# Review HistoryTheacrine alleviates sepsis-induced acute kidney injury by repressing the activation of NLRP3/Caspase-1 inflammasome

All reviews of published articles are made public. This includes manuscript files, peer review comments, author rebuttals and revised materials. Note: This was optional for articles submitted before 13 February 2023.

Peer reviewers are encouraged (but not required) to provide their names to the authors when submitting their peer review. If they agree to provide their name, then their personal profile page will reflect a public acknowledgment that they performed a review (even if the article is rejected). If the article is accepted, then reviewers who provided their name will be associated with the article itself.

## Summary

• The initial submission of this article was received on February 16th, 2022 and was peer-reviewed by 2 reviewers and the Academic Editor.
• The Academic Editor made their initial decision on April 5th, 2022.
• The first revision was submitted on May 10th, 2022 and was reviewed by 1 reviewer and the Academic Editor.
• A further revision was submitted on June 17th, 2022 and was reviewed by 2 reviewers and the Academic Editor.
• A further revision was submitted on August 16th, 2022 and was reviewed by the Academic Editor.
• A further revision was submitted on August 30th, 2022 and was reviewed by the Academic Editor.
• The article was Accepted by the Academic Editor on September 2nd, 2022.

## Version 0.5 (accepted)

Accept

We look forward to your next submission.

[# PeerJ Staff Note - this decision was reviewed and approved by Gwyn Gould, a PeerJ Section Editor covering this Section #]

## Version 0.4

Minor Revisions

There are still inconsistencies in the descriptions of N= 3 (or n=3) in the captions to Figures 3, 4 and 5.
Figure 3: N=3 per group（Data are presented as means ± standard error of mean (SEM) from three independent repetitions.）
Figure 4: n=3 per group（Data are presented as means ± standard error of mean (SEM) from independent repetitions.）
Figure 5: n=3 per group（Data are presented as means ± standard error of mean (SEM) from independent repetitions.）
Is there any difference between N and n? Why is "three" missing in the description of n? Clarification must be provided to enable the readers properly evaluate the outcome of the study.

## Version 0.3

Minor Revisions

1. If it is not originally drawn, provide citation for the source of the structure of Theacrine in Figure 1.
2. Provide clarification for the definition of N= 3 in Figures 3, 4 and 5.
3. Provide in the supplementary information ALL replicate full gels from which cropped gel shown in Figure 4 was obtained.

We look forward to your next submission.

### Reviewer 1 · Jun 24, 2022

#### Basic reporting

The basic reporting was improved, although I think that it is sad, that the authors claim that no clinical data on inflammasomes and AKI have been published.

#### Validity of the findings

none

Cite this review as

### Reviewer 2 · Jun 30, 2022

#### Basic reporting

The manuscript was improved and I recommend accept in the current form.

#### Experimental design

All experiments are realized adequately and the supplementary material support this.

#### Validity of the findings

The manuscript is novelty and the experiments support the obtained results.

Cite this review as

## Version 0.2

Minor Revisions

The reviewer suggests that you address issues relating to current knowledge about inflammasome, and typographic errors.

### Reviewer 1 · May 20, 2022

#### Basic reporting

Please find my comments on English language use and literature references below.

no comment

#### Validity of the findings

no comment

The manuscript is significantly improved. The methods' description and results presentation justify publication.

Nevertheless, there are two points that still need to be addressed:
1. Especially the current knowledge about inflammasome components in human AKI and human septic AKI should be discussed using up to date literature to underline/explain an importance of the research data presented in this manuscript. In general, it is preferable to find more than just one reference to support a statement.
I really want to stress, that the authors find relevant literature that assesses the topic in human AKI and human septic AKI (=patients). I do not assume that the authors' final aim of the research is the treatment of AKI/septic AKI in laboratory animals.

2. Please check again spelling and grammar, the revised paragraphs included.

Cite this review as

## Version 0.1 (original submission)

Major Revisions

The two expert reviewers gave thorough reviews and identified several key concerns. I urge you to address all of these concerns in your revised manuscript.

[# PeerJ Staff Note: Please ensure that all review and editorial comments are addressed in a response letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate. #]

### Reviewer 1 · Mar 19, 2022

#### Basic reporting

Basic reporting
1. The English used is acceptable, but could be improved.
Example Abstract:
there is no therapies – there are no therapies
after pretreatment with of theacrine
NLRP3 agonist damaged the effect

2. Literature references/background
It needs to be introduced that theacrine is found in tea. In the discussion the value of tea is mentioned, but the connection to theacrine is not made. What kind of tea, concentrations and some literature would support the case of theacrine use.
The introduction and discussion mention data on learning, memory, cognitive performance and liver injury. The authors need to explain more clearly why they suggest a role for theacrine in kidney injury/AKI.
Most important, as the role of inflammasomes is central in this manuscript, clearly more background on the role of inflammasome components in kidney disease is necessary. Especially the current knowledge about inflammasome components in human AKI and human septic AKI should be discussed using up to date literature to underline/explain an importance of the research data presented in this manuscript.
In general, it is preferable to find more than just one reference to support a statement.

3. Raw data. The Western blot raw data provided are not sufficient to support the quality of these data. The protein bands should not be presented as cut-out pieces and clearly a protein standard with kDa annotations should be visible.

#### Experimental design

Experimental design
4. The use of the TUNEL assay needs explanation to the reader.
a) explain the abbreviation
b) explain the principle and the conclusions that can be drawn from the experiments

5. please provide an exact description for all devices used. Example: Leica DM4 B & DM6 B upright digital research microscopes? Leica DM1000? Leica DM4 M & DM6 M? etc.

6. For the qPCR the beta-actin primers are missing. Was the annealing temperature for all primers identical?

8. Kidney function in mice: did the authors record urine volume or weight during the experiments?

9. In the results section and figures: For all statements about “expression” it should be specified, if gene expression or protein expression/protein amount was analyzed.

10. For all experiments (PCR, Western blot, TUNEL assay, tissue staining etc.) the number of experiments, animals, replicates etc. must be indicated. Example n=number of experiments in N=number of animals. For staining: representative example out of n= analyses.

#### Validity of the findings

11. The two major shortcomings in the experimental design are
a) The authors need to be exact about the difference between inflammasome expression, inflammasome priming and inflammasome activation. Changes in gene expression of IL-1beta and IL-18 only point to NFkappa-B involvement, which can contribute to the amount of mature IL-1beta and IL-18. Inflammasome activity/activation can only be concluded from an increase of mature IL-1beta and IL-18 protein in cells or after secretion/release.

b) The use of an NLRP3 activator (nigericin) does not really support the involvement of NLRP3 in the theacrine effect in AKI. The authors need to show the effect of any kind of inflammasome inhibition.

Cite this review as

### Reviewer 2 · Apr 5, 2022

#### Basic reporting

The manuscript by Maoxian Yang et al showed the effect of Theacrine in sepsis-induced AKI. The English in the manuscript is generally used well; however, an expert needs to revise again the grammar and spelling. The references are adequate and sufficient.
The article structure needs to improve. The results support the hypothesis

#### Experimental design

-The authors provide wb images, however they did not put which group each WB band belongs to. Please, add these data.
-The authors do not show all the N of the WB for the 6 per group.
- The changes they report in their graphs for figure 5C are not very evident, please provide better images. Please point out the changes with arrows on each representative image.
-Please mention in the figure caption the N you are using and what statistical test was applied.

#### Validity of the findings

The manuscript is novelty and the conclusions are well stated.

Cite this review as

All text and materials provided via this peer-review history page are made available under a Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.