PONE-D-24-12451Chlorogenic acid inhibits NLRP3 inflammasome activation through Nrf2 activation in diabetic nephropathyPLOS ONE

Dear Dr. Bao,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

 Specifically, please address the following points:

• Inclusion of complete western blot membranes with molecular weight markers important for evaluating pro- and cleaved forms of respective enzymes and cytokines. This may be included as supplementary information.
• Review and address concerns regarding the different dosing concentrations described throughout the manuscript.
• Review and address concerns regarding language some of the reviewers found confusing. For example, the statement regarding Nrf2 and its negative regulation of the NLRP3 inflammasome. You may consider addressing this by expanding the introduction and/or discussion.
• Address concerns regarding lack of detail in the methodology including, but not limited to, catalog numbers, dilution factors, etc.
• Address concerns regarding the presentation of figures including, but not limited to, inclusion of arrows to emphasize respective areas of figures, normalization of figure presentation, etc.
• Address recommendations regarding inclusion of NLRP3 inhibitors or NLRP3 KO mice, and potential explanations for mechanisms of NLRP3 activation of DN.

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PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. 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: Partly

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Partly

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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4. 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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: 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: This paper was mainly explored through two dimensions in vivo and in vitro. In vivo, the diabetic model rats were experimentally verified by ELISA, HE staining, PAS staining and Western blot. In vitro, HK-2 cells were used to explore the protein expression changes of inflammasome and Nrf2 pathway by Western blot, and the expression changes of inflammasome and Nrf2 pathway proteins were explored by Western blot after siRNA knockdown of Nrf2 expression, so as to verify that chlorogenic acid inhibits the activation of NLRP3 inflammasome in diabetic nephropathy by activating Nrf2.

The completion of this paper is good, the basic indicators have been verified, and the main problems are that the experimental grouping is thin, and the experimental design and the use of experimental methods are relatively simple and not novel enough. The content is relatively routine, and the verified mechanism has become universal. The main issues of the manuscript are as follows:

1. The weight of the Chinese drug group in Fig1 B was higher than that of the model, which was inconsistent with the description of the results.

2. It is recommended that the WB section provide the whole membrane；

3. Only one dose was used in the treatment group, and the angle was relatively simple. There was no experimental attempt to discuss the correlation between the drug dose and the treatment effect.

4. The content of the experimental design is relatively modeled, not novel enough, and there are some bright spots lacking in technology, so it is recommended to enrich the experimental design.

Reviewer #2: Notes on Review for PLOS One

“Chlorogenic acid inhibits NLRP3 inflammasome activation through Nrf2 activation in diabetic nephropathy”

Accept with Minor Revisions

Review Comments to Author:

This manuscript is technically sound, and the data supports all conclusions presented in the final document. Statistical Analysis has been performed and indicates that the results presented are statistically significant. The authors have also made it clear that all data underlying the findings in the manuscript are fully available.

Summary:

Diabetic neuropathy is a serious complication of both type 1 and type 2 diabetes, and it often affects the kidneys’ ability to remove waste products and extra fluid from the body. Inflammation caused by the NLRP3 inflammasome and further Nrf2 activation is a large contributing factor to the progression of diabetic nephropathy (DN). Chlorogenic acid (CGA) could activate Nrf2, which is found in nature. This study evaluated the ability of CGA to reduce inflammation. Findings showed that serum creatinine (Scr), blood urea nitrogen (BUN), and urinary protein excretions decreased after applying CGA. This occurs because CGA can activate the function of Nrf2 and halt the activity of NLRP3. Thus, this study demonstrated that chlorogenic acid could decrease DN progression and showed further that this impact is largely due to suppression of the NLRP3 inflammation and is modulated through the Nrf2 pathway. This research represents potential therapeutic implications for DN.

Abstract:

Comments: Overall, the abstract explains the background and premise of the article concisely and fully.

Major Revisions:

Minor Revisions:

1. In the abstract, you wrote: “CGA administration can active the Nrf2 pathway and inhibited NLRP3 inflammasome activation.” However, it should be noted that “CGA administration can activate the Nrf2 pathway and inhibit NLRP3 inflammasome activation.”

Introduction:

Comments:

The introduction concisely explains DN and how NLRP3 plays a role in developing the disease. It provides an adequate basis for the overall study.

Major Revisions:

1. The hypothesis should be rephrased; the way it is currently explained it does not explain how the Nrf2 negatively regulates NLRP3 inflammasome, particularly in this segment “negatively regulated the activation of NLRP3 inflammasome via regulation of the NLRP3 inflammasome.”

Minor Revisions:

None

Methods:

The methods described here were sufficient

Major Revisions: None

Minor Revisions:

I see that there are statistics performed on each of these experiments, but I could not find where there was a mention of how many times these experiments were completed. If they were done in duplicate or triplicate, please state this in the methods, particularly for the western blot analysis results and subsequent relative protein quantification.

Results:

Major Revisions: None

Minor Revisions: None

Though this is minor, I would make sure that all figures are uniform, there are a few that do not have the same borders which may make interpreting where certain bars are on the y axis confusing.

I think it was wise to quantify the relative levels of protein in western blot analysis.

Discussion:

Major Revisions: None

Minor Revisions:

1. The discussion mentions that “Growing evidence has demonstrated that some natural plant compounds or herbal products have therapeutic potential via regulation of inflammation to prevent and potentially treat DN[20-22]” but does not provide further details about where these compounds are found. This section would be improved in examples of where these compounds are found were also included.

2. Some small grammar repairs, consider changing “Chlorogenic acid (CGA) with a good Nrf2 activation effect possessed anti-inflammatory property. Little information concerning CGA is available on DN, and the underlying mechanism still remain to be further revealed.”

To “Chlorogenic acid (CGA) with a good Nrf2 activation effect possessed anti-inflammatory properties. Little information concerning CGA is available on DN, and the underlying mechanism still remains to be further revealed.”

Reviewer #3: The primary claim made by the authors of this submission is that the administration of Chlorogenic acid (CGA) demonstrates a renoprotective effect in the context of Diabetic Neuropathy (DN). Further the authors posit this effect is partially mediated through down-regulation of the NLRP3 inflammasome activation via the Nrf2 pathway. Reduction in the progression of kidney damage after treatment with CGA is significant due high rates of mortality despite treatment.

The authors cite multiple studies demonstrating the role inflammation reactions play in the development of DN as well as their own previous work demonstrating increased renal Nrf2 expression following CGA treatment.

Based on previous literature reports and their own previous studies on CGA treatment, the authors hypothesize that “Nrf2 negatively regulated the activation of NLRP3 inflammasome via regulation of the NLRP3 inflammasome in DN.” This statement is not written clearly and adds confusion to the mechanism the authors are targeting. The hypothesis of any research study should be clear and unambiguous as to the authors intended meaning. This statement should be re-written to explain their reasoning and scientific justification more clearly.

The authors used both an in-vitro model of cells supplemented with high levels of glucose as well as a high-fat/STZ induced diabetic rat model to replicate the Diabetes Mellitus condition. They clearly demonstrated their intended targets of study associated with NLRP3 inflammasome mechanism of action such as caspase-1, IL-1b, and IL-18. Additionally, they have identified a downstream protein of interest, heme oxgenase-1 (HO-1), which is associated with the Nrf2 pathway activation. The significance of these targets to clinical disease and oxidative stress were supported by previous literature. The methods associated with the experimental assays – histology, western blot, and blood and urine chemistries - were detailed and informative to allow for experiments to be reproduced.

Most of the results and data figures were clearly labeled and supported the conclusions stated by the authors. However, in section 3.1 the authors state that diabetic rats treated with CGA [DM + CGA group] had a lower body weight compared to untreated diabetic rats [DM group]. This is result is not what is represented in figure 1B – the DM + CGA treatment group are shown to have a higher body weight compared to the DM group. This result should be re-written to match the corresponding figure.

Overall, I felt this was a strong manuscript with a clearly defined objective and orthogonally designed experiments. The conclusions drawn by the authors are supported by the results and delve into the possible mechanisms associated with the benefits of CGA treatment in Diabetes Mellitus and inflammatory reactions. The hypothesis should be revised to clearly state their goal as well as address the conclusion drawn from figure 1B.

Reviewer #4: In this manuscript, Bao et al. explore the effects of chlorogenic acid (CGA) on diabetic nephropathy (DN). Using a diabetic rat model, they find that CGA has potential therapeutic implications for slowing the progression of DN by activating the Nrf2 pathway and inhibiting NLRP3 inflammasome activation. However, the data presented in the manuscript are sometimes not convincing and lack proper description. Addressing the following comments could help improve the quality of this study:

1. It would be better if providing histological quantification and using arrowheads to indicate pathological damage in Figure 2.

2. The catalog numbers of the antibodies used in the manuscript are not provided.

In all Western blot images, it is unclear whether IL-1β, IL-18, and caspase-1 proteins are in their pro or cleaved forms, since cleaved caspase-1 and cytokines (IL-1β, IL-18) are indicators of NLRP3 inflammasome activation. Providing additional data, such as ASC oligomerization or immunohistochemistry for NLRP3 and ASC proteins in the kidney or cells, could offer more solid evidence for NLRP3 activation. Alternatively, it would be best to use NLRP3-deficient mice to determine if NLRP3 is truly involved in the progression of DN would strengthen the findings. If knockout mice are unavailable, NLRP3 inhibitors could be used.

3. Please include molecular weight markers in all Western blot images.

4. The NLRP3 inflammasome activates the downstream effector caspase-1, leading to caspase-1-mediated pyroptosis. Since the authors have identified NLRP3 activation under their experimental conditions, it would be interesting to investigate whether CGA could prevent DN by inhibiting NLRP3 inflammasome activation and subsequently reducing pyroptosis,

5. The discussion section could be improved by exploring possible mechanisms of NLRP3 activation in DN and how Nrf2 is induced as part of the defense system against oxidative stress, as well as how NLRP3 inhibition occurs under these conditions.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Nicole Grigaitis-Esman

Reviewer #4: No

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Submitted filename: Notes on Review for PLOS One.docx

Chlorogenic acid inhibits NLRP3 inflammasome activation through Nrf2 activation in diabetic nephropathy

PONE-D-24-12451R1

Dear Dr. Yang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Jordan Robin Yaron, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

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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 #3: Yes

Reviewer #4: Yes

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

Reviewer #3: Yes

Reviewer #4: Yes

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: Yes

Reviewer #4: Yes

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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 #3: Yes

Reviewer #4: Yes

**********

6. 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 #3: The authors have addressed all of my comments and recommendations in initial review, including rewording their hypothesis to convey a more concise statement and addressing the errors in Figure 3.1.

Reviewer #4: The molecular weight of cleaved caspase-1 is not 45 kDa, as stated. The authors may need to review and correct this information to ensure accuracy.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: Yes: Nicole A. Grigaitis

Reviewer #4: No

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