Luteolin Enhances Endothelial Barrier Function and Attenuates Myocardial Ischemia–Reperfusion Injury via FOXP1-NLRP3 Pathway
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript addresses an important topic in cardiovascular research by exploring the protective role of luteolin against myocardial ischemia–reperfusion injury (MIRI) through the FOXP1–NLRP3 pathway. The study combines in vivo and in vitro approaches and provides compelling preliminary evidence for the mechanistic involvement of endothelial barrier protection. However, several methodological and presentation issues need clarification and strengthening to enhance the rigor and reproducibility of the findings.
Specific Reviewer Comments
The manuscript should clearly specify the number of experimental groups for both in vivo and in vitro studies (e.g., sham, MIRI, MIRI + luteolin, MIRI + MCC950). Please include group sizes, dosing regimen of luteolin (concentration, route, duration), and whether investigators were blinded during assessments.
2. While Evans blue staining and FITC-Dextran leakage assays are appropriate, the methodology lacks detail. Please describe how vascular permeability was quantified, how regions of interest were selected, and whether normalization methods were used.
3. The link between FOXP1 upregulation and NLRP3 inhibition is central to the study. However, mechanistic validation seems limited. Western blot or immunofluorescence data demonstrating direct interaction or downstream signaling changes (e.g., caspase-1 activation, IL-1β maturation) would strengthen the conclusions.
4. The manuscript states that FOXP1 siRNA abolished luteolin’s protective effect, but efficiency of knockdown needs to be shown quantitatively. Please include data on FOXP1 mRNA and protein reduction and address potential off-target effects or include scrambled siRNA controls.
5. Please provide detailed information regarding statistical methods: type of statistical tests used, correction for multiple comparisons, data distribution assessment, and how variability is represented (mean ± SD or SEM). Indicate the number of biological vs. technical replicates.
6. While the study is interesting, the background section should more clearly highlight gaps in current knowledge about FOXP1 in endothelial biology and MIRI. In the discussion, please elaborate on alternative pathways through which luteolin may exert effects and acknowledge limitations of the study.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript addresses the cardioprotective potential of luteolin, a natural flavonoid known for its antioxidant and anti-inflammatory properties. The study focuses on elucidating the mechanisms by which luteolin mitigates myocardial ischemia-reperfusion injury. However, while the study has potential, several aspects of the manuscript require substantial improvement:
- In Figure 1, it is not necessary to indicate statistical significance using a different symbol in comparison to Sham, as it is already visually clear from the graph which groups are being compared. It is recommended to use consistent and universal symbols for statistical significance, as done in Figures 2-4, to maintain clarity and uniformity throughout the manuscript.
- The Discussion section should be significantly expanded. The authors should provide a detailed explanation of all obtained results, critically comparing them with existing literature to highlight similarities, differences, and potential mechanisms.
- Additionally, the Discussion should address the significance of the study and clearly explain what new insights or contributions the work provides to the field. Strengthening these aspects will improve the scientific depth and relevance of the manuscript.
- The manuscript does not provide a clear explanation of how the doses of simvastatin was selected.
- In Section 4.4, Echocardiographic Assessment, the manuscript does not specify which parameters were monitored or measured.
- In Section 4.6, Detection of LDH, CK-MB, and IL-1β, the manuscript does not specify which commercial kits or assay sources were used for these measurements. This information is essential for reproducibility and comparability of the results.
- It is not clear whether the authors tested for normality of the data prior to applying statistical analyses. The manuscript should provide a detailed description of all statistical methods used, including tests for normality, the specific statistical tests applied for comparisons.
- The Conclusion section is not sufficiently informative. Considering that the study investigated the cardioprotective effects of luteolin in two experimental models, the conclusions should clearly summarize the main findings, their significance, and potential implications for future research or practical applications.
- In the References section, all instructional text or template guidance should be removed, leaving only the actual references cited in the manuscript.
- The Reference list should be formatted according to the journal’s guidelines. Currently, some entries do not follow the required style.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThis manuscript investigates the cardioprotective effects of luteolin in myocardial ischemia–reperfusion injury using both a rat LAD ligation model and OGD/R-challenged endothelial cells. The authors demonstrate that luteolin preserves endothelial barrier integrity, improves cardiac function, and suppresses inflammatory injury through modulation of the FOXP1–NLRP3 axis. The combined in vivo and in vitro approach, together with genetic and pharmacological interventions, provides mechanistic depth and translational relevance. However, major revisions are required before publication.
Comments for the authors
Comment 1. The introduction frames endothelial barrier dysfunction as a key contributor to MIRI but stops short of integrating recent mechanistic advances linking flavonoids to transcriptional control of inflammasome signaling in endothelial biology. Expand this section to incorporate up-to-date literature on flavonoid-mediated regulation of endothelial transcription factors and inflammasome modulation in cardiovascular injury, which will place the current study more clearly within the evolving field.
Comment 2. Given the focus on therapeutic potential, it would be helpful to briefly discuss how recent progress in flavonoid pharmacology—covering aspects such as metabolism, bioavailability, and formulation—contributes to flavonoid-based treatments in the introduction section. (See: Int. J. Mol. Sci. 26 (2025). 10.3390/ijms26062678).
Comment 3. The improvement in echocardiographic parameters is convincing, but infarct size quantification is absent. Add TTC or equivalent infarct analysis to correlate functional recovery with tissue-level myocardial protection directly.
Comment 4. Include normalized dye leakage measurement per tissue weight or area to support claims about changes in endothelial permeability in vivo.
Comment 5. ZO-1 and VE-cadherin restoration is interpreted as barrier recovery; however, junctional continuity versus total protein abundance is not clearly distinguished. Add quantitative junctional localization analysis to support functional barrier conclusions.
Comment 6. FOXP1 upregulation by luteolin is shown, but transcriptional activity is inferred. Include FOXP1 target gene analysis or promoter validation to confirm transcriptional engagement.
Comment 7. MCC950 phenocopying luteolin is interpreted as downstream validation; however, inflammasome activation markers beyond NLRP3 and IL-1β are limited. Include caspase-1 activation or ASC oligomerization to strengthen inflammasome-specific conclusions.
Comment 8. The discussion briefly acknowledges limitations but remains descriptive. Expand this section to critically evaluate whether luteolin primarily acts upstream at transcriptional regulation or downstream at inflammasome suppression, and discuss alternative pathways that may converge on the observed phenotype.
Comment 9. The limitations section acknowledges incomplete pathway coverage but remains descriptive. A more critical comparison with recent comprehensive flavonoid-focused analyses that map multi-pathway interactions would help delineate which aspects of luteolin signaling are truly unique versus reflective of conserved flavonoid biology. (See and incorporate: Int. J. Mol. Sci. 2025, 26(15), 7381; 10.3390/ijms26157381).
Comment 10. The manuscript contains frequent grammatical inconsistencies, awkward phrasing, and non-idiomatic scientific expressions across all sections. Conduct a thorough professional English language revision to ensure clarity, precision, and journal-level readability.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsXi et al. investigated the protective effects of the flavonoid luteolin on ischemia/reperfusion injury in a rat model and oxygen-glucose deprivation/reoxygenation in vitro in endothelial cells. They found that luteolin increased endothelial barrier function via the FOXP1-NLRP3 pathway.
Overall, the authors present a sound experimental study in which the underlying mechanism is elucidated using a variety of methods.
Comments:
- I feel that the use of the term ‘model’ is generally unfortunate. Why not simply use MIRI for in vivo experiments and OGD/R for in vitro experiments?
- Abstract: “In OGD/R-induced endothelial cells, luteolin restored barrier function and cell viability.” OGD/R-induced? -treated would be more appropriate here.
- Results, 2.1: “The model group's myocardial tissue architecture was severely disrupted by HE staining….” Was it disrupted by HE staining? Please revise.
- Results 2.1: This section lacks a few explanations. Why were CK-MD and LDH levels measured? Why was a statin used? This is not even mentioned...
- Legends: Actually, the significance symbols should all be superscripted.
- Fig. 2A: What does the bracket under the heart mean?
- Fig. 2B: N=3 is very few. Evans blue staining in tissue as a measure of vascular permeability should be evaluated quantitatively.
- 2.3: “…luteolin treatment may lower serum levels of the inflammatory cytokine IL-1ß….” Why maybe? The ELISA results show that.
- In the initial experiments, n=3 is specified for the immunostaining, which would be quite a small number of experiments. Apparently, more tests were carried out, as n=6 in the Western blots.
- Fig. 3, legend: “quantitative analysis of FOXP1 and CD31 in each group….” The authors state that a quantitative analysis was performed using IF for FOXP1 and CD31. However, this is not true, at least it is not shown... but it should be done. Here, too, I consider n=3 to be a very small sample size...
- Fig. 4F. Quantitative analysis on ZO-1 IF is missing.
- Results, 2.5: Why is the molecular interaction between FOXP1 and luteolin being investigated here? What is the rationale for this? Do the results provide any clues? If not, then this should be explained more clearly here.
- I think the discussion is simply too brief to discuss the issue in depth in light of the existing literature.
- M&M, 4.12: 104=104
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors have revised the manuscript and addressed all comments and concerns. I recommend accepting the paper in its present form.
Reviewer 4 Report
Comments and Suggestions for AuthorsNo further comments.
