No measurable effect of RU-486, the progesteronereceptor antagonist, was observed when compared to DMEM (NG) control group. In addition,nochanges were observed with GCH-1 with either group.
Fig2C and the next sentence demonstrate that GCH-1 mRNA levels are not changed.
Are the methods appropriate and well described?
If not, please specify what is required in your comments to the authors.
YesDoes the work include the necessary controls?
If not, please specify which controls are required in your comments to the authors.
YesAre the conclusions 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 statistical review is recommended, please specify what aspects require furtherassessment in your comments to the editors.
I am able to assess the statisticsQuality of written English
Please indicate the quality of language in the manuscript:
AcceptableDeclaration 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, funding, 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 from 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 that 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 be included
on my report to the authors and, if the manuscript is accepted for publication, my named reportincluding 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 CommonsCC-BY license (http://creativecommons.org/licenses/by/4.0/). I understand that any commentswhich I do not wish to be included in my named report can be included as confidential commentsto the editors, which will not be published.
I agree to the open peer review policy of the journal
assistant