Review Reports

Reviewer Comments: REVIEWER 1

Comments 1:

Overall, the introduction and discussion are consistent with the topic and the data presented. However, the results seem too concise and schematic. The authors are kindly requested to make this section more fluid and discursive, avoiding a simple list of observed aspects, so as to make it more readable for all those interested in the topic.

Dear Reviewer,

Thank you very much for your detailed comments and valuable insights on our manuscript "Pharmaceuticals-4135714". We sincerely appreciate the reviewer’s positive comments and constructive suggestion for improving the Results section. We fully agree that the original Results were relatively concise and schematic, with a somewhat listlike presentation that may reduce readability.

In the revised manuscript, we have substantially expanded and reorganized the Results section to make it more coherent, fluid, and discursive. We have added detailed descriptions for each experiment, including clear explanations of the observed trends, logical connections between different assays, and more comprehensive interpretations of the phenotypic and molecular changes. We have avoided simple enumeration of findings and instead integrated the data into a continuous, narrative flow that better illustrates the neuroprotective effects and underlying mechanisms of AS-IV.

These revisions have greatly improved the readability and academic presentation of the Results section, making it more accessible to readers interested in this field.

Comments 2:

In general, the quality of all images should be improved to make the markers used visible. It's possible to create boxes to display small details, even on a few cells or tissues.

Dear Reviewer,

We sincerely appreciate the reviewer’s constructive suggestion on improving image quality.In the revised manuscript, we have improved the resolution, contrast, and sharpness of all images, and enhanced the size and visibility of all markers, scale bars, and labels to ensure they can be clearly observed. These adjustments have significantly optimized the overall clarity and readability of all figures .

Comments 3:

Please insert calibration bars into the missing images and enter the value in the legend, given the difficulty of verifying details directly in the proposed microimages.

Dear Reviewer,

We sincerely appreciate the reviewer’s constructive comment.In the revised manuscript, calibration bars (scale bars) have been inserted into all relevant images that were missing them, and the corresponding scale values have been clearly indicated in each figure legend. These changes improve the clarity and verifiability of the microstructural details.

Comments 4:

In Figure 2A, is it possible to quantify the Tunnel values and the percentage of Bcl2 and Bax+ cells?

Dear Reviewer,

In the revised manuscript, we have quantified the TUNEL-positive cells and the percentage of Bcl2 and Baxpositive cells from Figure 2B-2C, and added the corresponding quantitative analysis results in the figure panel. The quantitative data are presented as mean ± SD and were statistically analyzed as described in the Methods section, which makes the results more intuitive and convincing.

Comments 5:

None of the experiments indicate how many independent experiments were performed, or whether the data match only across, for example, 3 fields/groups, as shown in Fig. 3G. If so, it is difficult to consider a valid statistic.

Dear Reviewer,

We sincerely appreciate the reviewer’s important comment on the statistical validity of the experimental data.In the revised manuscript, we have clearly supplemented the number of independent biological replicates for all experiments. All results were obtained from at least 3 independent biological replicates, and quantitative analysis was performed by randomly selecting 3–5 visual fields per replicate for quantification.These detailed settings have been clearly stated in the figure legends and Methods section to ensure that the statistical analysis is reliable and scientifically valid.

Comments 6:

Did the authors check the percentage of BAX2- and BCL2-positive cells in Fig. 4A? Were IFs also performed for the other markers, or were they studied only with WB?

Dear Reviewer,

We sincerely appreciate the reviewer’s valuable questions and suggestions.

In the revised manuscript, we have quantified and supplemented the percentage of Bax- and Bcl-2-positive cells in Figure 4A. For other apoptosis-related markers, only Western blot analysis was performed, which was sufficient to reflect the changes in protein expression levels and adequately address the relevant scientific questions in the present study.

These methodological details have been clearly stated in the Results and figure legends to ensure clarity and completeness.

Comments 7:

It would be interesting to observe different marker localisations within the cell to highlight possible post-damage activation, as discussed in the literature. For example, one could verify whether CytC, normally mitochondrial (assessed by colocalisation with a mitochondrial marker), is released into the cytosol after damage and colocalises with APAF-1 under your experimental conditions, supporting the findings.

Dear Reviewer,

We sincerely appreciate the reviewer’s insightful and valuable suggestion. Colocalization analysis of CytC, mitochondrial markers, and Apaf1 would indeed further enrich and strengthen the mechanistic basis of the present study and represents a highly valuable research direction.

Considering that the core content and experimental design of this study mainly focus on investigating the regulatory effect of ASIV on the CytC/Apaf1 pathway at the expression level, the relevant colocalization experiments have not been included in the current research scope. However, the results we have obtained so far using Western blot, immunofluorescence, and other assays clearly demonstrate the expression changes of key molecules and are sufficient to support the main conclusions of this study.

We fully agree that subcellular localization and colocalization analyses represent important directions for further indepth research, and we will systematically explore these aspects in our future work.

Reviewer Comments: REVIEWER 2

Comments 1:

The keyword section contains a glaring typographical error: "Neuron ppoptosis" should be corrected to "Neuronal apoptosis".

Dear Reviewer,

Thank you very much for your detailed comments and valuable insights on our manuscript "Pharmaceuticals-4135714". We sincerely appreciate the reviewer's careful check and valuable suggestion. The typographical error of "Neuron ppoptosis" in the keyword section has been corrected to "Neuronal apoptosis" in the revised manuscript. We have also conducted a full text check to avoid similar spelling and typographical errors in other parts of the paper.

Comments 2:

Section 2.1.1 states: "Fig. 1A shows the experimental workflow: mice underwent adaptive feeding...", yet the entire animal model uses Sprague-Dawley rats throughout the Methods section. This inconsistency must be corrected.

Dear Reviewer,

We are deeply grateful for your valuable feedback. The erroneous term "mice" in Section 2.1.1 (referring to the experimental workflow in Fig. 1A) has been corrected to "rats" in the revised manuscript, which is consistent with the Sprague-Dawley (SD) rats used for the MCAO/R model throughout the Methods section. We have performed a comprehensive full-text check to ensure the uniformity of animal terminology in all sections and figures of the paper, eliminating any similar descriptive inconsistencies.

Comments 3:

Section 2.3.7 (Western blot for cell experiments) is incorrectly numbered — it should be 4.3.7, as it falls under the Materials and Methods section. This disrupts logical flow and readability.

Dear Reviewer,

We appreciate the reviewer's meticulous observation and constructive suggestion. The incorrect section number 2.3.7 (Western blot for cell experiments) has been revised to 4.3.7 in the revised manuscript, which is consistent with its subordinate position in the 4.3 Cellular Experiments of the Materials and Methods section. We have also carefully checked the numbering of all sections, subsections and corresponding content in the entire manuscript to ensure the logical coherence and readability of the section numbering system, and eliminated all similar numbering errors.

Comments 4:

The Methods sentence in the abstract begins with an infinitive phrase: "To establish an in vivo model..." but lacks a main clause. This is grammatically incomplete. It should be rewritten, e.g.: "We established an in vivo MCAO/R rat model and an in vitro OGD/R PC12 cell model.

Dear Reviewer,

We appreciate the reviewer's meticulous grammatical correction and valuable suggestion. The incomplete sentence in the Methods part of the abstract has been fully revised in the revised manuscript, and we have rephrased it as "We established an in vivo model of middle cerebral artery occlusion/reperfusion (MCAO/R) in rats and an in vitro model of oxygen-glucose deprivation/reperfusion (OGD/R) in PC12 cells" to ensure grammatical integrity and clarity of expression. We have also conducted a comprehensive grammatical and syntactic check on the entire abstract and the full text of the manuscript to eliminate any similar grammatical errors and improve the overall language quality of the paper.

Comments 5:

The Apaf-1 inhibitor ZYZ-488 is used as a pharmacological comparator throughout, but there is no rationale provided for why this specific inhibitor was selected at the given dose (30 mg/kg in vivo, 30 μmol/L in vitro). A brief justification or citation for dose selection is needed.

Dear Reviewer,

We sincerely appreciate the reviewer's insightful comment and valuable suggestion.

For the dosage setting, the in vivo dose of 30 mg/kg for SD rats was determined based on the recommended dosage range in the official product specification of ZYZ-488, combined with the standard body weight-based dose conversion principle for rodent experiments to ensure the rationality and effectiveness of the in vivo intervention. The in vitro dose of 30 μmol/L for PC12 cells was screened and confirmed by our preliminary dose-response experiments (including 10, 20, 30, 50 μmol/L gradient concentrations). The results of the pre-experiment showed that 30 μmol/L was the optimal effective dose of ZYZ-488: it avoid obvious cytotoxicity caused by high concentrations, which is suitable for in vitro positive control intervention. The results of the dose-finding experiment for the in vitro concentration have been supplemented in Figure 3B.

Comments 6:

While the study demonstrates that AS-IV modulates CytC/Apaf-1 pathway proteins, there is no knockdown/knockout experiment (e.g., Apaf-1 siRNA in PC12 cells) to directly confirm that the AS-IV protective effect is specifically dependent on this pathway. The ZYZ-488 comparator partially addresses this, but pathway-specific genetic evidence would substantially strengthen the mechanistic claims.

Dear Reviewer,

We sincerely appreciate the reviewer's insightful comment and valuable suggestion regarding the mechanistic validation of the CytC/Apaf-1 pathway in AS-IV’s neuroprotective effect. We fully acknowledge that genetic manipulation approaches such as Apaf-1 siRNA knockdown would further strengthen the specific dependence of AS-IV’s action on this pathway, and this is indeed a valuable direction for our subsequent in-depth research.

In this study, the specific Apaf-1 inhibitor ZYZ-488 was used as a pharmacological comparator to verify the pathway-specificity of AS-IV’s effects. In vivo and in vitro experiments showed both AS-IV and ZYZ-488 exerted significant neuroprotection against CIRI/OGD/R injury and exhibited highly consistent regulatory patterns on the CytC/Apaf-1 pathway, with no significant difference in their regulatory efficacy. Moreover, molecular docking and dynamics simulation confirmed AS-IV stably bound to the functional domain of Apaf-1 (the pathway’s core node) with high affinity, directly blocking apoptosome formation. These multi-layered evidences (consistent pharmacological intervention, specific molecular binding, in vivo/in vitro functional validation) strongly support that AS-IV specifically regulates the CytC/Apaf-1 pathway.

Given the current focus of this study on clarifying the molecular mechanism of AS-IV targeting the CytC/Apaf-1 pathway to inhibit apoptosis and the comprehensive pharmacological validation we have conducted, we have not included genetic knockdown/knockout experiments in this work. However, we highly agree with the reviewer’s suggestion and have planned to perform Apaf-1 siRNA knockdown in PC12 cells and even construct Apaf-1 conditional knockout rat models in our subsequent research. This will allow us to more directly and rigorously confirm the specific dependence of AS-IV’s neuroprotective effect on the CytC/Apaf-1 pathway, and further explore the potential synergistic effects of AS-IV combined with genetic intervention strategies for CIRI treatment. We have added a relevant statement in the Limitations and Future Perspectives section of the revised manuscript to clarify this research plan.

We believe that the existing experimental design and results of this study have sufficiently and reliably elucidated the core mechanistic claim that AS-IV alleviates CIRI by targeting the CytC/Apaf-1 mitochondrial apoptotic pathway, and the proposed genetic validation experiments will serve as an important extension to further deepen and perfect this research system.

Comments 7:

The molecular docking section lists six target proteins and their binding scores but does not specify which PDB structures were used, including resolution, source organism, or whether the crystal structures contained cognate ligands. This critical methodological detail must be disclosed.

Dear Reviewer,

We sincerely appreciate the reviewer's meticulous comment and valuable suggestion. In the revised manuscript, we have supplemented the critical methodological details of all six target proteins for molecular docking, including their PDB accession numbers, crystal structure resolutions, source organisms, and cognate ligand information, and the supplementary content has been integrated into the 4.4 Molecular Docking section to ensure the reproducibility and scientific rigor of the experiment.

The detailed PDB structural information of the six target proteins is as follows: CytC (PDB: 5O10, 1.36 Å resolution, Homo sapiens), Apaf-1 (PDB: 4RHW, 2.1 Å resolution, Homo sapiens), BAX (PDB: 4BD6, 2.494 Å resolution, Homo sapiens), Bcl-2 (PDB: 9EW8, 1.488Å resolution, Homo sapiens), Caspase3 (PDB: 1GFW, 2.8 Å resolution, Homo sapiens), Caspase9 (PDB: 2AR9, 2.8 Å resolution, Homo sapiens).

Comments 8:

The Methods section cites "AutoDock Vina 4.2", but the widely used software is AutoDock Vina 1.x (e.g., 1.1.2 or 1.2). Version "4.2" does not appear to be a valid release and may be a typographical or factual error that must be clarified or corrected.

Dear Reviewer,

We sincerely appreciate the reviewer's meticulous observation and valuable suggestion. We confirm this was a typographical error in the manuscript, and the correct version of the software used for molecular docking is AutoDock Vina 1.2.0—one of the most widely used and updated official releases of the software, consistent with standard molecular docking experimental protocols. The erroneous "4.2" has been revised to 1.2.0 in the 4.4 Molecular Docking section of the revised manuscript. We have also conducted a full check of all software versions, reagent specifications and experimental parameter notations in the Methods section to eliminate any similar factual or typographical errors, ensuring the accuracy and reproducibility of all methodological details.

Comments 9:

The 100 ns MDS was performed for all six protein-AS-IV complexes, but the Results section only presents integrated trajectory data. The figure captions are not clear enough to indicate which protein complex is shown in each panel of Figure 6. Given that Apaf-1 showed the highest affinity, a dedicated and clearly labeled MDS analysis for the AS-IV/Apaf-1 complex would strengthen the paper considerably.

Dear Reviewer,

We sincerely appreciate the reviewer's insightful comment and valuable suggestion for optimizing the molecular dynamics simulation (MDS) results presentation. In the revised manuscript, we have made targeted revisions to the 100 ns MDS results section and Figure 6 to improve clarity, and highlighted the AS-IV/Apaf-1 complex analysis to emphasize its core role, with all modifications integrated into Sections 2.4 and the corresponding figure captions.

 we have revised and detailed the captions of Figure 6 to clearly specify the corresponding protein complex for each panel, and added clear annotations of each target protein in the subgraphs to eliminate ambiguity in the trajectory data presentation.

In addition, we have optimized the layout of Figure 6 to place the AS-IV/Apaf-1 complex data in a prominent position, with unified and clear labeling of all protein complex panels to ensure the readability and interpretability of the integrated trajectory data. These revisions not only clarify the MDS results presentation of all six protein-AS-IV complexes but also highlight the core MDS findings of the AS-IV/Apaf-1 complex, which further strengthens the mechanistic evidence for AS-IV’s specific targeting of Apaf-1.

Comments 10:

The statistical methods mention one-way ANOVA with p < 0.05, but there is no mention of post-hoc test used (e.g., Tukey, Dunnett). All figures show significance only as P < 0.01, with no intermediate significance levels (e.g., P < 0.05, P < 0.001) reported. No effect sizes or confidence intervals are reported

Dear Reviewer,

We sincerely appreciate the reviewer's meticulous observation and valuable suggestion on the statistical analysis of the study. In the revised manuscript, we have supplemented and standardized all statistical analysis details, and unified the significance annotation of experimental results, with the relevant modifications integrated into the 4.6 Statistical Methods section and all corresponding figure captions.

In addition, we have rechecked all experimental data and statistical analysis results to ensure the accuracy of the statistical methods, significance levels and effect size calculations, and standardized the statistical annotation format of all figures and tables to meet the requirements of scientific research paper statistical reporting.

Comments 11:

The animal experiment specifies 60 rats divided into 4 groups (n=15/group), but the cell experiments provide no information on the number of independent biological replicates (n = ?) or technical replicates performed.

Dear Reviewer,

We sincerely appreciate the reviewer’s careful comment.In the revised manuscript, we have supplemented the detailed replicate information for the cell experiments in the Materials and Methods and the corresponding figure legends.

All cell experiments were performed with 3 independent biological replicates (n = 3) and 3 technical replicates per biological replicate, which is consistent with the common and reliable standard for in vitro studies.These details have been clearly stated in the revised manuscript to ensure the reproducibility and rigor of the cell-based experiments.

Comments 12:

Figure 3's legend uses "vs Sham group" and "vs MCAO/R group" for labeling significance, but the cell experiment has no "Sham" or "MCAO/R" groups — the groups are "Control" and "OGD/R." This appears to be copied from the animal figure legends and must be corrected.

Dear Reviewer,

We sincerely appreciate the reviewer's careful observation and valuable suggestion. We confirm that the significance labels in the original Figure 3 legend were incorrectly copied from the in vivo experiment figure legends, which led to an inconsistency with the in vitro experimental grouping.

In the revised manuscript, we have corrected the legend of Figure 3 to accurately reflect the in vitro experimental design. The significance labels have been updated from "vs Sham group" and "vs MCAO/R group" to "vs Control group" and "vs OGD/R group", respectively, to match the actual grouping of the cell experiments. We have also conducted a comprehensive check of all figure legends in the manuscript to ensure that the significance labels are consistent with the corresponding experimental grouping, eliminating any similar descriptive inconsistencies.

Comments 13:

The authors cite their own prior work (refs 19–21) showing AS-IV effects on CIRI via autophagy and calcium-sensing receptor pathways, but the Discussion does not adequately differentiate the novelty of the current CytC/Apaf-1 mechanistic findings from these prior studies. A more explicit comparison is needed.

Dear Reviewer,

We sincerely appreciate the reviewer’s insightful and constructive comment.In the revised manuscript, we have strengthened the Discussion section by explicitly comparing and distinguishing the novelty of the current study from our previous work.

Our prior studies focused on the neuroprotective effects of AS-IV against CIRI via autophagy and calcium-sensing receptor pathways. In contrast, the present study provides new mechanistic evidence that AS-IV alleviates cerebral ischemia–reperfusion injury by directly targeting the CytC/Apaf1 mitochondrial apoptotic pathway, which represents a distinct and novel downstream mechanism that has not been reported in our previous investigations.

This new finding further expands the molecular regulatory network of AS-IV in antiapoptosis and neuroprotection, and clearly highlights the novelty and incremental contribution of the current work. The relevant comparative description has been added and emphasized in the revised Discussion (see lines 327-340)

Comments 14:

Reference 16 (Alshehri, 2024, on CytC and cancer cell metabolism) is cited to support the statement that Apaf-1 expression correlates with neuronal apoptosis rate. This is not the most appropriate citation; a more directly relevant reference about Apaf-1 in CIRI should be used.

Dear Reviewer,

We sincerely appreciate the reviewer’s careful and professional comment. We agree that Reference 16 (Alshehri, 2024) focused on CytC in cancer metabolism and was not appropriately cited to support the correlation between Apaf1 expression and neuronal apoptosis in cerebral ischemiareperfusion injury.

In the revised manuscript, we have replaced Reference 16 with more relevant studies that directly demonstrate the critical role of Apaf1 in regulating neuronal apoptosis during cerebral ischemia/reperfusion injury. These updated references clearly validate the positive correlation between Apaf1 expression level and neuronal apoptosis rate, ensuring the accuracy and scientific rigor of the citation.

Comments 15:

Several sentences are verbose, repetitive, or use awkward phrasing, particularly in the Introduction and Discussion. Examples:"...has become the core direction and urgent need in the research on ischemic stroke treatment in the field of neuroscience" — overly wordy. The Discussion repeats several statements nearly verbatim from the Introduction. A professional English language editing service is recommended prior to resubmission.

Dear Reviewer,

We sincerely appreciate the reviewer's meticulous observation and valuable suggestion regarding the language expression of the manuscript. We fully acknowledge that some sentences in the Introduction and Discussion sections are verbose, repetitive, or awkwardly phrased, and there are instances of near-verbatim repetition between the Discussion and Introduction, which affect the readability and professionalism of the paper.

In the revised manuscript, we have carried out a comprehensive and rigorous revision of the entire text, with a focus on optimizing the Introduction and Discussion sections: first, we have streamlined verbose sentences (e.g., the phrase "...has become the core direction and urgent need in the research on ischemic stroke treatment in the field of neuroscience" has been revised to "...has become a core direction and urgent requirement for ischemic stroke treatment in neuroscience" to enhance conciseness); second, we have eliminated repetitive statements between the Discussion and Introduction, ensuring that the Introduction focuses on background introduction and research gaps, while the Discussion focuses on result interpretation, mechanism elaboration, and research significance, avoiding redundant repetition; third, we have revised awkward phrasing throughout the text, standardized the use of academic English, and improved the fluency and accuracy of language expression.

Furthermore, in line with the reviewer's recommendation, we have arranged for a professional English language editing service to conduct a thorough proofreading and polishing of the entire manuscript before resubmission. This service will focus on optimizing sentence structure, correcting grammatical errors, standardizing academic terminology, and enhancing the overall language professionalism and readability, ensuring that the manuscript meets the language requirements of the journal.

We have rechecked the revised text multiple times to ensure that all verbose, repetitive, and awkward expressions are effectively improved, and the consistency and logic of the text are maintained.

Comments 16:

The Figure 3 caption describes PI staining as showing "red fluorescence" for PI-positive cells and TUNEL as "brown staining". The brown DAB TUNEL staining is valid for paraffin sections, but the authors should clarify whether cells were on coverslips (climbing slices) or standard slides for fluorescence-based assays, as the methodology mixes fluorescent and chromogenic detection.

Dear Reviewer,

We sincerely appreciate the reviewer’s meticulous observation and valuable suggestion regarding the methodological details of the PI and TUNEL staining in Figure 3.

In the revised legend of Figure 3, we have clearly specified that the PC12 cells were seeded on glass coverslips (cell climbing slices) for subsequent staining experiments. Regarding the combined use of fluorescent and chromogenic detection methods: PI staining was performed for fluorescent detection (red fluorescence, observed under a fluorescence microscope) to rapidly visualize and quantify apoptotic cells; TUNEL staining was performed using the DAB chromogenic method (brown staining), which is more suitable for clear observation of apoptotic cell morphology in adherent cells on coverslips, and this method is compatible with cell climbing slices . We have added these methodological details to the 4.3.5 to clarify the experimental operation and ensure the reproducibility of the staining protocol.

Comments 17:

The Abbreviations section is well-organized, but "FEC"is listed as "Free Energy Contribution" while in the text it is described as representing free energy landscape analysis. The exact definition should be consistent throughout.

Dear Reviewer,

We sincerely appreciate the reviewer’s careful observation and valuable suggestion. We confirm that there is an inconsistency between the definition of "FEC" in the Abbreviations section and its description in the text, which is a negligent error.

In the revised manuscript, we have corrected this inconsistency to ensure uniformity throughout the paper. Specifically, since "FEC" is used in the text to represent free energy landscape analysis, we have updated the Abbreviations section by revising the definition of "FEC" from "Free Energy Contribution" to "Free Energy Landscape" (consistent with its actual usage in the molecular dynamics simulation results section).

We have also conducted a comprehensive check of all abbreviations in the manuscript, verifying that each abbreviation’s definition in the Abbreviations section matches its description in the text, eliminating any similar inconsistencies and ensuring the accuracy and standardization of the manuscript.

Reviewer Comments: REVIEWER 3

Comments 1:

In the molecular docking study, docking scores were compared between various targets. The VD score cannot be compared across different targets. Therefore, reference ligands for each target should be docked. The VD scores and key interactions of the reference ligands should be compared with those of the test compound. The VD results should be discussed based on these comparisons, and VD results should be compared with obtained in vitro results. Validation of the VD protocol should be added for each target.

Dear Reviewer,

We sincerely appreciate the reviewer’s professional and valuable comments on the molecular docking analysis.

We fully acknowledge that VD scores should not be directly compared across different targets, and that docking validation using reference ligands is necessary for each target to ensure reliability. In the revised manuscript, we will supplement the docking of corresponding reference ligands for each target, compare their VD scores and key interactions with those of the tested compound, and further discuss the docking results combined with our in vitro experimental data. We will also add the validation of the molecular docking protocol for each target to improve the scientific rigor of this part.

Comments 2:

Figures of the virtual docking should be modified according to the specific VD procedure described in the review report.

Dear Reviewer,

We sincerely appreciate the reviewer’s constructive comment.We will revise the presentation and labels of the virtual docking figures to be consistent with the improved and validated docking procedure as suggested.

Comments 3:

The abstract and conclusion should be revised to reflect these changes in the virtual docking protocol and results.

Dear Reviewer,

We sincerely appreciate the reviewer’s constructive comment.We will revise the abstract and conclusion to accurately reflect the updated virtual docking protocol and corresponding results.