Distinct and Overlapping Neuroprotective Efficacy of Silk Lutein and Sericin-Derived Oligopeptides from Yellow Silk Cocoons in Rodent Models of Aβ-Induced and Age-Related Cognitive Decline

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents a comparative investigation of the cognitive and neurobiological effects of silk lutein (SL) and sericin-derived oligopeptides (SDO) in two rodent models, combining behavioral assessments, electrophysiological recordings, and histological analyses to explore their potential mechanisms of action in Aβ-induced and age-related cognitive impairment.

The study would benefit from several clarifications and minor methodological refinements to strengthen transparency, precision, and reproducibility.

Page 2, lines 49–56. The discussion of oxidative stress and Aβ toxicity is thorough but slightly repetitive. Please condense the mechanistic explanation of ROS generation and Aβ aggregation to improve flow and avoid redundancy. Merging lines 49–53 and 53–56 into a more concise paragraph would enhance readability without reducing scientific depth.

Page 2, lines 83–91. The link between antihypertensive effects and cognitive health is plausible but somewhat indirect in the context of the present study. I suggest adding one sentence acknowledging that the vascular–cognitive link is inferential here (e.g., “Although indirect, these vascular effects may contribute to improved cerebral perfusion and resilience”).

Page 3–4, lines 115–130 (Table 1). Units are inconsistent (mg/mL vs μg/mL). The authors should standardize units across compounds or explicitly state in the table heading that the difference reflects relative potency. This will improve clarity and comparability.

Page 9, lines 294–303. The interpretation becomes assertive when introducing the term “synaptoprotective mechanism.” Please revise “Consequently, the efficacy of SL and SDO in this model points towards a synaptoprotective mechanism…” to “Consequently, the efficacy of SL and SDO in this model is consistent with preservation of synaptic function…” to better align the conclusion with the functional data presented.

Page 19, lines 552–565. The statement concluding that the compounds converge on a shared synaptoprotective mechanism and are “reinforcing their potential as multi-target agents” is slightly strong. Please replace “reinforcing their potential as multi-target agents” with “supporting their potential as multi-target candidates” to maintain a balanced tone.

Page 20, lines 595–600. In the final paragraph of the Discussion, the sentence stating that the compounds “exert significant neuroprotective and cognitive-enhancing effects” may be somewhat overstated given that overt neurodegeneration was not demonstrated. Please consider revising to “exert significant cognitive-enhancing effects and are associated with improved synaptic plasticity.”

Page 22, line 684. There is a minor typographical error: “Fourty-eight” should be corrected to “Forty-eight.”

Page 21–22, lines 680–692 (Section 3.3 Animals). Although ethical approval, strain, age, housing conditions, and ARRIVE compliance are clearly described, there is no explicit statement indicating how animals were randomized into treatment groups, whether behavioral testing was performed under blinded conditions, or whether electrophysiological (LTP) recordings and analyses were conducted blind to treatment allocation. Please add a brief clarification specifying the method of group allocation and whether experimenters were blinded during behavioral and electrophysiological assessments to strengthen internal validity and reporting transparency.

Page 23, lines 724–727 (Intracerebroventricular surgery). The injection depth (2.5 mm) is reported; however, the anteroposterior (AP) and mediolateral (ML) stereotaxic coordinates relative to bregma are not provided. Since the text states that coordinates were precisely targeted relative to bregma, the exact AP and ML values should be included to ensure full reproducibility of the i.c.v. procedure.

Page 23, lines 745–751 (TUNEL assay). The Methods indicate that TUNEL-positive cells were quantified under a light microscope; however, the Results section presents the findings largely descriptively. Please clarify how quantification was performed (e.g., cells per defined hippocampal area, number of sections analyzed per animal, subregions evaluated) and whether the analysis was conducted under blinded conditions.

Author Response

We sincerely thank Reviewer 1 for the constructive feedback and the time invested in reviewing our manuscript. We appreciate the reviewer’s insightful suggestions regarding methodological transparency and scientific precision, specifically the inclusion of exact stereotaxic coordinates and blinding procedures, as well as the refinement of our terminology to ensure our conclusions remain strictly aligned with the functional data presented. We have addressed each of your comments below and have incorporated the corresponding changes into the revised manuscript. Please find the detailed responses below and the corresponding revisions highlighted in the re-submitted files.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors! You have addressed a pressing issue in fundamental and clinical medicine, namely, the development of new nootropic agents for the treatment of cognitive dysfunction. The following comments arose during the analysis of your study: 1. The introduction does not describe the basic mechanisms of cognitive-mnestic impairment in neurodegenerative pathology. 2. The introduction does not describe the characteristics of basic nootropic agents and their shortcomings. It is unclear why it is necessary to develop new nootropics. 3. Please describe in detail the animal randomization method used so that the reader can easily reproduce it. For example: how were the animals labeled for assignment of numbers, what type of output data was obtained by the random number generator (which one?), and how was it compared with the numbered animals, if applicable. Examples of randomization methods include block randomization, stratified randomization, covariance adaptive randomization, etc., used to assign subjects to different experimental groups. 4. Describe the inclusion and exclusion criteria, or indicate that exclusion criteria were not predetermined, and whether any animals (and how many) were excluded based on the exclusion criteria or died during the experiments. If any animals were excluded, indicate whether they were replaced, how many, in which group(s), and at what stage of the experiment. 5. Indicate whether an outlier test (and which one) was performed on the data, and indicate whether any data points were excluded. Or, indicate that an outlier test was not performed. 6. For all data sets, please provide full statistical reports (e.g., for ANOVA, provide degrees of freedom, F-value, and actual P-value; for t-test, provide degrees of freedom, t-value, and actual P-value; for Mann-Whitney U-test, provide U-value and actual P-value; for Kruskal-Wallis test, provide H-statistic, degrees of freedom, and P-value). This information may be presented in the text of the results, figure legends, or as a separate table of all analyses, but must be presented in one of these ways. 7. No clear study objective 8. Methods of obtaining and standardizing test samples? 9. Do low temperatures and storage durations affect structural integrity and pharmacological activity? 10. Bacteriological contamination? 11. Blinding of the experiment at various stages 12. Did you administer the peptides orally? How accurate and logical is this in terms of digestion processes in the stomach? 13. There is no explanation for conducting in vitro studies. What did the authors hope to obtain using these methods? What do these parameters provide in terms of the nootropic effect of the test systems? 14. Please describe the open-field technique more clearly. Illumination zones, penumbra, indicating the light intensity. 15. I did not see a good discussion in the manuscript. 16. There is no unified cohesive model for the pathogenesis of cognitive-mnestic impairments in neurodegenerative diseases. There are no clear targets for pharmacological intervention. 17. The novelty of the work is not clearly articulated. 18. The authors did not provide a reliable methodological basis for the preclinical evaluation of the nootropic activity of the test samples. 19. The authors should clearly distinguish between nootropic activity and neuroprotective activity. 20. There are no study limitations.

Author Response

We sincerely thank Reviewer 2 for the exceptionally thorough and rigorous evaluation of our manuscript. We appreciate the detailed feedback provided, particularly regarding the transparency of our animal randomization and blinding procedures, the necessity for comprehensive statistical reporting, and the clarification of our mechanistic targets and study limitations. These insights have been invaluable in strengthening the methodological foundation and the overall scientific impact of our study. We have addressed each of your comments below and have incorporated these revisions into the updated manuscript to ensure the highest standards of clarity and reproducibility. Please find the detailed responses below and the corresponding revisions highlighted in the re-submitted files.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

SL and SDO study review

The paper under review contains novel and very promising experimental data which stated that the Bombyx mort derived substances possess the biological activity. It was demonstrated as the amelioration of behavioral defects in respective tests addressed to memory  processes. The problem of even modest symptomatic relief in CNS state stays as important one and the need for search of new therapy approaches is obvious.

The authors used two experimental models in rodents for testing the effects of new substances : an amyloid-beta (Aβ₂₅₋₃₅)-induced amnesia model  in mice and a natural aging model in rats. Morris water maze test and Novel object recognition as well as the indices of hippocampal LTP were analysed.  It was presumably obvious that the treatment applied in this study aimed to affect the damage of oxidative stress and of destructive action of Aβ peptides. Both -  carotenoid silk lutein and sericin-derived oligopeptides of Bombyx mori proved to possess the clear cut “improving” effects on CNS function.

The paper is well illustrated and demonstrate that authors are “in a pitch” of the whole problem of neurodegeneration.

The only remark should be done – we recommend authors to give the general outline of neurodegenerative experimental models indicating that many of them could be used to further elucidate the direct effects of substances  used in this study.

Author Response

We sincerely thank Reviewer 3 for the positive assessment of our work and for recognizing the novelty and clinical relevance of our findings regarding Bombyx mori-derived bioactives. We appreciate the encouraging comments regarding the quality of our illustrations and our approach to the problem of neurodegeneration. Please find the detailed responses below and the corresponding revisions highlighted the re-submitted files.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors! You have done a superb job of revising the manuscript. I recommend your manuscript for publication. I wish you success in conquering scientific heights.

Author Response

We sincerely thank Reviewer 2 for the constructive feedback in our manuscript. We appreciate the reviewer’s insightful suggestions.