Dear Dr. Sun,

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.

Please submit your revised manuscript by Jan 15 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Songyun Zhao

Academic Editor

PLOS ONE

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2. Thank you for stating the following financial disclosure: [This research was supported by the Liaoning Provincial Science and Technology Program (2022-MS-329), the General Program of National Natural Science Foundation of China�82071911�, and the Science and Technology Innovation Fund of Dalian�2021JJ12SN38�.]. Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

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5. Please include a caption for figure 8.

6. Please ensure that you refer to Figure 7 in your text as, if accepted, production will need this reference to link the reader to the figure.

Additional Editor Comments (if provided):

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #1: Partly

Reviewer #2: Yes

**********

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

Reviewer #1: No

Reviewer #2: Yes

**********

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

The PLOS Data policy

Reviewer #1: No

Reviewer #2: Yes

**********

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

Reviewer #1: No

Reviewer #2: Yes

**********

Reviewer #1: This study examines the role of chloride intracellular channel 1 (CLIC1) in oxidative stress and inflammation during cerebral ischemia–reperfusion injury (CIRI) using an oxygen and glucose deprivation/reoxygenation (OGD/R) model in HT22 hippocampal neurons. The results show that CLIC1 is significantly upregulated under OGD/R conditions, leading to neuronal damage by suppressing the Nrf2/HO-1 signaling pathway. This suppression exacerbates oxidative stress, inducing apoptosis and NLRP3 inflammasome-mediated pyroptosis. Silencing CLIC1 alleviates these effects, reducing oxidative stress markers, inflammasome activity, and cell death. The findings highlight CLIC1's dual role in apoptosis and pyroptosis and suggest its potential as a therapeutic target to mitigate CIRI-induced neuronal injury by modulating the Nrf2/HO-1 pathway. I recommend publishing this manuscript with the inclusion of the following significant corrections, elaborated below, to further enhance its clarity, robustness, and scientific rigor.

1. Figures: Figures need higher resolution (300–600 dpi, per PLOS ONE guidelines) and more detailed, self-explanatory legends to improve clarity and interpretability.

2. Heatmaps: Gene expression and correlation heatmaps require better labeling for easier understanding.

3. Abbreviation Use: Abbreviations (e.g., NLRP3, GSDMD) should be defined at first mention and used consistently throughout the text.

4. Reference Formatting: The reference list does not fully adhere to journal guidelines. Full journal names (e.g., "Biomedicine & Pharmacotherapy" instead of "Biomed Pharmacother") and consistent DOI formatting are required.

5. Language and Grammar: Minor grammatical issues (e.g., “to promoted apoptosis” instead of “to promote apoptosis”) and wordiness reduce readability and need correction.

6. Novelty: The study needs to better emphasize its novel contribution, particularly the regulatory role of CLIC1 in the Nrf2/HO-1 pathway and its implications.

7. Mechanistic Clarity: While the study links CLIC1 to pyroptosis/apoptosis via Nrf2/HO-1, it does not explore alternative pathways or contributing factors, such as other stress-response mechanisms.

8. Experimental Design:

- Control Groups: Details on control groups, including sham-treated groups, are insufficient, raising concerns about baseline comparisons.

- Sample Size: With only three replicates per group, conclusions may lack statistical robustness. A power analysis is needed.

9. Pathway Validation: The evidence for Nrf2/HO-1 inhibition or silencing requires stronger validation, such as co-immunoprecipitation or experiments using pathway-specific inhibitors.

10. Methods: Experimental details, including validation of viral silencing and OGD/R treatment conditions, are inadequately described, which may hinder reproducibility.

Addressing these issues will significantly improve the manuscript’s clarity, scientific rigor, and alignment with journal guidelines.

Reviewer #2: In this manuscript, the authors use a well-established model (OGD/R-treated HT22 cells) and robust methodologies, including real-time PCR, western blotting, flow cytometry, and ELISA, to investigate apoptosis and pyroptosis. The study links CLIC1 overexpression with oxidative stress-induced apoptosis and NLRP3-mediated pyroptosis, providing mechanistic evidence via modulation of the Nrf2/HO-1 pathway. To enhance the study's rigor, several improvements could be made:

1. Lack of In Vivo Validation: While the cell model findings are significant, they may not fully translate to in vivo systems. Including animal studies would strengthen the conclusions. Please add in vivo experimental verification if conditions permit.

2. Some sentences are overly complex, leading to occasional ambiguity (e.g., in the introduction and discussion).

3. Several grammatical errors and awkward sentence structures are present. For example: "And this process led to an exacerbation of apoptosis..." → Avoid starting sentences with "And." "Upon CLIC1 silencing, all the mentioned indicators above exhibited an inverse trend..." → Simplify to: "Silencing CLIC1 reversed these effects." Repetition: The phrase "CLIC1 silencing" is overused. Vary terminology to improve readability.

4. In the discussion section, while the discussion highlights the importance of the findings, it occasionally reiterates results rather than expanding on their broader implications.

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what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Dear Dr. Sun,

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.

Please submit your revised manuscript by Apr 07 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Songyun Zhao

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

**********

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

Reviewer #2: Yes

**********

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

Reviewer #2: Yes

**********

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

The PLOS Data policy

Reviewer #2: Yes

**********

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

Reviewer #2: Yes

**********

Reviewer #2:  The author have addressed my concerns properly.

However, some of the references in the article are dated. It is recommended to add recently published studies on related topics in the field.

Please add the following references

1. Zhao, S., Zhuang, H., Ji, W. et al. Identification of Disulfidptosis-Related Genes in Ischemic Stroke by Combining Single-Cell Sequencing, Machine Learning Algorithms, and In Vitro Experiments. Neuromol Med 26, 39 (2024). https://doi.org/10.1007/s12017-024-08804-2.

2. Qian, Y., Yang, L., Chen, J. et al. SRGN amplifies microglia-mediated neuroinflammation and exacerbates ischemic brain injury. J Neuroinflammation 21, 35 (2024). https://doi.org/10.1186/s12974-024-03026-6.

3. Zhuang H, Lei W, Wu Q, Zhao S, Zhao Y, Zhang S, Zhao N, Sun J, Liu Y. Overexpressed CD73 attenuates GSDMD-mediated astrocyte pyroptosis induced by cerebral ischemia-reperfusion injury through the A2B/NF-κB pathway. Exp Neurol. 2025 Jan 18;386:115152. doi: 10.1016/j.expneurol.2025.115152.

**********

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 #2: No

**********

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org

CLIC 1 down-regulates Nrf2/HO-1 signalling pathway promoting the apoptosis and pyroptosis in OGD/R-treated HT22 cells

PLOS ONE

Dear Dr. Sun,

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 all the points raised during the review process.

Please submit your revised manuscript by Jul 26 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Mária A. Deli, M.D., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments:

Unfortunately none of the original 2 reviewers were available for the re-review of the manuscript. A third independent reviewer was asked to evaluate the second revision, who very carefully and critically read the paper and suggested several improvements. While no further experiments are needed, many small corrections, explanations need to be done. Importantly, the text also needs English editing!

Comments from PLOS Editorial Office: We note that one or more reviewers has recommended that you cite specific previously published works in an earlier round of revision. As always, we recommend that you please review and evaluate the requested works to determine whether they are relevant and should be cited. It is not a requirement to cite these works and you may remove them before the manuscript proceeds to publication. We appreciate your attention to this request.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: (No Response)

**********

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

Reviewer #3: Partly

**********

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

Reviewer #3: Yes

**********

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

The PLOS Data policy

Reviewer #3: No

**********

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

Reviewer #3: No

**********

Reviewer #3: This study demonstrates that manipulating the expression of CLIC1 may affect the development of inflammation and oxidative stress in an in vitro model of cerebral ischemia-reperfusion injury. However there are a few major gaps in the conclusions of this study.

1) How do the authors reconcile the fact that manipulating the same molecule CLIC1 may lead to two distinct cell death processes – apoptosis and pyroptosis that can not occur at the same time. Is there a preferential pathway to one or the other, or a state of the cell or a time window?

2) What is the rationale for the CLIC1 multiple roles in inflammation and oxidative stress in ischemic damage. Using the term “cellular adaptor“ (Discussion- line 5) will not do.

3) It is never disclosed what is the basis of the hypothesis that CLIC1 would suppress Nrf2/HO-1. In fact, it is not clearly disclosed what is the initial downstream pathway for the many CLIC1 effects.Only in Fig 1 “ARE“ is indicated without further mentioning or even explaining the abbreviation.

4) How do the authors reconcile their proposal of therapy by supressing CLIC1 and apoptosis/pyroptosis with their stating that CLIC1 has a cellular defensive mechanism (p. 16, 2pgph line 2-3) or that it stabilizes cation fluctuations during stress conditions and cytoplasmic oxidation (p17 last line, p18 lines 1-2) as well as with the sentence “Apoptosis aids in clearing infected cells, whereas pyroptosis facilitates the removal of intracellular pathogens and the activation of immune responses.“

In addition:

The experiment in Fig. 4 using activated NLRP3 after silencing CLIC1 to determine if NLRP3 mediates pyroptosis in OGD/R seems forced and superfluous since in the same Figure it was shown that NLRP3 is activated by OGD/R itself followed by pyroptosis markers. It is also merely strange to state in the same Fig legend (line 4-5) that activation of NLRP3 upregulates itself!

Page 4, 2nd pgph, line 5 : “CLIC1 ...removing excessive negative charge via anion channel“ However CLIC1 IS an anion channel

Legend to Fig. 1 is missing

First pgph on page 14 should belong to the part of text with Figure 4 .

There are a lot of typos and English is below acceptance level (eg “A recent study discovered that in umbilical vein endothelial cells, CLIC1 high-expressed and impaired the ability of vascular cells to resist oxidative stress to speed up cellular senescence, whose inhibition conversely perform a protective effect on preventing cellular aging and disorder“)

The authors claim that all relevant data are within the ms but this is not the case – jus the means and errors can be found.

Why do the authors disclose ethical review of animal welfare when they used no animals in their experiments? Only commercially available animal cell line was used

**********

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: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Dear Dr. Sun,

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.

Please submit your revised manuscript by Sep 19 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Mária A. Deli, M.D., Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: All comments have been addressed

**********

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

Reviewer #3: Yes

**********

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

Reviewer #3: Yes

**********

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

The PLOS Data policy

Reviewer #3: No

**********

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

Reviewer #3: Yes

**********

Reviewer #3: Comments are addressed thoroughly and successfully. Nevertheless, it would be more appropriate in the revised ms to use examples from CNS and not from cancer (Discussion Pgph 6) or vascular and bronchial tissues (Introduction pgph 3).

In addition, text in Duscussion pgph 2 5th line from the end should be changed to read "...highlighting its role in the apoptotic process..."

**********

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:  Pavle Andjus

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org

CLIC 1 down-regulates Nrf2/HO-1 signalling pathway promoting the apoptosis and pyroptosis in OGD/R-treated HT22 cells

PONE-D-24-49743R4

Dear Dr. Sun,

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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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Mária A. Deli, M.D., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

I thank the authors to do the final round of amendments. Since the last reviewer already accepted the paper, no need for further rounds of review, I consider the manuscript scientifically acceptable for publishing.

Reviewers' comments: