PONE-D-25-32780Electroacupuncture Alleviates Pain-Like Behaviors Through Modulating DNMT3a/MOR Signaling Pathway in CCI RatsPLOS ONE

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Reviewers' comments:

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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: Yes

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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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

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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: This research investigates the mechanism by which electroacupuncture (EA) at Zusanli (ST36) and Yanglingquan (GB34) alleviates neuropathic pain in CCI rats, finding that EA relieves pain-like behaviors by inhibiting DNMT3a expression and restoring MOR expression in the dorsal root ganglion (DRG). The study is well-designed and the results are clearly stated. I suggest to accept the manuscript for publication after clarifying the following minor questions.

Q1. In the Abstract, the description of the duration of EA treatment is somewhat ambiguous, and the wording should be revised to enhance readability. Also, the full name should be indicated when an acronym appears for the first time.

Q2. Add explanations of the other detected indicators (Fos, ERK1/2, pERK1/2, CD11b, Iba1, and GFAP) in the Introduction section. This will help readers understand the relevance of these indicators to neuropathic pain and the study's mechanistic framework, thereby enhancing the logical coherence of the introduction.

Reviewer #2: Dear Editor,

Thank you for the opportunity to review this manuscript entitled “Electroacupuncture Alleviates Pain-Like Behaviors Through Modulating DNMT3a/MOR Signaling Pathway in CCI Rats.” The authors investigated the potential mechanism by which electroacupuncture alleviates CCI-induced pain in rats, focusing on the DNMT3a/MOR signaling pathway. This study holds certain scientific value and research significance. Below are my detailed comments and suggestions for the authors and editors to consider:

1. Overall evaluation

This manuscript focuses on pain mechanisms induced by chronic constriction injury (CCI) in rats, exploring the therapeutic effect of electroacupuncture. The authors combine RT-qPCR, immunofluorescence staining, Western blot, and behavioral pain tests to investigate whether electroacupuncture may attenuate pain by downregulating DNMT3a expression and upregulating MOR expression in DRG. Overall, the manuscript has a relatively complete structure, and the experimental design is clear, with some data being convincing. However, several issues remain. I recommend that the authors revise by adding experimental details, improving image quality, and strengthening logical interpretation before the manuscript is considered for publication.

2. Major issues and suggestions

(1) In Figure 2, the authors examined the dynamic changes of Fos mRNA in DRG and SCDH under the CCI model, providing molecular-level evidence for activation of the pain pathway. Notably, they found that transcriptional levels were significantly increased on the ipsilateral side in the CCI group and that electroacupuncture intervention could inhibit this effect, which is a valuable finding. However, relying solely on Fos mRNA levels as evidence of neuronal activation has clear limitations:

① mRNA elevation does not necessarily translate into increased functional c-Fos protein expression, which is the widely accepted marker for neuronal activation.

② The current detection method cannot localize the specific activated cell types (neurons vs. glial cells) or spatial distribution (e.g., specific laminae of the spinal dorsal horn).

Therefore, I strongly recommend that the authors add a double immunofluorescence co-labeling experiment (e.g., c-Fos/NeuN) to confirm the inhibitory effect of electroacupuncture on pain-related neuronal activation, and to specify its location and quantity. If experimental conditions do not allow this, please address in the discussion section:

① clearly state that the conclusions are based only on transcriptional data;

② interpret the results cautiously (e.g., “Increased Fos mRNA suggests possible neuronal activation, which needs further protein validation”);

③ explicitly acknowledge the limitation of not detecting protein expression and cell-type localization.

(2) The fluorescence images in Figure 3C and Figure 4H are not sufficiently clear. Please provide higher-resolution or clearer images to ensure accurate representation of the results.

(3) In the mechanistic diagram (Figure 7), the authors propose that CCI can induce upregulation of DNMT3a expression in DRG, along with increased transcription of Oprm1 and Kcna2. However, no experimental data or figures supporting these two targets are provided in the manuscript. Please supplement with relevant data or figures. Additionally, the manuscript mentions a “cascade reaction,” but this should be substantiated experimentally; otherwise, the term should be revised or used more cautiously.

(4) The discussion section is somewhat lengthy and parts of it lack clear structure. It is recommended to restructure this section by adding subheadings or adjusting the logical flow to improve clarity and coherence.

3. Minor issues and suggestions

(1) Please add the full name of DNMT3a in the abstract to help readers better understand the study background.

(2) Figure 1 lacks a figure number in the manuscript. Please check and correct this. Also, the rat model illustration in Figure 1 appears rather rough; it is recommended to redesign it for better clarity and professionalism. In Figure 2, please add scatter points on the bar charts to indicate the sample size and distribution, which would enhance the transparency of the data.

(3) Figure 5E and Figure 5F lack serial number labels. Please check and revise accordingly.

4. Recommendation

Major revision (Revision required before reconsideration)

I hope these comments will help the authors to improve the manuscript. With additional experimental support and enhancements in figure presentation and logical structure, the study may reach the publication standard.

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Reviewer #1: No

Reviewer #2: No

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Electroacupuncture Alleviates Pain-Like Behaviors Through Modulating DNMT3a/MOR Signaling Pathway in CCI Rats

PONE-D-25-32780R1

Dear Dr. Zhao,

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.

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Kind regards,

Yung-Hsiang Chen, Ph.D.

Academic Editor

PLOS ONE

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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 #2: 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 #2: Yes

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

Reviewer #2: 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 #2: 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 #2: Yes

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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 #2: All comments have been addressed, and the manuscript has been improved significantly. I have no more concerns.

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Reviewer #2: Yes:  Diansan Su

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