Title: Remimazolam alleviates hepatic ischemia-reperfusion injury by activating FOXO1/3
signaling
Version: 2 Date: 20 Feb 2025
Reviewer:  Anwar Ashraf Abouelnasr
Reviewer's report:
Dear Authors, please address the following points below
-The introduction would benefit from a more concise statement of the study’s specific aim.
Although the hypothesis is clear, a more direct mention of the research question—whether
RMZL protects against HIRI via FOXO1/3 activation—would create a stronger transition from
background information to study goals.
-While the introduction does a good job of discussing prior research on RMZL and FOXO
signaling, the connections between these studies could be more explicitly stated. For example, it
would be helpful to highlight the gap in knowledge about RMZL’s potential role in HIRI more
directly, and the need to investigate the molecular mechanism involving FOXO proteins.
-While the mouse model is well-detailed, there could be more information on the use of age-
matched controls and the rationale behind the chosen dosages of RMZL (20 mg/kg). Providing
more justification for the chosen treatment regimen would enhance the rigor of the experimental
design. Additionally, the duration of reperfusion could be varied to assess the temporal dynamics
of RMZL's protective effects.
-The hypoxia/reoxygenation (H/R) protocol used for the LO2 cells is outlined, but more
information on the rationale behind the duration and oxygen concentrations could help further
justify the experimental setup. Additionally, clarifying whether this model mimics conditions
similar to those observed in vivo would be beneficial for contextualizing the cell-based results.
-It is stated that inflammatory cytokines such as IL-1#, TNF-#, IL-6, and IL-10 will be measured
in both mouse serum and LO2 cell supernatants using ELISA kits. However, more specific
details on the time points for collection, as well as the expected changes in these cytokines
following RMZL treatment, could help provide context to the findings.
-While the methodology mentions 24-hour reperfusion for mouse experiments, it would be
helpful to clarify the time points for in vitro experiments as well. For instance, is the assessment
of oxidative stress, apoptosis, or inflammation being measured at specific intervals after RMZL
treatment?
-The connection between RMZL and FOXO1/3 is an important finding, and the manuscript
clearly explains how these proteins regulate oxidative stress and inflammation. However, the
specific molecular mechanisms through which RMZL elevates FOXO1 and FOXO3 expressions
could be explored in more detail. For example, is RMZL acting directly on the FOXO1/3
pathway, or is it an indirect effect mediated by other molecules? Providing more detailed insight
into the signaling pathway would enhance the mechanistic depth of the discussion.
It might also be helpful to briefly mention any key downstream effectors or pathways that RMZL
might activate via FOXO1/3, as this would strengthen the link between the molecular findings
and their functional consequences.
-While the manuscript does a good job of outlining the therapeutic potential of RMZL, it
would benefit from a more explicit discussion of the study's limitations. For example, while the
results in animal models are promising, how generalizable are these findings to human clinical
scenarios? Are there any concerns regarding RMZL’s pharmacokinetics or long-term safety that
need further investigation?
-The authors mention the need for "extensive preclinical and clinical trials," which is a crucial
point. However, it would strengthen the discussion if the authors could identify specific areas
for future research, such as optimal dosing, the impact of RMZL in different stages of HIRI, and
potential interactions with other drugs or anesthetics.
-While the authors mention RMZL’s potential for clinical use, a more explicit discussion of its
advantages over other anesthetics or treatment options for HIRI would be valuable. How does
RMZL compare to existing therapies (e.g., propofol) in terms of its effectiveness, safety profile,
or ease of use?
-Additionally, it might be useful to consider the broader implications of RMZL’s use in
different surgical settings and whether its anesthetic effects might influence other aspects of
liver recovery, such as metabolic function or the immune response.
Title: Remimazolam alleviates hepatic ischemia-reperfusion injury by activating FOXO1/3
signaling
Version: 2 Date: 20 Feb 2025
Reviewer:  Anwar Ashraf Abouelnasr
Reviewer's report:
Dear Authors, please address the following points below
-The introduction would benefit from a more concise statement of the study’s specific aim.
Although the hypothesis is clear, a more direct mention of the research question—whether
RMZL protects against HIRI via FOXO1/3 activation—would create a stronger transition from
background information to study goals.
-While the introduction does a good job of discussing prior research on RMZL and FOXO
signaling, the connections between these studies could be more explicitly stated. For example, it
would be helpful to highlight the gap in knowledge about RMZL’s potential role in HIRI more
directly, and the need to investigate the molecular mechanism involving FOXO proteins.
-While the mouse model is well-detailed, there could be more information on the use of age-
matched controls and the rationale behind the chosen dosages of RMZL (20 mg/kg). Providing
more justification for the chosen treatment regimen would enhance the rigor of the experimental
design. Additionally, the duration of reperfusion could be varied to assess the temporal dynamics
of RMZL's protective effects.
-The hypoxia/reoxygenation (H/R) protocol used for the LO2 cells is outlined, but more
information on the rationale behind the duration and oxygen concentrations could help further
justify the experimental setup. Additionally, clarifying whether this model mimics conditions
similar to those observed in vivo would be beneficial for contextualizing the cell-based results.
-It is stated that inflammatory cytokines such as IL-1#, TNF-#, IL-6, and IL-10 will be measured
in both mouse serum and LO2 cell supernatants using ELISA kits. However, more specific
details on the time points for collection, as well as the expected changes in these cytokines
following RMZL treatment, could help provide context to the findings.
-While the methodology mentions 24-hour reperfusion for mouse experiments, it would be
helpful to clarify the time points for in vitro experiments as well. For instance, is the assessment
of oxidative stress, apoptosis, or inflammation being measured at specific intervals after RMZL
treatment?
-The connection between RMZL and FOXO1/3 is an important finding, and the manuscript
clearly explains how these proteins regulate oxidative stress and inflammation. However, the
specific molecular mechanisms through which RMZL elevates FOXO1 and FOXO3 expressions
could be explored in more detail. For example, is RMZL acting directly on the FOXO1/3
pathway, or is it an indirect effect mediated by other molecules? Providing more detailed insight
into the signaling pathway would enhance the mechanistic depth of the discussion.
It might also be helpful to briefly mention any key downstream effectors or pathways that RMZL
might activate via FOXO1/3, as this would strengthen the link between the molecular findings
and their functional consequences.
-While the manuscript does a good job of outlining the therapeutic potential of RMZL, it
would benefit from a more explicit discussion of the study's limitations. For example, while the
results in animal models are promising, how generalizable are these findings to human clinical
scenarios? Are there any concerns regarding RMZL’s pharmacokinetics or long-term safety that
need further investigation?
-The authors mention the need for "extensive preclinical and clinical trials," which is a crucial
point. However, it would strengthen the discussion if the authors could identify specific areas
for future research, such as optimal dosing, the impact of RMZL in different stages of HIRI, and
potential interactions with other drugs or anesthetics.
-While the authors mention RMZL’s potential for clinical use, a more explicit discussion of its
advantages over other anesthetics or treatment options for HIRI would be valuable. How does
RMZL compare to existing therapies (e.g., propofol) in terms of its effectiveness, safety profile,
or ease of use?
-Additionally, it might be useful to consider the broader implications of RMZL’s use in
different surgical settings and whether its anesthetic effects might influence other aspects of
liver recovery, such as metabolic function or the immune response.
Please review this paper.
user
Title: Remimazolam alleviates hepatic ischemia-reperfusion injury by activating FOXO1/3
signaling
Version: 2 Date: 20 Feb 2025
Reviewer:  Anwar Ashraf Abouelnasr
Reviewer's report:
Dear Authors, please address the following points below
-The introduction would benefit from a more concise statement of the study’s specific aim.
Although the hypothesis is clear, a more direct mention of the research question—whether
RMZL protects against HIRI via FOXO1/3 activation—would create a stronger transition from
background information to study goals.
-While the introduction does a good job of discussing prior research on RMZL and FOXO
signaling, the connections between these studies could be more explicitly stated. For example, it
would be helpful to highlight the gap in knowledge about RMZL’s potential role in HIRI more
directly, and the need to investigate the molecular mechanism involving FOXO proteins.
-While the mouse model is well-detailed, there could be more information on the use of age-
matched controls and the rationale behind the chosen dosages of RMZL (20 mg/kg). Providing
more justification for the chosen treatment regimen would enhance the rigor of the experimental
design. Additionally, the duration of reperfusion could be varied to assess the temporal dynamics
of RMZL's protective effects.
-The hypoxia/reoxygenation (H/R) protocol used for the LO2 cells is outlined, but more
information on the rationale behind the duration and oxygen concentrations could help further
justify the experimental setup. Additionally, clarifying whether this model mimics conditions
similar to those observed in vivo would be beneficial for contextualizing the cell-based results.
-It is stated that inflammatory cytokines such as IL-1#, TNF-#, IL-6, and IL-10 will be measured
in both mouse serum and LO2 cell supernatants using ELISA kits. However, more specific
details on the time points for collection, as well as the expected changes in these cytokines
following RMZL treatment, could help provide context to the findings.
-While the methodology mentions 24-hour reperfusion for mouse experiments, it would be
helpful to clarify the time points for in vitro experiments as well. For instance, is the assessment
of oxidative stress, apoptosis, or inflammation being measured at specific intervals after RMZL
treatment?
-The connection between RMZL and FOXO1/3 is an important finding, and the manuscript
clearly explains how these proteins regulate oxidative stress and inflammation. However, the
specific molecular mechanisms through which RMZL elevates FOXO1 and FOXO3 expressions
could be explored in more detail. For example, is RMZL acting directly on the FOXO1/3
pathway, or is it an indirect effect mediated by other molecules? Providing more detailed insight
into the signaling pathway would enhance the mechanistic depth of the discussion.
It might also be helpful to briefly mention any key downstream effectors or pathways that RMZL
might activate via FOXO1/3, as this would strengthen the link between the molecular findings
and their functional consequences.
-While the manuscript does a good job of outlining the therapeutic potential of RMZL, it
would benefit from a more explicit discussion of the study's limitations. For example, while the
results in animal models are promising, how generalizable are these findings to human clinical
scenarios? Are there any concerns regarding RMZL’s pharmacokinetics or long-term safety that
need further investigation?
-The authors mention the need for "extensive preclinical and clinical trials," which is a crucial
point. However, it would strengthen the discussion if the authors could identify specific areas
for future research, such as optimal dosing, the impact of RMZL in different stages of HIRI, and
potential interactions with other drugs or anesthetics.
-While the authors mention RMZL’s potential for clinical use, a more explicit discussion of its
advantages over other anesthetics or treatment options for HIRI would be valuable. How does
RMZL compare to existing therapies (e.g., propofol) in terms of its effectiveness, safety profile,
or ease of use?
-Additionally, it might be useful to consider the broader implications of RMZL’s use in
different surgical settings and whether its anesthetic effects might influence other aspects of
liver recovery, such as metabolic function or the immune response.
assistant