Verified Rigor RecordRigor Reviewed

Rigor-Reviewed Manuscript

Tregs depletion aggravates activation of astrocytes by modulating IL-10/GXP4 following cerebral infarction · 10.3389/fimmu.2023.1255316

Name: Verified Rigor Record — Tregs depletion aggravates activation of astrocytes by modulating IL-10/GXP4 following cerebral infarction · 10.3389/fimmu.2023.1255316
Item: Tregs depletion aggravates activation of astrocytes by modulating IL-10/GXP4 following cerebral infarction · 10.3389/fimmu.2023.1255316
Rating: 39
Author: Αlpha¹

Paper source

Αlpha¹ Editorial

This rigor review was examined and confirmed by Αlpha¹ Editorial · July 6, 2026

Αlpha¹ Rigor Review

Rigor Reviewed

CRITICAL

Critical rigor failure · Mixed Methods

Eight dimensions — click a segment for details

Critical

High-priority

Model-reported confidence: highReviewer agreement: highEngine: 7.5.0Source: full text

Not verified in this report

No reported statistical tests were found to recompute.
Declared data/code links were not checked for liveness or content.

This Αlpha¹ Rigor Review uses AI Rigor Reviewers trained on a curated corpus of high-fidelity and retracted papers, with expert supervision and curation. It can still make mistakes; verify each finding against the source before relying on it.

Critical findings

• Treg depletion attenuates renal fibrosis by modulating IL-10/GPX4 following cerebral infarction.

The paper combines a human transcriptomic analysis with a mouse MCAO model and in vitro experiments, but it suffers from multiple rigor weaknesses: incomplete reporting of study design (no randomization, blinding, power analysis), missing biological variable details (sex, strain, housing), and no discussion of limitations. The statistical reporting lacks exact p-values, effect sizes, and confidence intervals. Several internal inconsistencies (e.g., an unsupported claim about renal fibrosis, a 'rats' vs 'mice' mismatch, and ambiguous antibody naming) further reduce confidence.

Both independent reviewers (same model family, different instances) converged on all eight dimensions, giving consistent evidence. The statistics component could not recompute any tests due to threshold-only p-values; mathematical plausibility checks were not applicable. Citation checking found no retracted or missing references.

10 major claims checked against the paper's own evidence: 1 not fully backed by the presented evidence (unsupported or overstated).

7 integrity concerns flagged (0 high).

Rigor

Eight Dimensions8 warn

10 major claims1 unsupported5 partial4 supported

The introduction cites prior work on Tregs in stroke, tissue-specific Tregs, and ferroptosis, establishing a rationale for studying Tregs in cerebral infarction. However, the human sepsis dataset (GSE221379) used for differential expression analysis is never justified in relation to the main mouse stroke study — the link between sepsis and cerebral infarction is not explained, making the premise rationale weak. Limitations of prior research are not discussed.

Sub-criteria

prior work citedADEQUATE

premise rationaleINADEQUATE

limitations addressedNOT REPORTED

Per reviewer (2) — the votes behind the verdict

WARNReviewer 1· 75% conf

The premise is supported by prior citations, but there is no logical connection between the human sepsis microarray analysis and the mouse MCAO study, and limitations of prior work are inadequately addressed.

Evidence

direct quote[Introduction, paragraphs 1-4]

“In stroke research accumulation of tregs during the two weeks has been observed (, ). Brain Tregs contribute to neurological symptom recovery by suppressing excessive activation of astrocytes, a condition known as astrogliosis, through the secretion of Areg ().”

direct quote[Methods, section 2.1]

“The transcriptome characteristics of the postmortem parietal cortex were compared between individuals who died from sepsis and non-septic critically ill individuals in the GEO database GSE221379.”

absence[Introduction, entire]

No discussion of limitations of prior studies is present.

WARNReviewer 2· 75% conf

The premise linking Tregs, astrocytes, and renal fibrosis after stroke is introduced, but the rationale is incomplete and prior limitations are not systematically addressed.

Evidence

paraphrase[Introduction, paragraphs 1-3]

“The introduction cites studies on Treg accumulation in stroke, brain Tregs and astrogliosis, IL-2 post-stroke, and ferroptosis.”

paraphrase[Introduction, entire section]

“The introduction discusses Tregs and ferroptosis separately but does not build a clear logical bridge to renal fibrosis or the specific IL-10/GPX4 mechanism tested.”

absence[Introduction, entire section]

No discussion of limitations of prior research is provided.

For the animal experiments, randomization method, blinding, power analysis, and pre-specified inclusion/exclusion criteria are not reported. 'A total of 66 mice were successfully induced with MCAO and included in further experiments. However, 15 mice died within 24 hours following reperfusion and were subsequently excluded from the study' — this is a post-hoc exclusion without a pre-specified rule. The distinction between biological and technical replicates is unclear (n=4 is stated for immunofluorescence but replicate type is not defined). Controls are present (sham group). For the computational part, dataset selection (GSE221379) is described but without sample size justification. Independent replication is not reported.

Sub-criteria

controlsADEQUATE

power analysisNOT REPORTED

blinding levelsNOT REPORTED

outlier handlingNOT REPORTED

randomization unitNOT REPORTED

inclusion exclusionINADEQUATE

randomization methodNOT REPORTED

replicate distinctionNOT REPORTED

independent replicationNOT REPORTED

Per reviewer (2) — the votes behind the verdict

WARNReviewer 1· 80% conf

The mouse study lacks a power analysis and pre-specified inclusion/exclusion criteria, and randomization/blinding are not described; the computational analysis has no pre-registration.

Evidence

absence[Methods, section 2.4]

Randomization method is not described.

direct quote[Methods, section 2.4]

“A total of 66 mice were successfully induced with MCAO and included in further experiments. However, 15 mice died within 24 hours following reperfusion and were subsequently excluded from the study”

direct quote[Methods, section 2.4]

“For sham-operated mice, the same surgical procedures were performed, with the exception of filament insertion.”

WARNReviewer 2· 80% conf

The animal experiment part lacks essential rigor details: no randomization, blinding, power analysis, or pre-specified inclusion/exclusion criteria are reported.

Evidence

absence[Methods, Section 2.4]

No randomization method or unit is mentioned for the animal experiments.

paraphrase[Methods, Section 2.4]

“"A total of 66 mice were successfully induced with MCAO and included ... However, 15 mice died within 24 hours ... and were subsequently excluded from the study."”

absence[Methods, Section 2.4 and 2.10]

Power analysis is not reported.

Mice are described as '9-10 weeks old, weighing 25-30 g' in the microglia depletion section, but age/weight are not stated for the main MCAO experiment. Sex is never mentioned — unclear whether male or female mice were used, and no justification is given if single-sex. The species is stated as 'mice' but not specified (likely C57BL/6 from supplier context, but not explicit). Housing conditions (light cycle, temperature, enrichment) are not reported. Source is implied by the diet supplier (Research Diets) but not explicitly stated for the mice themselves.

Sub-criteria

demographicsN/A

sex reportedNOT REPORTED

sex justifiedNOT REPORTED

age weight healthINADEQUATE

housing conditionsNOT REPORTED

species strain sourceINADEQUATE

Per reviewer (2) — the votes behind the verdict

WARNReviewer 1· 85% conf

Sex is not reported, and age/weight are partially reported for some experiments but not all; species/strain/source are adequately reported; housing conditions are not described.

Evidence

absence[Methods, section 2.6]

Sex is not reported.

direct quote[Methods, section 2.6]

“To deplete microglia and macrophages, the mice (9-10 weeks old, weighing 25-30 g) were provided with PLX5622 in their diet”

absence[Methods, entire]

Housing conditions are not described.

WARNReviewer 2· 85% conf

Mouse sex is reported (inadequate justification for single sex), age/weight are given, strain/source are partially reported, but housing conditions are missing.

Evidence

absence[Methods, Section 2.6]

Sex of mice is not reported.

direct quote[Methods, Section 2.6]

“"the mice (9-10 weeks old, weighing 25-30 g)"”

absence[Methods, entire section]

Housing/husbandry conditions are not described.

The ethics statement names the approving body: 'Clinical Ethical Committee of Xinhua Hospital Affiliated with Shanghai Jiao Tong University School of Medicine. (No. (2022) 189)' — this is adequate for an IACUC-equivalent statement. However, regulatory compliance is only mentioned as 'conducted in accordance with the local legislation and institutional requirements' without naming a specific framework (e.g., Guide for the Care and Use of Laboratory Animals). The mention of 'written informed consent was obtained from the owners for the participation of their animals' is non-standard and potentially inadequate for animal research. For the human GEO data, no ethics statement is provided, but these are publicly available de-identified data, so n/a applies.

Sub-criteria

iacuc statementADEQUATE

informed consentN/A

irb ethics statementN/A

regulatory complianceINADEQUATE

Per reviewer (2) — the votes behind the verdict

WARNReviewer 1· 80% conf

Ethics approval is stated for the animal study, but the statement uses 'written informed consent was obtained from the owners for the participation of their animals' which is unusual and may indicate a misunderstanding, and regulatory compliance is vague.

Evidence

direct quote[Ethics statement, after Data availability]

“The animal studies were approved by Clinical Ethical Committee of Xinhua Hospital Affiliated with Shanghai Jiao Tong University School of Medicine. (No. (2022) 189).”

direct quote[Ethics statement]

“The studies were conducted in accordance with the local legislation and institutional requirements.”

direct quote[Ethics statement]

“Written informed consent was obtained from the owners for the participation of their animals in this study.”

WARNReviewer 2· 80% conf

An animal ethics statement is present and adequate, but an IACUC statement with a named body and protocol number is provided. However, a separate statement for human data (GEO) is missing, and the use of public human data does not strictly require it (n/a).

Evidence

direct quote[Ethics statement]

“"The animal studies were approved by Clinical Ethical Committee of Xinhua Hospital Affiliated with Shanghai Jiao Tong University School of Medicine. (No. (2022) 189)."”

direct quote[Ethics statement]

“"The studies were conducted in accordance with the local legislation and institutional requirements."”

Primary antibodies are identified by vendor (AiFang Biological) and catalog number, but lot numbers are missing. Cell lines are not used (primary astrocytes), so cell_line_authentication and mycoplasma_testing are n/a. The organism (mouse) is not fully specified (strain not given). Key reagents: diphtheria toxin dose is given (0.05 mg/g) but manufacturer/catalog not stated; PLX5622 is identified by supplier (Research Diets) and concentration. Software: GraphPad Prism 9.0 is named with version, but R packages (DESeq2, ggplot2, pheatmap) are mentioned without version numbers. The custom code or analysis pipeline is not shared.

Sub-criteria

mycoplasma testingN/A

reagents identifiedINADEQUATE

organisms identifiedINADEQUATE

antibodies identifiedINADEQUATE

cell line authenticationN/A

software tools identifiedINADEQUATE

Per reviewer (2) — the votes behind the verdict

WARNReviewer 1· 85% conf

Antibodies are identified with catalog numbers but without lot numbers; cell lines are not authenticated; software tools lack version numbers; the investigational (diphtheria toxin, IL-10) reagents are partially identified.

Evidence

direct quote[Methods, section 2.5]

“Primary antibodies, including anti-2 (1:100, AiFang Biological, AF14722), anti-IL-10 (1:100, AiFang Biological, AF06580), anti-IL-2(1:100, AiFang Biological, AF02481), anti-IL-6 (1:100, AiFang Biological, AF06790), anti-FSP1 (1:100, AiFang Biological, AF14902), and anti-GPX4 (1:100, AiFang Biological, AF06580)”

paraphrase[Methods, section 2.6]

“Mice are only referred to as 'mice' without specifying strain (e.g., C57BL/6).”

direct quote[Methods, sections 2.1 and 2.10]

“Statistical analyses were conducted using GraphPad Prism 9.0.”

WARNReviewer 2· 80% conf

Antibodies and reagents are partially identified with catalog numbers, but cell line authentication and mycoplasma testing are missing, and software versions are given but not all with URLs/RRIDs.

Evidence

direct quote[Methods, Section 2.5]

“"anti-2 (1:100, AiFang Biological, AF14722), anti-IL-10 (1:100, AiFang Biological, AF06580)..."”

absence[Methods, Section 2.7]

Cell line authentication and mycoplasma testing are not reported.

paraphrase[Methods, Section 2.6]

“Mice are described only as 'mice (9-10 weeks old, weighing 25-30 g)' – no strain or source.”

Tests named: Student's t-test, χ2 test, Kruskal-Wallis test. Normality and homogeneity of variance were tested (stated in methods). Bonferroni correction for multiple comparisons was used, but no details on which comparisons or adjusted p-values. Exact p-values are not reported; significance is indicated by asterisks (*, **, ***, ****) with threshold set at p<0.05. Data presentation shows bar graphs with error bars, but individual data points are not shown; per-group n is given (N=4). No effect sizes or confidence intervals are reported. Mathematical plausibility is not verifiable from the reported summary statistics (bar graphs without numeric means/SDs in text).

Sub-criteria

tests namedADEQUATE

exact p valuesNOT REPORTED

effect sizes ciNOT REPORTED

data presentationINADEQUATE

software identifiedADEQUATE

assumptions verifiedINADEQUATE

mathematical plausibilityN/A

Per reviewer (2) — the votes behind the verdict

WARNReviewer 1· 70% conf

Statistical tests are named but key assumptions are not verified for the mouse experiments; exact p-values are reported as thresholds; effect sizes and CIs are absent; mathematical plausibility cannot be fully verified from available data.

Evidence

direct quote[Methods, section 2.10]

“The Student’s t-test and χ 2 test were used to analyze the normally distributed data... Non-normally distributed data was analyzed using the Kruskal-Wallis test... Normality and homogeneity of variance were tested, and appropriate adjustments for multiple comparisons were made using Bonferroni correction”

direct quote[Results, figure legends]

“with a sample size of N=4 and significance level of ***P<0.005, and ****P<0.001”

absence[Results, figure legends]

Error bars in figures are not defined.

WARNReviewer 2· 75% conf

Statistical tests are named and software is identified, but multiple comparisons correction is reported without detail, exact p-values are not always given (stars used), and effect sizes/confidence intervals are missing.

Evidence

direct quote[Methods, Section 2.10]

“"The Student’s t-test and χ2 test were used ... Non-normally distributed data was analyzed using the Kruskal-Wallis test ... A significance threshold of p<0.05 was set."”

paraphrase[Results, Figures 4-8]

“Figures are described as showing bar graphs with error bars and significance stars; per-group n is 4.”

absence[Results and Methods]

Effect sizes and confidence intervals are not reported.

The data availability statement: 'The original contributions presented in the study are included in the article/. Further inquiries can be directed to the corresponding authors.' This is vague — it does not specify a repository or accession number for the mouse data. The GEO dataset GSE221379 is mentioned in methods but not in the data availability statement. No code repository is provided for the bioinformatics analysis. For a study that includes computational analysis and animal experiments, the lack of deposited data (other than the publicly available GEO dataset) and code is inadequate. The statement meets the bare minimum but is rated 'reported_but_inadequate' as it only promises data on request without a mechanism.

Sub-criteria

code sharingNOT REPORTED

accession numbersINADEQUATE

repository depositNOT REPORTED

data availability statementINADEQUATE

Per reviewer (2) — the votes behind the verdict

WARNReviewer 1· 80% conf

A data availability statement exists but is vague ('included in the article/'); no code or protocol is shared; the GEO accession for the human dataset is provided.

Evidence

direct quote[Methods, section 2.1]

direct quote[Data availability statement]

“The original contributions presented in the study are included in the article/. Further inquiries can be directed to the corresponding authors.”

absence[Entire paper]

No code repository is provided.

WARNReviewer 2· 80% conf

A data availability statement is present but vague ('available on request'), and no code or data is deposited in a repository.

Evidence

direct quote[Data availability statement]

“"The original contributions presented in the study are included in the article/. Further inquiries can be directed to the corresponding authors."”

direct quote[Methods, Section 2.1]

“"the GEO database GSE221379"”

absence[Data availability statement and Methods]

No code repository is mentioned.

Methods are described in moderate detail for the MCAO and in vitro experiments, but key elements (randomization, blinding, exact sample sizes) are missing. No reporting guideline (ARRIVE, CONSORT) is mentioned. Negative results are not systematically reported — only positive findings are shown. Limitations are not discussed in a dedicated section; the Discussion does not address study weaknesses. The conclusion 'tregs depletion aggravates activation of astrocytes by modulating IL-10/GXP4 following cerebral infarction' is supported by the data, but the claim that this 'provides new therapeutic directions for the prevention and treatment of MCAO' is an overstatement given the preliminary nature of the study. Funding sources are listed; conflicts of interest are stated.

Sub-criteria

funding coiADEQUATE

trial registrationN/A

reporting guidelineNOT REPORTED

methods completenessINADEQUATE

all outcomes reportedINADEQUATE

limitations discussedNOT REPORTED

conclusions proportionalINADEQUATE

Per reviewer (2) — the votes behind the verdict

WARNReviewer 1· 75% conf

Methods are partially described but lack replication detail; no trial registration or reporting guideline is followed; limitations are not discussed; conclusions are partially proportional but overstated in linking human sepsis data to mouse stroke.

Evidence

absence[Discussion, entire]

No limitations section or discussion of study weaknesses.

direct quote[Conclusion, final sentence]

“provides new therapeutic directions for the prevention and treatment of MCAO”

direct quote[Funding Statement and Conflict of Interest sections]

“Emergency Specialty - Department of Emergency Medicine (YXMZK005); General Discipline at TongRen Hospital (tr2023xk36). The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.”

WARNReviewer 2· 75% conf

Methods are partially complete, no trial registration (n/a), no reporting guideline mentioned, limitations not discussed, conclusions are somewhat proportional, and funding/COI are reported.

Evidence

absence[Methods, entire section]

No reporting guideline is mentioned.

absence[Discussion, entire section]

Limitations of the study are not discussed.

direct quote[Funding Statement and Conflict of Interest]

“"Emergency Specialty - Department of Emergency Medicine (YXMZK005); General Discipline at TongRen Hospital (tr2023xk36)." and "The authors declare that the research was conducted in the absence of any commercial or financial relationships..."”

21 copyedit issues flagged (4 major): mostly consistency, typo, clarity.

Checked 41 references: 41 verified.

Αlpha¹ Rigor Action ItemsFinal pass before journal submission

As a post-publication audit, this paper shows concerning reporting gaps. An informed reader should note the absence of randomization/blinding, the unsupported claim of renal fibrosis attenuation, and the lack of exact p-values and effect sizes. A correction or independent re-analysis is warranted to address the internal contradictions and verify the direction of effects claimed in the abstract versus the results.

1.
CRITICALreporting
Remove or substantiate the claim that 'Treg depletion attenuates renal fibrosis by modulating IL-10/GPX4 following cerebral infarction' in the Abstract and Conclusion with actual data or remove it; the paper presents no renal fibrosis data.
This claim is unsupported and contradicts the paper's focus on brain astrocytes, constituting a major integrity concern that could require a correction.
2.
CRITICALreporting
Resolve the internal contradiction between the Abstract (which states Treg depletion protects against astrocyte activation) and the Results/Conclusion (which state Treg depletion promotes astrocyte activation); correct the Abstract to match the data.
An opposite-direction claim in the Abstract is a serious reporting error that misleads readers and requires a correction.
3.
HIGHrigor
Describe the randomization method and unit for animal group allocation in Methods Section 2.4 (e.g., random number generator).
Without randomization, the risk of selection bias is unquantifiable, undermining the validity of the animal experiments.
4.
HIGHrigor
State whether investigators were blinded to group assignment during surgery or outcome assessment in Methods Section 2.4.
Blinding prevents observer bias; its absence is a major methodologic weakness that should be declared.
5.
HIGHrigor
Report the sex of all mice used and, if only one sex was used, provide a scientific justification (not cost/convenience) in Methods Section 2.6.
Sex is a fundamental biological variable; omission limits reproducibility and generalizability.
6.
HIGHreporting
Report exact p-values (e.g., P=0.023) instead of threshold-based asterisks for all statistical comparisons in Results and figure legends.
Exact p-values allow readers to assess evidence strength; threshold-only reporting is imprecise and outdated.
7.
HIGHreporting
Add effect sizes (e.g., Cohen's d) and 95% confidence intervals alongside p-values for every comparison in the Results.
Effect sizes and CIs convey the magnitude and precision of effects, which are critical for interpretation and meta-analysis.
8.
HIGHdata code
Deposit raw data from animal and in vitro experiments in a public repository (e.g., Figshare) and update the data availability statement with a DOI; include the GEO accession in the statement.
Without deposited data, the findings cannot be verified or reused, violating transparency norms.
9.
HIGHdata code
Share custom analysis code (e.g., for the bioinformatics pipeline) in a public repository like GitHub and cite it.
Code sharing is essential for reproducibility of the computational components.
10.
HIGHreporting
Add a 'Limitations' paragraph in the Discussion addressing the lack of randomization/blinding, small sample sizes, single sex, and the disconnect between the human sepsis data and the mouse stroke model.
Acknowledging limitations demonstrates scientific self-awareness and helps readers appropriately weigh the findings.
11.
HIGHreporting
Clarify the logical connection between the human sepsis dataset (GSE221379) and the mouse MCAO model in the Introduction, or remove the human data analysis if it is tangential.
The rationale for including the human dataset is currently unexplained, weakening the scientific premise.
12.
MEDIUMcopyedit
Standardize 'Tregs' (uppercase T, consistent) throughout the manuscript; fix instances of 'tregs', 'regs', and 'Treg cells'.
Inconsistent capitalization of a key term appears unprofessional and may confuse readers.
13.
MEDIUMcopyedit
Correct the antibody name 'anti-2' (Methods Section 2.5) to the specific target (e.g., anti-GFAP or anti-IL-2) as intended.
An incomplete antibody name prevents reproducibility of the immunohistochemistry experiments.
14.
MEDIUMcopyedit
Replace the erroneous 'rats' in Methods Section 2.6 with 'mice' and verify no other animal-species inconsistencies remain.
A species mismatch is a serious confusion that could undermine trust in the methodology.
15.
MEDIUMcopyedit
Remove the statement 'Written informed consent was obtained from the owners for the participation of their animals' from the Ethics Statement, as it is non-standard for animal research.
This phrase implies substandard ethics understanding and may raise unnecessary concerns.
16.
MEDIUMrigor
Specify the mouse strain (e.g., C57BL/6J) and supplier in Methods Section 2.6.
The strain and source are essential for reproducibility of the animal model.
17.
MEDIUMrigor
Describe housing conditions for mice (light cycle, temperature, enrichment, group size) in Methods Section 2.4.
Housing conditions can affect behavioral and physiological outcomes and must be reported.
18.
MEDIUMstatistics
Define whether the reported n=4 for immunofluorescence represents biological or technical replicates, and clarify the distinction throughout.
Ambiguity about replicate type affects the interpretation of statistical tests and conclusions.
19.
MEDIUMreporting
Include a statement referencing the ARRIVE guidelines for animal research in the Methods and submit the checklist as supplementary material.
ARRIVE adherence is increasingly required by journals and improves reporting completeness.
20.
LOWcopyedit
Fix the section number '38.' to '3.8.' in Section 3.8 title and correct the grammar 'astrocytes ferroptosis' to 'astrocyte ferroptosis'.
These typos disrupt readability but do not affect scientific integrity.

Full text (30,810 chars)Analyzed: Jul 6, 2026

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