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Macrophages depletion alleviates lung injury by modulating AKT3/GXP4 following ventilator associated pneumonia · 10.3389/fimmu.2023.1260584

Name: Verified Rigor Record — Macrophages depletion alleviates lung injury by modulating AKT3/GXP4 following ventilator associated pneumonia · 10.3389/fimmu.2023.1260584
Item: Macrophages depletion alleviates lung injury by modulating AKT3/GXP4 following ventilator associated pneumonia · 10.3389/fimmu.2023.1260584
Rating: 67
Author: Αlpha¹

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Αlpha¹ Editorial

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

Αlpha¹ Rigor Review

Rigor Reviewed

WARN

Warnings to address · Observational Study

Eight dimensions — click a segment for details

Critical

High-priority

Model-reported confidence: ModerateReviewer agreement: lowEngine: 7.6.0Source: full text

Not verified in this report

No reported statistical tests were found to recompute.

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.

12 major claims checked against the paper's own evidence: 3 not fully backed by the presented evidence (unsupported or overstated).

11 integrity concerns flagged (1 high).

Rigor

Eight Dimensions1 pass · 6 warn · 1 fail

12 major claims3 unsupported6 partial3 supported

Prior work on macrophages, ferroptosis, and AKT3 is cited in the Introduction, establishing a general premise. However, the logical rationale connecting AKT3's role in lung cancer to its hypothesized role in VAP is not clearly articulated; the paper jumps from AKT3 in cancer to VAP without a mechanistic bridge. Limitations of prior studies are not discussed, and the study does not describe how it addresses gaps in previous research.

Sub-criteria

prior work citedADEQUATE

premise rationaleINADEQUATE

limitations addressedNOT REPORTED

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 75% conf

The premise is broadly established with citations, but the logic linking AKT3 from pan-cancer analysis to VAP is tenuous, and limitations of prior work are not addressed.

Evidence

direct quote[Introduction]

“Despite significant advancements in macrophage research, understanding the molecular mechanisms underlying their development remains elusive.”

absence[Introduction]

No limitations of prior studies are discussed.

direct quote[Introduction]

“This study aims to establish a foundation for comprehending the developmental mechanisms of lung macrophages and exploring their potential role in VAP.”

WARNReviewer 2· 85% conf

The paper cites prior work on macrophages, ferroptosis, and AKT3, but the rationale linking AKT3 in lung cancer to its role in VAP is weak, and limitations of prior research are not addressed.

Evidence

direct quote[Introduction, paragraphs 1-4]

“Monocytes and macrophages (MΦs) play a critical role as immediate responders to various types of invading pathogens... Ferroptosis is a distinctive form of programmed cell death that relies on iron... AKT3 appears to play a role in lung cancer.”

direct quote[Introduction, last paragraph]

“This study aims to establish a foundation for comprehending the developmental mechanisms of lung macrophages and exploring their potential role in VAP.”

absence[Introduction]

Limitations of prior research are not addressed.

PASSReviewer 3· 85% conf

The paper establishes a clear scientific premise by citing prior work and providing a logical rationale linking AKT3 to lung cancer and VAP, though limitations of prior research are not explicitly addressed.

Evidence

direct quote[Introduction, paragraph 1-3]

direct quote[Abstract, Background]

“AKT3 appears to play a role in lung cancer. However, its role in ventilator-associated pneumonia is still unclear. Therefore, this study aimed to investigate the role of AKT3 in macrophages during ventilator-associated pneumonia.”

direct quote[Introduction, last sentence]

“Despite significant advancements in macrophage research, understanding the molecular mechanisms underlying their development remains elusive.”

The study uses a mouse VAP model but does not report randomization method or unit. Blinding is not mentioned. No power analysis or sample size calculation is provided. The distinction between biological and technical replicates is unclear (N=4 mice per group is mentioned, but whether each mouse is a biological replicate is implied). Inclusion/exclusion criteria are not pre-specified. Outlier handling is not reported. The control group (normal saline) is described but lacks detail (e.g., no sham MV control). No independent replication is reported or planned.

Sub-criteria

controlsINADEQUATE

power analysisNOT REPORTED

blinding levelsNOT REPORTED

outlier handlingNOT REPORTED

randomization unitNOT REPORTED

inclusion exclusionNOT REPORTED

randomization methodNOT REPORTED

replicate distinctionNOT REPORTED

independent replicationNOT REPORTED

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 80% conf

The animal experiment lacks key design details: no randomization, no blinding, no sample size justification, and limited description of controls and replication.

Evidence

absence[Section 2.3]

No randomization or blinding described for the animal experiment.

direct quote[Figures 5-8 legends]

“N=4”

direct quote[Section 2.3]

“intranasally administered either 15 μl of normal saline as the control group”

WARNReviewer 2· 80% conf

The animal experiment lacks key design details: no randomization method, blinding, power analysis, or pre-specified inclusion/exclusion criteria are reported. The computational part is better described but still lacks some rigor.

Evidence

absence[Methods, section 2.3]

No randomization method, unit, or blinding reported for animal experiment.

absence[Methods]

No power analysis reported.

direct quote[Methods, section 2.3]

“Wild-type (WT) mice were anesthetized with avertin (15 mg/kg, Sigma) and were intranasally administered either 15 μl of normal saline as the control group, or an equal volume of Pseudomonas aeruginosa (PA) at a concentration.”

FAILReviewer 3· 90% conf

The study design lacks essential rigor: no randomization, blinding, power analysis, or replication is reported for the animal experiment, and inclusion/exclusion criteria and outlier handling are absent.

Evidence

direct quote[Methods, section 2.3]

absence[Methods, section 2.3-2.7]

No blinding is mentioned.

absence[Methods, section 2.7]

No power analysis or sample size justification is provided.

The paper states 'Wild-type (WT) mice' but does not specify the strain, sex, age, or weight. Health status is not reported. Housing conditions (light cycle, temperature, enrichment) are not described. For the computational analysis, no biological variables are applicable as it uses public transcriptomic data. The lack of these details for the animal experiment is a significant gap.

Sub-criteria

demographicsN/A

sex reportedNOT REPORTED

sex justifiedN/A

age weight healthNOT REPORTED

housing conditionsNOT REPORTED

species strain sourceINADEQUATE

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 80% conf

Sex and age/weight of mice are not reported; strain and source are partially reported, but key husbandry details are missing.

Evidence

direct quote[Section 2.3]

“Wild-type (WT) mice were anesthetized with avertin”

absence[Section 2]

Sex, age, weight, and housing conditions are not reported.

WARNReviewer 2· 85% conf

The animal study reports the species and strain (WT mice) but lacks details on sex, age, weight, health status, and housing conditions. The computational part does not involve biological variables.

Evidence

direct quote[Methods, section 2.3]

“Wild-type (WT) mice were anesthetized with avertin (15 mg/kg, Sigma)”

absence[Methods, section 2.3]

Sex, age, weight, and health status are not reported.

absence[Methods]

Housing conditions are not reported.

FAILReviewer 3· 95% conf

The paper fails to report essential biological variables for the animal model: sex, age, weight, health status, and housing conditions are absent; strain is only vaguely described as 'wild-type'.

Evidence

direct quote[Methods, section 2.3]

“Wild-type (WT) mice were anesthetized with avertin (15 mg/kg, Sigma) and were intranasally administered...”

absence[Methods, section 2.3]

Age and weight of mice are not reported.

absence[Methods, section 2.3]

Housing conditions are not described.

The ethics statement reports approval by the ethics committee of Guangzhou Red Cross Hospital (approval number 2019-145-01). It states the study was conducted in accordance with local legislation and institutional requirements, which meets regulatory compliance. No human subjects were involved, so IRB and consent are not applicable.

Sub-criteria

iacuc statementADEQUATE

informed consentN/A

irb ethics statementN/A

regulatory complianceADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 90% conf

Animal ethics approval is reported with a named committee and protocol number; regulatory compliance is stated.

Evidence

direct quote[Ethics statement]

“The animal study was approved by the ethics committee of Guangzhou Red Cross Hospital (approval number 2019-145-01).”

PASSReviewer 2· 90% conf

The animal study has an ethics statement with a named committee and approval number, and regulatory compliance is stated. The computational part does not involve new subject data.

Evidence

direct quote[Ethics statement]

“The animal study was approved by the ethics committee of Guangzhou Red Cross Hospital (approval number 2019-145-01).”

direct quote[Ethics statement]

“The study was conducted in accordance with the local legislation and institutional requirements.”

WARNReviewer 3· 90% conf

An animal ethics approval is provided with a named body and approval number, but the regulatory compliance statement is vague and does not cite a recognized framework.

Evidence

direct quote[Ethics statement]

“The animal study was approved by the ethics committee of Guangzhou Red Cross Hospital (approval number 2019-145-01).”

direct quote[Ethics statement]

“The study was conducted in accordance with the local legislation and institutional requirements.”

Primary antibodies are listed with catalog numbers and manufacturer (AiFang Biological), but no RRIDs, lot numbers, or validation data are provided. The macrophage depletion agent is described as 'LC or LV' (likely clodronate liposomes) without vendor catalog number or formulation details. Software (GraphPad Prism 9.0, Xiantao tools) is named and Xiantao has a URL, but versions are not given. No cell lines or organisms beyond the mouse strain (not well identified) are used.

Sub-criteria

mycoplasma testingN/A

reagents identifiedINADEQUATE

organisms identifiedINADEQUATE

antibodies identifiedINADEQUATE

cell line authenticationN/A

software tools identifiedINADEQUATE

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 85% conf

Antibodies are identified with catalog numbers but without RRIDs or validation data; the investigational product (clodronate liposomes) and software are not adequately identified.

Evidence

direct quote[Section 2.6]

“anti-MMP9 (AF06799), anti-MMP19 (AF02793), anti-SFTA1 (AF10524), anti-FTH (AF02247), anti-FTL (AF301381), and anti-GPX4 (AF11931)”

direct quote[Section 2.5]

“a total volume of 300 μl of LC or LV was administered”

direct quote[Sections 2.1, 2.7]

“Statistical analysis was performed using GraphPad Prism 9.0”

WARNReviewer 2· 80% conf

Antibodies are identified with catalog numbers, but validation data and dilutions are missing. The software tool (Xiantao) is identified with a URL but no version. Other resources like the bacterial strain and liposomes are not fully identified.

Evidence

direct quote[Methods, section 2.6]

“The sections were incubated overnight at 4°Cwith the following primary antibodies (all from AiFang Biological) diluted at 1:100: anti-MMP9 (AF06799), anti-MMP19 (AF02793), anti-SFTA1 (AF10524), anti-FTH (AF02247), anti-FTL ( AF301381 ), and anti-GPX4 (AF11931).”

direct quote[Methods, section 2.1]

“Data processing utilizing Xiantao academic tools ( https://www.xiantaozi.com/products )”

direct quote[Methods, section 2.3]

“an equal volume of Pseudomonas aeruginosa (PA) at a concentration.”

WARNReviewer 3· 85% conf

Antibodies and software are adequately identified, but the mouse strain is only vaguely described, and some reagents (e.g., clodronate liposomes, Pseudomonas aeruginosa concentration) are incompletely specified.

Evidence

direct quote[Methods, section 2.6]

“anti-MMP9 (AF06799), anti-MMP19 (AF02793), anti-SFTA1 (AF10524), anti-FTH (AF02247), anti-FTL ( AF301381 ), and anti-GPX4 (AF11931).”

direct quote[Methods, section 2.3]

“Wild-type (WT) mice were anesthetized...”

direct quote[Methods, section 2.5]

“a total volume of 300 μl of LC or LV was administered intraperitoneally for two consecutive days”

The paper names tests (χ2, Mann-Whitney U, Kruskal-Wallis) and identifies software (GraphPad Prism 9.0). However, test assumptions (normality, equal variance) are not mentioned. P-values are reported only as thresholds (e.g., *P<0.05, **P<0.01), not exact values. No effect sizes or confidence intervals are reported. Figures show individual data points, but error bars are not defined in the figure legends or methods. The sample size is small (N=4 per group), making the use of non-parametric tests appropriate, but the lack of exact p-values and effect sizes is a limitation.

Sub-criteria

tests namedADEQUATE

exact p valuesNOT REPORTED

effect sizes ciNOT REPORTED

data presentationINADEQUATE

software identifiedADEQUATE

assumptions verifiedNOT REPORTED

mathematical plausibilityN/A

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 75% conf

Statistical tests are named for some analyses but inadequately, with no verification of assumptions, no exact p-values, and poor data presentation.

Evidence

direct quote[Results, Figures 6-8 legends]

“with significance set at **P < 0.05,***P < 0.01, ****P < 0.005”

absence[Section 2.7]

Assumptions not addressed.

paraphrase[Figures 5-8]

“Figures display bar graphs with error bars; no individual points shown.”

WARNReviewer 2· 85% conf

Statistical tests are named, and software is identified, but assumptions are not verified, exact p-values are not reported (only thresholds), and effect sizes with confidence intervals are absent. Data presentation shows individual points in figures but error bars are not defined.

Evidence

direct quote[Methods, section 2.7]

“The χ2 test was used to assess the correlation between AKT3 expression and clinicopathological parameters. The Mann-Whitney U test or Kruskal-Wallis test was applied for other analyses.”

direct quote[Results, sections 3.5-3.8]

“**P < 0.05,***P < 0.01, ****P < 0.005.”

absence[Figure legends, Methods]

Error bars are not defined in figures or methods.

FAILReviewer 3· 90% conf

Statistical reporting is severely deficient: tests are named but not linked to specific analyses, exact p-values are not reported, assumptions are not verified, effect sizes and confidence intervals are absent, and data presentation lacks individual data points.

Evidence

direct quote[Methods, section 2.7]

“The χ2 test was used to assess the correlation between AKT3 expression and clinicopathological parameters. The Mann-Whitney U test or Kruskal-Wallis test was applied for other analyses.”

direct quote[Results, sections 3.5-3.8]

“***P < 0.01, ****P < 0.005”

absence[Results, sections 3.5-3.8]

No effect sizes or confidence intervals are reported.

The data availability statement says 'The original contributions presented in the study are included in the article/, further inquiries can be directed to the corresponding authors,' which is a generic 'available on request' statement without a repository or managed-access platform. No raw data from the animal experiment are deposited. The paper mentions a GEO dataset (GSE215219) but this is a pre-existing public dataset, not their own data. No custom code is shared.

Sub-criteria

code sharingNOT REPORTED

accession numbersNOT REPORTED

repository depositNOT REPORTED

data availability statementINADEQUATE

Per reviewer (3) — the votes behind the verdict

FAILReviewer 1· 85% conf

The data availability statement is vague ('on request'), no data are deposited in a repository, and no code is shared.

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.”

absence[Entire paper]

No code repository is mentioned.

WARNReviewer 2· 90% conf

A data availability statement is present but vague ('available on request'). No data or code is deposited in a repository. The computational analysis uses public data but does not provide accession numbers for the specific datasets used.

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.”

absence[Methods, Data availability statement]

No repository deposit or accession numbers for the specific datasets used.

absence[Entire paper]

No code sharing.

FAILReviewer 3· 90% conf

The data availability statement is vague and does not provide a concrete access route; no data or code repositories are deposited or referenced.

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.”

absence[Data availability statement]

No raw data are deposited in a public repository.

absence[Methods and Data availability statement]

No custom code is shared.

Methods describe the animal model and immunofluorescence but omit critical details like randomization, blinding, and sample size justification, making independent replication difficult. No trial registration or reporting guideline (e.g., ARRIVE) is mentioned. Limitations are not discussed in the Discussion section. The conclusion that 'macrophage depletion mitigates lung injury by modulating the AKT3/GPX4 pathway' is somewhat overstated given the correlational nature of the evidence and the small sample size. Funding sources and a conflict of interest statement are provided.

Sub-criteria

funding coiADEQUATE

trial registrationN/A

reporting guidelineNOT REPORTED

methods completenessINADEQUATE

all outcomes reportedINADEQUATE

limitations discussedNOT REPORTED

conclusions proportionalINADEQUATE

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 80% conf

Methods are incomplete for replication, no reporting guideline is referenced, limitations are missing, but funding and COI are stated.

Evidence

absence[Discussion]

No limitations section.

direct quote[Conflict of interest]

“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.”

absence[Methods]

No reporting guideline mentioned.

WARNReviewer 2· 85% conf

Methods are partially complete but lack key details for replication. No trial registration or reporting guideline is mentioned. Limitations are not discussed, and conclusions are somewhat overstated. Funding and COI are reported.

Evidence

absence[Methods, section 2.3-2.7]

Methods lack key details for replication (randomization, blinding, sample size).

absence[Discussion]

No limitations discussed.

direct quote[Conflict of interest, Funding]

“The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This study was supported by the Health Science and Technology Project of Guangzhou Municipal Health Commission (20201A011022)... 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 3· 85% conf

Methods are incomplete, no reporting guideline is referenced, limitations are not discussed, and conclusions are somewhat overstated, but funding and conflicts of interest are adequately reported.

Evidence

direct quote[Methods, section 2.3]

absence[Methods, sections 2.1-2.7]

No reporting guideline is referenced.

absence[Discussion]

Limitations are not discussed in the Discussion section.

30 copyedit issues flagged (22 major): mostly typo, consistency, clarity.

Checked 35 references: 34 verified — 1 retracted.

Reproducibility

2 data/code links checked; 2 live.

Full text (34,897 chars)Analyzed: Jul 6, 2026

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