PONE-D-24-15025Immunohistochemical analyses reveal FoxP3 expressions in spleen and colorectal cancer in mice treated with AOM/DSS, and their suppression by glycyrrhizinPLOS ONE

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Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The present manuscript focuses on the effects of glycyrrhizin (GL) on inflammation and carcinogenesis in a murine model of colorectal cancer (CC). Wang and colleagues examine the role of regulatory T cells (Tregs) in this process and the potential mechanisms by which GL exerts its effects. The authors conducted experiments using ICR mice divided into four groups: control, GL alone, CC alone, and CC with GL treatment (CC+GL). After inducing colorectal cancer using azoxymethane (AOM) and dextran sodium sulfate (DSS), they measured various parameters such as spleen weight, white and red pulp areas, as well as different immune cell populations in splenic and colorectal tissues. The study reveals that during AOM/DSS-induced colon carcinogenesis, spleen enlargement and accumulation of Tregs and CD8+ cells (CTL) occur through the HMGB1-TLR4/RAGE NF-κB signaling pathway. GL inhibits these

signaling pathways by targeting HMGB1, potentially impeding carcinogenesis processes.

However, the following issues must be addressed.

1. Did the authors measure the CD4 T cell population in the red pulp? Does GL affect CD4 T effector cells in CC and CC+GL mice? If the authors had used flow cytometry, they might have obtained more precise quantitative data regarding immune cell populations. This represents one of the limitations of the study.

2. GL treatment alone increases the basal levels of CD8 and CD11c in the spleen and Foxp3 and RAGE expression in tumor tissue. Although the authors noted weak staining, could this be due to an overdose or potential side effects of GL? Explain.

3. Is there any difference in the expression of FoxP3 and RAGE in the CC+GL mice group between CC1 and CC2 regions?

4. The method section is not clearly written. How do the authors normalize spleen weights? Do they use tibia lengths?

5. Throughout the manuscript, the authors measured cell counts or semi-quantitative staining grading to assess immune cell populations in IHC staining. They could have quantified the intensity of staining and normalized the total area, potentially avoiding human errors and bias.

6. While the study suggests mechanisms involving Tregs and HMGB1, more detailed mechanistic studies are needed to fully understand GL's effects.

7. Understanding the effects of GL and Tregs in other tissues related to colorectal cancer progression would provide a more comprehensive picture.

8. Using additional colorectal cancer models, such as genetically engineered models or patient-derived xenografts, would provide a more comprehensive understanding of GL's potential effects across different cancer contexts.

9. Further studies are needed to determine the clinical relevance of these findings in human colorectal cancer patients. The authors should mention the limitations of the manuscript.

10. Authors must thoroughly check their manuscript for typos and spacing errors.

Reviewer #2: Authors in this manuscript discuss most possible mechanisms by which Glycyrrhizin might suppress colon cancer development. The study focuses on the role of inflammation and immune response in the process.

The authors made a good colon cancer mice model via Inflammation induced by chemicals (AOM/DSS) damages DNA and promotes colon cancer. Resulting damage triggers a chain reaction involving molecules like NO, HMGB1, and signaling pathways.

Splenomegaly phenotype is seen due to the accumulation of immune cells CTLs, Tregs, DCs responding to inflammation and potential cancer.

Glycyrrhizin appears to disrupt this process by binding to HMGB1 and potentially affecting other signaling pathways, ultimately suppressing Tregs and cancer cell growth.

The data highlights the role of specific molecules HMGB1, NO and immune cells CTLs, Tregs in the process. Mechanism of GL's anticancer effect is proposed with supporting evidence from previous studies and present work.

The exact mechanism by which GL suppresses STAT3 and FoxP3 needs further investigation.

Overall, the manuscript provides a reasonable explanation of a potential mechanism for GL's anticancer properties. It highlights the complex interplay between inflammation, immune response, and cancer development. Further research is needed to validate the proposed mechanism fully. Though not completely new, this study adds a decent piece of knowledge to the existing literature on Glycyrrhizin`s possible mechanism of action.

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Reviewer #1: Yes: Suman Asalla

Reviewer #2: No

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Immunohistochemical analyses reveal FoxP3 expressions in spleen and colorectal cancer in mice treated with AOM/DSS, and their suppression by glycyrrhizin

PONE-D-24-15025R1

Dear Dr. Murata,

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Academic Editor

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Comments to the Author

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Reviewer #1: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

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4. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: (No Response)

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

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Reviewer #1: Yes: Suman Asalla

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