Ageratina adenophora causes spleen toxicity by inducing oxidative stress and pyroptosis in mice

Ageratina adenophora is an invasive weed with potent toxicological effects on livestock. Oxidative stress and pyroptosis play a pivotal role in regulating animal or human health and disease. The object of this study was to determine the mechanism underlying splenic toxicity induced by A. adenophora in a mouse model. Ageratina adenophora significantly increased the levels of reactive oxygen species and malondialdehyde, but decreased the antioxidants like catalase, superoxide dismutase, glutathione and glutathione peroxidase. In addition, the activity of the antioxidant enzymes was also decreased upon A. adenophora treatment. The induction of the pyroptosis pathway was evaluated in terms of the expression levels of Nod-like receptor protein 3, nuclear factor-κB, caspase-1, gasdermin-D and interleukin-1β, all of which were significantly elevated by A. adenophora. These findings suggest that A. adenophora impairs spleen function in mice through oxidative stress damage and pyroptosis.


Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? No

Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
The article is very interesting, and demonstrates the mechanisms involving the toxic effects of Ageratina adenofora in the spleen of treated mice. Some considerations should be examined. In the abstract the authors stated that the data indicate that A. adenofora impairs spleen function, however there is no evidences for this, despite the oxidative stress and pyroptosis. Maybe change the word indicate by suggest. Please confirm the age of mice -48 weeks?

21-May-2019
Dear Dr Hu, The editors assigned to your paper ("Ageratina adenophora causes spleen toxicity by inducing oxidative stress and pyroptosis in mice") have now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Associate Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Your manuscript has been reviewed by 3 reviewers. While all reviewers agree that the work is interesting, several points of concerns were brought up. In particular there is a lack of description/details in some parts and the placement into context of broader field could be done more thoroughly. I recommend a major revision with a point-by-point response to each concern before considering the manuscript any further.

Comments to Author:
Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) The manuscript by Sun et al., describes an experiment where 40 mice divided into groups of ten and then fed either control, 10%, 20%, or 30% A. adenophora in the form of a pelleted diet for 42 days. At the end of the study, the mice were sacrificed, spleens removed, and assays were performed. Comments: Introduction General comment: What motivated the investigators to test pyroptosis? More detail would be helpful to understand why the work was performed. Perhaps this could be used to better organize the introduction. Line 32, what regions worldwide? Line 33, what is a grave impact? More detail please. Line 36-40, please provide more detail about each species and what pathology this plant causes. Lines 64-65, Due to the lack of detail I cannot assess the validity of the statement "that it is unclear whether the toxic effects of A. adenophora on splenocyte involve pyroptosis." Materials and methods General comment, for the sake of the reader who isn't an immunologist please try to limit your use of abbreviations or at least define them. Maybe a sentence or two about what a technique does. For example, what is cell quest software?
Line 79-81, How exactly was the plant material stored? Was a voucher specimen obtained and deposited in a herbarium? How exactly was this plant material incorporated in to a pellet? Was a chemical characterization of this plant material performed? Line 152, what exactly is "mechanical method"? Lines 154 -158, The authors describe an ANOVA but no post-test. Why wasn't a post-test used?

Results
The authors should consider combining the results and discussion section to guide the reader through the data. This would allow for greater detail. For example, how exactly is NRRP3 an immune system damage sensor? The authors mention dose-dependency, a chemical characterization of the plant material and specific concentrations of plant compounds would provide greater support to this statement. Discussion The authors set out to investigate if A. adenophora causes oxidative stress and pyroptosis. A figure illustrating the pyroptocic pathway and a point by point discussion of this pathway with regard to the experimental results would improve the discussion.

Reviewer: 2
Comments to the Author(s) The authors present an interesting study of the spleen toxicity caused by Ageratina adenophora. This work provided a new insight into the mechanism of the toxicity of A. adenophora in a mice model. I agree to publish by Royal Society Open Science. But I have some comments: 1. Line 20: "Glutathione peroxidase" should be abbreviated to GPx. And abbreviations should be unified in line 94, 162 and 218. 2. The manuscript has some grammatical issues, and the authors should correct and improve them. The tenses of sentences should be consistent. Such as in lines 17-20, "the decrease…" should be corrected as "decreased the …" 3. Lines 113: space should be between number and unit. Same errors should corrected in Figure 6. 4. Line 75: The source format of reagent is generally expressed as "city, province and country", however, the sentence of line 75 lacks "province". 5. Line 123: replace "μl" with "μL". 6. Lines 190-191: What are "20% and 30% groups"? 7. Line 212: Please correct the expression of this sentence. 8. Although the manuscript is generally written clearly. Pay attention to minor errors. For Figure  5 caption, some words misspelled, which is confusing to read. 9. In figure 3A, high resolution picture should be used so that the reader could see the data clearly. 10. There are some unscientific expression. For instance, the "p" in line 199, 201 and 201 should be expressed in italics. The whole Ms should be revised. 11. Please modify the format of references according to magazine's requirements.

Reviewer: 3
Comments to the Author(s) The article is very interesting, and demonstrates the mechanisms involving the toxic effects of Ageratina adenofora in the spleen of treated mice. Some considerations should be examined. In the abstract the authors stated that the data indicate that A. adenofora impairs spleen function, however there is no evidences for this, despite the oxidative stress and pyroptosis. Maybe change the word indicate by suggest. Please confirm the age of mice -48 weeks?
Author's Response to Decision Letter for (RSOS-190127.R0) See Appendix A.

Recommendation?
Accept as is

25-Jun-2019
Dear Dr Hu, I am pleased to inform you that your manuscript entitled "Ageratina adenophora causes spleen toxicity by inducing oxidative stress and pyroptosis in mice" is now accepted for publication in Royal Society Open Science.
You can expect to receive a proof of your article in the near future. Please contact the editorial office (openscience_proofs@royalsociety.org and openscience@royalsociety.org) to let us know if you are likely to be away from e-mail contact. Due to rapid publication and an extremely tight schedule, if comments are not received, your paper may experience a delay in publication.
Royal Society Open Science operates under a continuous publication model (http://bit.ly/cpFAQ). Your article will be published straight into the next open issue and this will be the final version of the paper. As such, it can be cited immediately by other researchers. As the issue version of your paper will be the only version to be published I would advise you to check your proofs thoroughly as changes cannot be made once the paper is published.
On behalf of the Editors of Royal Society Open Science, we look forward to your continued contributions to the Journal. Consistent with this, our previous study has approved that ≥ 20% dose of A. adenophora increased the liver weight and caused extensive inflammation, in addition to decreasing antioxidant activity, increasing the production of reactive oxygen species (ROS) ." Lines 64-65, Due to the lack of detail I cannot assess the validity of the statement "that it is unclear whether the toxic effects of A. adenophora on splenocyte involve pyroptosis."

Response:
We have revised this statement in lines 62-66 to clarify the purpose to investigate pyroptosis, which may be a new mechanism involved in the toxic effects caused by A. adenophora

Materials and methods
General comment, for the sake of the reader who isn't an immunologist please try to limit your use of abbreviations or at least define them. Maybe a sentence or two about what a technique does.
For example, what is cell quest software?

Response:
In "Materials and methods" section, we have detailed the abbreviation of immunological related factors. Moreover, additional explanations are given for the techniques used in this study.
Line 79-81, How exactly was the plant material stored? Was a voucher specimen obtained and deposited in a herbarium?

Response:
We have revised in lines 81-83: "The collected leaves were cleaned, grounded and screened to make dry power. The power was stored in shade condition with ambient temperature at 20±2 ℃." A. adenophora specimen is not deposited in a herbarium but in our lab.
How exactly was this plant material incorporated in to a pellet?

Response:
We have revised in lines 83-85: "For the preparation of 10%, 20% and 30% diet pellet, A. adenophora and mice feed were homogenized in water solution by the ratio of 1:9, 1:8 and 1:7, respectively. Then the diet was cast in the form of cylinders and dried at 27 ℃ for 48 h." Was a chemical characterization of this plant material performed?
Response: Plant material chemical characterization was not performed in this study. But the major active compound related to A. adenophora was researched by our lab in previous study. The detail information could be obtain from the papers entitled "Anti-NDV activity of 9-oxo10,11-dehydroageraphorone extracted from Eupatorium adenophorum Spreng in vitro", "Clinical efficacy of 9-oxo-10,11-dehydroageraphorone extracted from Eupatorium adenophorum against Psoroptes cuniculi in rabbits" et al.
Line 152, what exactly is "mechanical method"?

Results
The authors should consider combining the results and discussion section to guide the reader through the data. This would allow for greater detail. For example, how exactly is NRRP3 an immune system damage sensor?
Response: Lines 178-179, "To test the effects of A. adenophora on pyroptosis pathway in vivo, we measured the protein levels of pyroptosis-related factors." was added in the result section. And we have detailed information in lines 218-223: "Inflammasomes are assembled by sensing a variety of tissue injury signals. Diverse stimuli could promote the release of inflammatory cytokines, including IL-1β. NLRP3 is currently the best characterized one, which plays an important role in the immune system as damage sensor. In addition, NLRP3 can also sense and be activated by ROS produced in the cell. It is known that GSDMD is a key pyrotosis executor [48].
GSDMD can be cleaved into GSDMD-N and GADMD-C by caspase-1. GSDMD-N promotes cell lysis and IL-1β release through forming pores by binding to cell membrane. Inflammasomes are assembled by sensing a variety of tissue injury signals. Diverse stimuli could promote the release of inflammatory cytokines, including IL-1β. NLRP3 is currently the best characterized one, which plays an important role in the immune system as damage sensor. In addition, NLRP3 can also sense and be activated by ROS produced in the cell. It is known that GSDMD is a key pyrotosis executor [48]. GSDMD can be cleaved into GSDMD-N and GADMD-C by caspase-1. GSDMD-N promotes cell lysis and IL-1β release through forming pores by binding to cell membrane." The authors mention dose-dependency, a chemical characterization of the plant material and specific concentrations of plant compounds would provide greater support to this statement.

Response:
A. adenophora was ground to uniform power and then homogenized with feed to produce different levels of diet. The results showed that the degree of oxidative stress and pyroptosis aggravated with the increased levels of A. adenophora-administration. Plant material chemical characterization was not performed in this study, but could be found in our previous studies related to study on the biological properties of active compounds from Ageratina adenophora (Title: Euptox A induces G1 arrest and autophagy via p38 MAPK-and PI3K/Akt/mTOR-mediated pathways in mouse splenocytes).

Discussion
The authors set out to investigate if A. adenophora causes oxidative stress and pyroptosis. A figure illustrating the pyroptosic pathway and a point by point discussion of this pathway with regard to the experimental results would improve the discussion.
Response: A schematic diagram of A. adenophora causes oxidative stress and pyroptosis ( Figure   8) was added in this revised version manuscript. It will be helpful for understanding pyroptosic pathway. And we have revised the discussion section in lines 218-223: "Inflammasomes are assembled by sensing a variety of tissue injury signals. Diverse stimuli could promote the release of inflammatory cytokines, including IL-1β. NLRP3 is currently the best characterized one, which plays an important role in the immune system as damage sensor. In addition, NLRP3 can also sense and be activated by ROS produced in the cell. It is known that GSDMD is a key pyrotosis executor [48]. GSDMD can be cleaved into GSDMD-N and GADMD-C by caspase-1. GSDMD-N promotes cell lysis and IL-1β release through forming pores by binding to cell membrane.
Inflammasomes are assembled by sensing a variety of tissue injury signals. Diverse stimuli could promote the release of inflammatory cytokines, including IL-1β. NLRP3 is currently the best characterized one, which plays an important role in the immune system as damage sensor. In addition, NLRP3 can also sense and be activated by ROS produced in the cell. It is known that