Title: Investigation of the Mechanism by which miR-223-3p Inhibits Reflux Esophagitis
through Targeting the NLRP3 Inflammasome
Version: 3 Date: 20 Jan 2025
Reviewer:  Anwar Ashraf Abouelnasr
Reviewer's report:
Dear Authors, please address the following points below
-Consider expanding on the relationship between reflux esophagitis and Barrett’s esophagus,
particularly how chronic inflammation may lead to this condition. This could provide a better
connection to the clinical relevance of your study.
=The contradiction about miR-223-3p expression in reflux esophagitis (with an increase in
acute stages and a decrease in chronic stages) could be further explored. Clarifying the possible
underlying mechanisms contributing to this pattern and its implications for the study would be
beneficial.
-In 2.4 Cell Transfection, the sequences for miR-223-3p mimics, inhibitors, and NC (negative
control) are given, but it might be helpful to explain the rationale behind the specific sequences
chosen. Why were these particular sequences selected over others? A brief explanation could
enhance the clarity of the experimental design.
-Consider discussing the potential off-target effects of miR-223-3p mimics and inhibitors. How
do you plan to control this?
-The NLRP3 overexpression section is well described, but you might want to explain why a
lentiviral system was chosen over other overexpression methods, such as plasmid transfection
or CRISPR activation.
-In 2.7 ELISA Detection of IL-1# and IL-18 Levels, it would be helpful to clarify how these
cytokines will be analyzed and the rationale for selecting these particular markers. Are these
the main inflammatory mediators implicated in reflux esophagitis, or is this more of a general
inflammation marker panel?
-For 2.10 Western Blot, more detail on the specific markers or pathways assessed (e.g., caspase-1
activation, NLRP3 oligomerization, or GSDMD cleavage) would be useful for readers to
understand how these markers directly correlate with NLRP3 inflammasome activation and
pyroptosis.
-When discussing cell cycle analysis (2.6), it may be worth specifying which phases (e.g., G1/S,
S/G2, G2/M) are of particular interest. If specific changes are expected, mentioning this would
improve the clarity of the analysis.
-While the authors correctly point out the need for in vivo studies, they could be more explicit
in their suggestions for the types of animal models or clinical samples that would be most
informative. For instance, what specific animal models of reflux esophagitis or gastric acid/bile
reflux could be used to better replicate human disease conditions? Also, would it be useful to
explore longitudinal studies to assess the long-term effects of miR-223-3p modulation?
-The authors mention that miR-223-3p might intervene at key nodes in the activation of the
NLRP3 inflammasome, but they could elaborate more on the potential molecular mechanisms
involved. For example, the NF-#B and K+ efflux signaling pathways are mentioned, but it would
be useful to discuss whether miR-223-3p could regulate these pathways directly or indirectly.
How does miR-223-3p modulate NF-#B signaling, and could other inflammasomes (e.g., AIM2)
be involved?
-Although the authors refer to IL-1# and IL-18 as key inflammatory markers, a more
comprehensive cytokine profiling would help to better understand the broader anti-inflammatory
effects of miR-223-3p. Other markers of inflammation, such as TNF-#, IL-6, IL-8, and
chemokines like CXCL1, could provide additional insights into how miR-223-3p regulates
inflammation in reflux esophagitis.
-The discussion regarding the multi-target nature of miR-223-3p is somewhat general. It would
benefit from a more detailed exploration of the potential off-target effects of miR-223-3p
modulation. Given that miR-223-3p regulates a number of immune and inflammatory genes,
what are the potential risks of overexpressing or inhibiting miR-223-3p in vivo? Are there any
known miR-223-3p targets that could lead to adverse effects or unwanted immune responses?
-The authors mention that miR-223-3p is downregulated in the reflux esophagitis model but
do not delve into the regulatory mechanisms behind this downregulation. What is responsible
for this decrease in miR-223-3p? Are bile acids, acidic conditions, or transcription factors
(e.g., NF-#B, AP-1) involved in regulating miR-223-3p expression in reflux esophagitis?
Understanding these regulatory mechanisms could enhance the therapeutic potential of
miR-223-3p modulation.
Title: Investigation of the Mechanism by which miR-223-3p Inhibits Reflux Esophagitis
through Targeting the NLRP3 Inflammasome
Version: 3 Date: 20 Jan 2025
Reviewer:  Anwar Ashraf Abouelnasr
Reviewer's report:
Dear Authors, please address the following points below
-Consider expanding on the relationship between reflux esophagitis and Barrett’s esophagus,
particularly how chronic inflammation may lead to this condition. This could provide a better
connection to the clinical relevance of your study.
=The contradiction about miR-223-3p expression in reflux esophagitis (with an increase in
acute stages and a decrease in chronic stages) could be further explored. Clarifying the possible
underlying mechanisms contributing to this pattern and its implications for the study would be
beneficial.
-In 2.4 Cell Transfection, the sequences for miR-223-3p mimics, inhibitors, and NC (negative
control) are given, but it might be helpful to explain the rationale behind the specific sequences
chosen. Why were these particular sequences selected over others? A brief explanation could
enhance the clarity of the experimental design.
-Consider discussing the potential off-target effects of miR-223-3p mimics and inhibitors. How
do you plan to control this?
-The NLRP3 overexpression section is well described, but you might want to explain why a
lentiviral system was chosen over other overexpression methods, such as plasmid transfection
or CRISPR activation.
-In 2.7 ELISA Detection of IL-1# and IL-18 Levels, it would be helpful to clarify how these
cytokines will be analyzed and the rationale for selecting these particular markers. Are these
the main inflammatory mediators implicated in reflux esophagitis, or is this more of a general
inflammation marker panel?
-For 2.10 Western Blot, more detail on the specific markers or pathways assessed (e.g., caspase-1
activation, NLRP3 oligomerization, or GSDMD cleavage) would be useful for readers to
understand how these markers directly correlate with NLRP3 inflammasome activation and
pyroptosis.
-When discussing cell cycle analysis (2.6), it may be worth specifying which phases (e.g., G1/S,
S/G2, G2/M) are of particular interest. If specific changes are expected, mentioning this would
improve the clarity of the analysis.
-While the authors correctly point out the need for in vivo studies, they could be more explicit
in their suggestions for the types of animal models or clinical samples that would be most
informative. For instance, what specific animal models of reflux esophagitis or gastric acid/bile
reflux could be used to better replicate human disease conditions? Also, would it be useful to
explore longitudinal studies to assess the long-term effects of miR-223-3p modulation?
-The authors mention that miR-223-3p might intervene at key nodes in the activation of the
NLRP3 inflammasome, but they could elaborate more on the potential molecular mechanisms
involved. For example, the NF-#B and K+ efflux signaling pathways are mentioned, but it would
be useful to discuss whether miR-223-3p could regulate these pathways directly or indirectly.
How does miR-223-3p modulate NF-#B signaling, and could other inflammasomes (e.g., AIM2)
be involved?
-Although the authors refer to IL-1# and IL-18 as key inflammatory markers, a more
comprehensive cytokine profiling would help to better understand the broader anti-inflammatory
effects of miR-223-3p. Other markers of inflammation, such as TNF-#, IL-6, IL-8, and
chemokines like CXCL1, could provide additional insights into how miR-223-3p regulates
inflammation in reflux esophagitis.
-The discussion regarding the multi-target nature of miR-223-3p is somewhat general. It would
benefit from a more detailed exploration of the potential off-target effects of miR-223-3p
modulation. Given that miR-223-3p regulates a number of immune and inflammatory genes,
what are the potential risks of overexpressing or inhibiting miR-223-3p in vivo? Are there any
known miR-223-3p targets that could lead to adverse effects or unwanted immune responses?
-The authors mention that miR-223-3p is downregulated in the reflux esophagitis model but
do not delve into the regulatory mechanisms behind this downregulation. What is responsible
for this decrease in miR-223-3p? Are bile acids, acidic conditions, or transcription factors
(e.g., NF-#B, AP-1) involved in regulating miR-223-3p expression in reflux esophagitis?
Understanding these regulatory mechanisms could enhance the therapeutic potential of
miR-223-3p modulation.
Please review this paper.
user
Title: Investigation of the Mechanism by which miR-223-3p Inhibits Reflux Esophagitis
through Targeting the NLRP3 Inflammasome
Version: 3 Date: 20 Jan 2025
Reviewer:  Anwar Ashraf Abouelnasr
Reviewer's report:
Dear Authors, please address the following points below
-Consider expanding on the relationship between reflux esophagitis and Barrett’s esophagus,
particularly how chronic inflammation may lead to this condition. This could provide a better
connection to the clinical relevance of your study.
=The contradiction about miR-223-3p expression in reflux esophagitis (with an increase in
acute stages and a decrease in chronic stages) could be further explored. Clarifying the possible
underlying mechanisms contributing to this pattern and its implications for the study would be
beneficial.
-In 2.4 Cell Transfection, the sequences for miR-223-3p mimics, inhibitors, and NC (negative
control) are given, but it might be helpful to explain the rationale behind the specific sequences
chosen. Why were these particular sequences selected over others? A brief explanation could
enhance the clarity of the experimental design.
-Consider discussing the potential off-target effects of miR-223-3p mimics and inhibitors. How
do you plan to control this?
-The NLRP3 overexpression section is well described, but you might want to explain why a
lentiviral system was chosen over other overexpression methods, such as plasmid transfection
or CRISPR activation.
-In 2.7 ELISA Detection of IL-1# and IL-18 Levels, it would be helpful to clarify how these
cytokines will be analyzed and the rationale for selecting these particular markers. Are these
the main inflammatory mediators implicated in reflux esophagitis, or is this more of a general
inflammation marker panel?
-For 2.10 Western Blot, more detail on the specific markers or pathways assessed (e.g., caspase-1
activation, NLRP3 oligomerization, or GSDMD cleavage) would be useful for readers to
understand how these markers directly correlate with NLRP3 inflammasome activation and
pyroptosis.
-When discussing cell cycle analysis (2.6), it may be worth specifying which phases (e.g., G1/S,
S/G2, G2/M) are of particular interest. If specific changes are expected, mentioning this would
improve the clarity of the analysis.
-While the authors correctly point out the need for in vivo studies, they could be more explicit
in their suggestions for the types of animal models or clinical samples that would be most
informative. For instance, what specific animal models of reflux esophagitis or gastric acid/bile
reflux could be used to better replicate human disease conditions? Also, would it be useful to
explore longitudinal studies to assess the long-term effects of miR-223-3p modulation?
-The authors mention that miR-223-3p might intervene at key nodes in the activation of the
NLRP3 inflammasome, but they could elaborate more on the potential molecular mechanisms
involved. For example, the NF-#B and K+ efflux signaling pathways are mentioned, but it would
be useful to discuss whether miR-223-3p could regulate these pathways directly or indirectly.
How does miR-223-3p modulate NF-#B signaling, and could other inflammasomes (e.g., AIM2)
be involved?
-Although the authors refer to IL-1# and IL-18 as key inflammatory markers, a more
comprehensive cytokine profiling would help to better understand the broader anti-inflammatory
effects of miR-223-3p. Other markers of inflammation, such as TNF-#, IL-6, IL-8, and
chemokines like CXCL1, could provide additional insights into how miR-223-3p regulates
inflammation in reflux esophagitis.
-The discussion regarding the multi-target nature of miR-223-3p is somewhat general. It would
benefit from a more detailed exploration of the potential off-target effects of miR-223-3p
modulation. Given that miR-223-3p regulates a number of immune and inflammatory genes,
what are the potential risks of overexpressing or inhibiting miR-223-3p in vivo? Are there any
known miR-223-3p targets that could lead to adverse effects or unwanted immune responses?
-The authors mention that miR-223-3p is downregulated in the reflux esophagitis model but
do not delve into the regulatory mechanisms behind this downregulation. What is responsible
for this decrease in miR-223-3p? Are bile acids, acidic conditions, or transcription factors
(e.g., NF-#B, AP-1) involved in regulating miR-223-3p expression in reflux esophagitis?
Understanding these regulatory mechanisms could enhance the therapeutic potential of
miR-223-3p modulation.
assistant