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After revisions, this manuscript has been obviously improved. All comments have been addressed. This manuscript is suitable for publication.
[# PeerJ Staff Note - this decision was reviewed and approved by Vladimir Uversky, a PeerJ Section Editor covering this Section #]
After revision, this paper has been significantly improved.
There are still some minor concerns need to be addressed:
1. Please provide how you extracted the nuclear fraction in method section greater in details.
2. Please provide Molecular marker (e.g TLR4 96kDa) in Figure 1 A, C.
3. As suggested by reviewer, please explain unnaturally high MSS.
The grammar and sentences in the manuscript has been improved a lot since last version. The manuscript is now well-written with good readability. All the comments have been addressed.
The experiments are well designed, all the comments have been addressed in this revision.
The results were well organized and written. The strength, limitations and future directions were well discussed. All the comments have been addressed.
This reviewer reviewed the revised version of the manuscript entitled "Toll-like Receptor 4 damages the intestinal epithelial cells by activating endoplasmic reticulum stress in septic rats” submitted to PeeJ (Manuscript ID#102147). Thank you very much for your effort and time spent on this revision. This manuscript is now of publication quality, pending some revisions which are listed below:
NFκB (nuclear factor kappa-B) is the nuclear protein, as it is named. Did you use the nuclear fraction? If so, please provide how did you extract the nuclear fraction in method section greater in details.
Figure 1 A, C
Molecular marker (e.g TLR4 96kDa) is missing. Please provide.
Figure 2F
The murine sepsis scores (MSS) are unnaturally high.
According to Shrum, fifty-seven percent of mice that reached a score of 15 died or had to be euthanized (in accordance with guidelines set by the Animal Use Subcommittee) within 1 hour, and 86% of mice that reached a score of 15 died within 2 hours. An MSS of 3 had a specificity of 100% for predicting onset of septic shock and death within 24 hours.
Your result show that even control rat had MSS of 5, and Sepsis and treatment group had MSS of 17. This reviewer thinks the differences of the species alone between mouse and rat does not explain these discrepancies.
Thank you for your effort.
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1. While the manuscript is generally well-written, some sections could benefit from more concise wording to enhance readability. Please carefully proofread the manuscript and correct any typographical or grammatical errors (e.g., "I thanks" in the acknowledgments section).
2. In Methods:
a) Include the relevant date of the ethical approval and specify when the experiments were conducted.
b) Provide details on how the sample size was determined, including any a priori sample size calculations or the rationale for not performing them.
c) Describe the histological analysis protocol in greater detail.
d) Provide the method used to generate the randomization sequence for dividing the rats into groups.
e) Describe the strategies used to minimize potential confounders and who was aware of group allocations at different stages of the experiment.
3. Results:
a) Include scale bars in the histological images.
b) Clearly define the number of independent replications for each analysis in the figure legends, reporting the exact value of n for each experimental group.
4. Discussion:
a) Comment on the study's limitations, including potential sources of bias, limitations of the animal model, and imprecision associated with the results.
b) Discuss the strengths of your experimental approach.
c) Address the generalizability of the findings to other species or experimental conditions, including any relevance to human biology.
d) Expand on the future research direction in more detail, after the limitations section.
5. Please clarify how the p-value was calculated for Figure 2 E-G, as mentioned in the manuscript.
**PeerJ Staff Note:** It is PeerJ policy that additional references suggested during the peer-review process should only be included if the authors agree that they are relevant and useful.
Clarity and Language: The manuscript is written in clear and professional English, making it accessible to an international audience. However, some sections, such as the detailed descriptions of methods and results, could benefit from more concise wording to enhance readability. For example, in acknowledgement section, “I thanks” should be “I thank”.
Introduction and Background: The introduction provides a comprehensive overview of the research context, highlighting the significance of studying AGI in sepsis and the role of TLR4 in MODS. The background on TLR4 and its impact on Lgr5+ cells and goblet cells is well established, providing a solid foundation for the study.
Literature References: The manuscript is well-referenced, citing relevant and up-to-date literature to support the research rationale and findings. The references are appropriate and contribute to the credibility of the study.
Structure and Figures: The structure of the manuscript conforms to PeerJ standards. The figures included are relevant, of high quality, and effectively support the data presented. However, figure legends could be more detailed to provide better context without referring back to the main text.
Raw Data: The authors have provided the raw data as per PeerJ policy, which supports the transparency and reproducibility of the study.
Original Research: This study presents original primary research within the scope of the journal. It addresses a significant gap in understanding the impact of TLR4 on intestinal epithelial cells in septic conditions.
Research Question: The research question is well-defined, relevant, and meaningful. The study aims to investigate the effect of TLR4 on Lgr5+ cells and goblet cells in septic rats and explores the underlying mechanisms involving ER stress.
Methodology: The experimental design is rigorous, employing appropriate models (cecal ligation and puncture) and interventions (TLR4 inhibitor Tak-242). The methods are described in sufficient detail to allow replication. However, additional details on the statistical methods used for some of data analysis could enhance the reproducibility of the results. For example, the method for p-value calculation in Figure 2 E-G.
Ethical Considerations: The study involves the use of vertebrate animals, and the authors have provided an ethical approval statement.
Data and Statistical Soundness: Most of the underlying data provided are robust, statistically sound, and well-controlled. However, there are some part where the statistics were not clarified. For example, In Figure 2 E-G, it is unclear how the p-value was calculated, as stated in the manuscript “Moreover, the level of improved in the Tak-242 group(p<0.0001) (Fig2.E-G)”.
Conclusions and Research Questions: The conclusions drawn are well stated and directly linked to the original research question. The findings support the hypothesis that TLR4 activation leads to damage in intestinal epithelial cells through ER stress, affecting Lgr5+ cells and goblet cells.
Impact and Novelty: While the manuscript does not explicitly assess impact and novelty, the study's findings contribute valuable insights into the mechanisms of septic AGI and potential therapeutic interventions. The replication of results and the exploration of TLR4 inhibition as a treatment strategy are well justified.
1. The study could benefit from a more detailed discussion of limitations, such as potential variability in animal models and the scope of TLR4 inhibition effects. Additionally, suggesting future research directions could provide a more comprehensive perspective on the study's implications.
2. While the results are comprehensive, the study could benefit from more detailed discussion on transcriptome-wide effects of TLR4 on Lgr5+ cells and goblet cells. For example, are there other genes that are affected, are there any different impacts on the two cell types.
I reviewed the article by Wu et al. entitled "Toll-like Receptor 4 damages the intestinal epithelial cells by activating endoplasmic reticulum stress in septic rats" submitted to PeerJ (Manuscript ID: #102147). In this preclinical study, the authors sought to determine the role of Toll-like Receptor 4 (TLR4) on leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) + cells and goblet cells in septic rats.
The authors found that structure of the intestinal mucosa was destroyed, Lgr5+ cells and goblet cells count was reduced, and the secretory function of goblet cells also decreased in the septic animals. The authors also observed that sepsis increased activating transcription factor-6(ATF6), XBP1, ER chaperone (Bip) and CHOP levels, all of which was at least partially mediated by TLR pathway. From these observations, they claimed that inhibition of TLR4-mediated ER stress may be a promising therapy of sepsis induced acute gastrointestinal injury. The research topic is important and methodology used here seems to be appropriate. The conclusion drawn from the results of the experiments seems to be reasonable. This reviewer thinks these results are of interests for the readers of this journal. However, there are several concerns that need to be addressed before publication. The comments from this reviewer are listed below:
General
Keep abbreviations to a minimum. Do not use non-standard abbreviations unless they appear at least three times in the text.
Methods
1
Ethical approval section should include the relevant date of the approval.
2
When these experiments were conducted?
3
Explain how the sample size was decided. Provide details of any a priori sample size calculation, if done. if not, please provide the rationale why you have not determined a priori sample size calculation.
4
Histological analysis protocol should be described greater in details.
5
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The rats were randomly divided...
Please provide the method used to generate the randomization sequence.
6
Please describe the strategy used to minimize potential confounders, such as the order of treatments and measurements, time (night/date), researcher who conducted the experiments or animal/cage location. If confounders were not controlled, state this explicitly.
7
Describe who was aware of the group allocation at the different stages of the experiment (during the allocation, the conduct of the experiment, the outcome assessment, and the data analysis.
Results
8
The histological images should include the scale bar.
9
Figure legends
For each analysis, please report the exact value of n in each experimental group. The authors should clearly define the number of independent replications in each figure legends.
10
This manuscript contains several typological errors. See, for example, Figure 1 and Figure 4. action should be actin. There are some other similar errors, that should be amended.
Additional experiments
This reviewer think this manuscript can be strengthen in several places.
11
NF κB is the key regulator of the TLR 4 pathway, which is indispensable when discussing the signal pathway. TLR4 mRNA and protein levels were determined in this study, but TLR4 upregulation itself does not always mean the inflammatory signal activation. The authors therefore should confirm the NFκB activity by conducting the additional experiments. The authors can solve this problem by conducting ELISA or western blot. Following article may aid you to address this comment. If pertinent, please also consider to cite these articles in your revised version of your manuscript.
Ono Y, Maejima Y, Saito M, et al. TAK-242, a specific inhibitor of Toll-like receptor 4 signaling, prevents endotoxemia-induced skeletal muscle wasting in mice. Sci Rep 2020;10:694.
Ono Y, Sakamoto K. Lipopolysaccharide inhibits myogenic differentiation of C2C12 myoblasts through the Toll-like receptor 4-nuclear factor-κB signaling pathway and myoblast-derived tumor necrosis factor-α. PLoS One 2017;12:e0182040.
12
Can the authors provide the differences of sepsis severity among control, sepsis, and treatment group? This can be achieved by comparing body temperature, food intake, body weight change, or the murine sepsis score (Shrum B, et al. BMC Res Notes. 2014). This reviewer thinks food intake and body weight change is especially important outcome measures in mice with sepsis induced gastrointestinal injury.
Shrum B, Anantha RV, Xu SX, et al . A robust scoring system to evaluate sepsis severity in an animal model. BMC Res Notes. 2014;7:233.
13
Can the author furnish the survival curve among these three groups? Is TLR4 specific inhibitor TAK242 useful to improve the survival rate?
14
The authors did not measure the inflammatory cytokine levels, which is the major weakness of this study. The authors should investigate the inflammatory cytokines (i.e. IL-5, TNFα, IL-1β) by conducting qRT-PCR of intestinal track among control, sepsis, and treatment group. Similarly, systemic inflammatory response should be also confirmed by measuring plasma cytokines (i.e. IL-5, TNFα, IL-1β) by ELISA or some other methods control, sepsis, and treatment group. These experiments would be beneficial to the readers of this journal.
Discussion
15
Comment on the study limitations including potential sources of bias, limitations of the animal model, and imprecision associated with the results.
16
What is the strength of your experimental approach? Please clarify.
17
Comment on whether, and how, the findings of this study are likely to generalize to other species or experimental conditions, including any relevance to human biology (where appropriate).
18
Please indicate future research direction more in details, after the limitation section.
Although the number of criticisms listed above, I should however state that it is laudable that this work is derived from huge efforts made by the authors, who are working as the frontline researchers. This reviewer respects the authors’ time and effort spent on this manuscript, and the authors ‘patience and professionalism in dealing with my comments. This reviewer is looking forward to read the revised version of your manuscript with some new results. This reviewer will recommend publication if the authors can address my comment #11-#14 by conducting additional experiments.
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