Microwave-Assisted Propolis Extract Attenuates Oxidative-Stress- and Replicative Senescence via NRF2 and Wnt/β-Catenin–TERT Activation in Human Dermal Fibroblasts

Round 1

Reviewer 1 Report

The manuscript entitled presents an in vitro study investigating the anti-senescent and regenerative effects of a propolis extract in human dermal fibroblasts. The authors examine two complementary models—H₂O₂-induced senescence and replicative senescence—and report that MAPE reduces oxidative stress, suppresses senescence-associated markers, restores extracellular matrix (ECM) homeostasis, and activates Wnt/β-catenin signaling with induction of TERT expression.

The study is conceptually interesting and addresses a relevant topic in redox biology and skin aging. The experimental design is structured logically, moving from extract characterization to oxidative stress assays, senescence markers, ECM remodeling, wound healing, and mechanistic pathway analysis. The use of both oxidative and replicative senescence models strengthens the biological relevance. Inclusion of EGCG and CHIR99021 comparators is appropriate and supports interpretation of antioxidant and Wnt-related effects.

Major comments

Activation of Nrf2 is evaluated through the expression of target genes (NQO1, GCLM), and activation of Wnt/β-catenin signaling is based on mRNA changes and β-catenin localization. Direct functional validation is lacking. The manuscript would be substantially strengthened by pharmacological inhibition of Nrf2 to demonstrate causality at least in ROS production. The induction of TERT mRNA is interpreted as telomerase reactivation and partial rejuvenation, but no telomerase activity assay or telomere analysis is presented.

Cytotoxicity or proliferation controls should be provided to exclude the possibility that reductions in senescence markers result from altered cell survival or growth dynamics.

The phytochemical characterization of MAPE, while including HPLC analysis of selected flavonoids and total phenolic content, remains limited for a complex natural extract. A more comprehensive chemical fingerprint would improve reproducibility and clarity regarding active constituents.

Finally, “rejuvenation” term is not appropriate because the study only refers to in vitro data and should be revised to reflect attenuation rather than reversal of senescence.

Minor Comments

“dual protection of redox balance and structural homeostasis is likely…” (Section 3.6) → sentence fragment; revise.

Some minor spacing inconsistencies around percentages and µM.

Statistical Reporting: indicate exact n values for each experiment. Dots graph are appreciated

Discussion: address potential oncogenic risks of chronic Wnt/TERT activation in the context of cosmetic use.

Terminology: ensure consistent gene/protein formatting (italic for genes, normal for proteins).

Author Response

Major comments

▶Activation of Nrf2 is evaluated through the expression of target genes (NQO1, GCLM), and activation of Wnt/β-catenin signaling is based on mRNA changes and β-catenin localization. Direct functional validation is lacking. The manuscript would be substantially strengthened by pharmacological inhibition of Nrf2 to demonstrate causality at least in ROS production. The induction of TERT mRNA is interpreted as telomerase reactivation and partial rejuvenation, but no telomerase activity assay or telomere analysis is presented.

Response: We thank the reviewer for this insightful comment and agree that pharmacological inhibition experiments would provide stronger causal validation of Nrf2-mediated signaling.

In the present study, activation of the Nrf2 pathway was inferred from the upregulation of canonical Nrf2 target genes (NQO1 and GCLM) together with a marked reduction in intracellular ROS levels, which is consistent with activation of an Nrf2-associated antioxidant response. While these findings support the involvement of Nrf2 signaling in the antioxidant effects of MAPE, we acknowledge that pharmacological or genetic inhibition of Nrf2 would further strengthen the mechanistic validation. Due to the scope of the current study, such experiments were not performed and will be addressed in future investigations.

Regarding the reviewer’s comment on telomerase activation, we agree that increased TERT mRNA expression alone does not directly demonstrate telomerase enzymatic activity. Accordingly, we have revised the manuscript to avoid overinterpretation and now describe this observation as suggesting potential involvement of telomerase-related regulatory pathways rather than definitive telomerase reactivation. We have also added a statement in the Discussion clarifying that additional studies assessing telomerase activity and telomere dynamics will be required to further validate this mechanism.

These revisions have been incorporated into the revised manuscript (Discussion section, Lines 639–649).

▶Cytotoxicity or proliferation controls should be provided to exclude the possibility that reductions in senescence markers result from altered cell survival or growth dynamics.

Response: We thank the reviewer for this important comment. To exclude the possibility that the observed reductions in senescence-associated markers were due to altered cell survival or growth dynamics, we evaluated the cytotoxicity of MAPE in replicatively senescent human dermal fibroblasts across the tested concentration range (0.002–0.02%). MAPE did not induce detectable cytotoxicity after 5 days of treatment. These data are now provided as Supplementary Figure S2, and the corresponding description has been added to the Results section of the revised manuscript, as described in Section 3.9 (Lines 489–492).

▶The phytochemical characterization of MAPE, while including HPLC analysis of selected flavonoids and total phenolic content, remains limited for a complex natural extract. A more comprehensive chemical fingerprint would improve reproducibility and clarity regarding active constituents.

Response: We thank the reviewer for highlighting the importance of comprehensive phytochemical characterization of complex natural extracts. To address this comment, we expanded the phytochemical profiling of MAPE by conducting a screening of 20 major phenolic compounds, which are now listed in the revised Section 2.1 (Lines 126–135).

None of these commonly reported phenolic standards were detected in MAPE under the present analytical conditions, suggesting that the extract possesses a phytochemical profile distinct from the screened phenolic standards. This is illustrated in the HPLC chromatograms provided in Supplementary Figure S1, where the chromatographic profiles of the 20 phenolic standards are compared with that of MAPE.

To further establish a reproducible chemical fingerprint of the extract, we quantified three representative propolis flavonoids—chrysin, pinocembrin, and galangin—which were used as marker compounds for extract standardization. These results have been incorporated into the revised Section 3.1 (Lines 253–259).

In addition, the full list of the 20 screened compounds together with their retention times is now provided in Supplementary Table S1. These additions provide a clearer chemical fingerprint of MAPE and improve the transparency and reproducibility of the extract characterization.

▶Finally, “rejuvenation” term is not appropriate because the study only refers to in vitro data and should be revised to reflect attenuation rather than reversal of senescence.

Response: We thank the reviewer for this valuable comment. We agree that the term “rejuvenation” may overstate the interpretation of the present in vitro findings. Accordingly, the manuscript has been revised to replace “rejuvenation” with more appropriate terminology such as “attenuation of cellular senescence” or “anti-senescent effects.” In addition, several related statements have been slightly toned down to ensure that the interpretation accurately reflects attenuation rather than reversal of senescence. These revisions were implemented throughout the manuscript, including the section titles, Results descriptions, figure legends, and Discussion.

 

Detailed comments

▶ “dual protection of redox balance and structural homeostasis is likely…” (Section 3.6) → sentence fragment; revise.

Response: We apologize for the grammatical error. As suggested by the reviewer, we have corrected the sentence fragment in Section 3.6 by adding a formal subject and ensuring a complete sentence structure. The revised sentence now reads: "This dual protection of redox balance and structural homeostasis is likely to underlie the anti-senescent phenotype and improved dermal homeostasis observed in our in vitro skin-aging model." (Section 3.6, Lines 404).

▶Some minor spacing inconsistencies around percentages and µM.

Response: We apologize for the inconsistencies in spacing. As pointed out by the reviewer, we have performed a comprehensive review of the entire manuscript, including all figures and tables, to standardize the spacing around units. All inconsistent notations have been corrected.

▶Statistical Reporting: indicate exact n values for each experiment. Dots graph are appreciated

Response: We thank the reviewer for this helpful suggestion. Following this comment, we carefully reviewed all statistical reporting and ensured that the exact sample size (n = 3 independent biological replicates) is clearly indicated in the figure legends for each experiment throughout the manuscript.

Regarding the suggestion to include dot plots, we agree that visualization of individual data points can improve transparency of the data distribution. However, to maintain visual consistency and clarity across all figures in the manuscript, we retained the current bar graph format. Importantly, the error bars (mean ± SD) and exact n values are clearly provided in the revised figure legends to facilitate interpretation of biological variability and statistical significance. These clarifications have been incorporated into the revised manuscript.

▶Discussion: address potential oncogenic risks of chronic Wnt/TERT activation in the context of cosmetic use.

Response: We thank the reviewer for this important comment. In response, we have added a paragraph in the Discussion addressing safety considerations related to Wnt/β-catenin and TERT modulation in the context of cosmetic use. Specifically, we clarify that canonical Wnt signaling is a physiological pathway involved in skin homeostasis, fibroblast function, and tissue repair, and that the present study reports transcriptional modulation observed in vitro rather than sustained proliferative signaling. We also note that topical exposure in cosmetic applications and the presence of the skin barrier are expected to limit persistent pathway activation. These points have been incorporated into the revised Discussion section (Lines 628–636).

▶Terminology: ensure consistent gene/protein formatting (italic for genes, normal for proteins).

Response :We appreciate the reviewer’s careful attention to the nomenclature. Following the international guidelines for scientific writing, we have thoroughly reviewed and standardized the formatting of all biological markers throughout the manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

This study reports that a standardized microwave-assisted propolis extract (MAPE) attenuates oxidative stress- and replication-driven senescence in human dermal fibroblasts by reducing ROS, suppressing SASP markers, restoring ECM-related gene expression, and improving wound closure. Mechanistically, the authors propose that MAPE activates Nrf2-dependent antioxidant signaling and reactivates Wnt/β-catenin–TERT signaling, with effects comparable to CHIR99021, suggesting potential cosmeceutical and therapeutic anti-aging applications in both acute and replicative senescence models. Overall, the manuscript is promising and of potential interest; however, several issues should be addressed before it can be considered for acceptance.

1.Compared with the background provided for the Wnt/β-catenin pathway, the introduction of Nrf2 signaling is relatively limited (Lines 51–53). Please expand this section to provide sufficient context on the biological role and relevance of Nrf2 signaling （Please refer to references: doi:10.7150/thno.98457; DOI:10.15212/bioi-2025-0142）.
2. The manuscript proposes that MAPE exerts anti-senescent effects through activation of Nrf2 and Wnt/β-catenin–TERT signaling; however, the current evidence appears largely correlative. Please provide more direct loss-of-function or pathway-interference evidence (e.g., Nrf2 inhibition/knockdown, β-catenin inhibition/knockdown, or TERT perturbation) to demonstrate whether these pathways are necessary for the observed anti-senescent effects of MAPE.
3.Because MAPE is a natural extract with potentially variable composition, the manuscript would benefit from a more rigorous description of extract standardization. Please clarify how batch consistency was ensured, provide the quantitative ranges (and preferably batch variability) of major constituents (e.g., chrysin, pinocembrin, galangin, total phenolics), and indicate whether the reported biological effects were reproduced across independent MAPE batches.
4.You mentioned that IL-6 mRNA and protein secretion, representative components of the senescence-associated secretory phenotype. Could you please provide supporting references for this statement? I was unable to identify an appropriate reference that substantiates this point.

Author Response

Major comments

This study reports that a standardized microwave-assisted propolis extract (MAPE) attenuates oxidative stress- and replication-driven senescence in human dermal fibroblasts by reducing ROS, suppressing SASP markers, restoring ECM-related gene expression, and improving wound closure. Mechanistically, the authors propose that MAPE activates Nrf2-dependent antioxidant signaling and reactivates Wnt/β-catenin–TERT signaling, with effects comparable to CHIR99021, suggesting potential cosmeceutical and therapeutic anti-aging applications in both acute and replicative senescence models. Overall, the manuscript is promising and of potential interest; however, several issues should be addressed before it can be considered for acceptance.

 

Detailed comments

▶1.Compared with the background provided for the Wnt/β-catenin pathway, the introduction of Nrf2 signaling is relatively limited (Lines 51–53). Please expand this section to provide sufficient context on the biological role and relevance of Nrf2 signaling （Please refer to references: doi:10.7150/thno.98457; DOI:10.15212/bioi-2025-0142）.

Response: We thank the reviewer for the helpful suggestion. We have expanded the description of the Nrf2 signaling pathway in the Introduction section and incorporated the recommended references. (Introduction, Lines 51-60).

▶2. The manuscript proposes that MAPE exerts anti-senescent effects through activation of Nrf2 and Wnt/β-catenin–TERT signaling; however, the current evidence appears largely correlative. Please provide more direct loss-of-function or pathway-interference evidence (e.g., Nrf2 inhibition/knockdown, β-catenin inhibition/knockdown, or TERT perturbation) to demonstrate whether these pathways are necessary for the observed anti-senescent effects of MAPE.

Response: We thank the reviewer for this insightful comment regarding the mechanistic validation of the signaling pathways involved in the anti-senescent effects of MAPE.

In the present study, activation of the Nrf2 pathway was inferred from the upregulation of canonical Nrf2 target genes (NQO1 and GCLM) together with a marked reduction in intracellular ROS levels, which is consistent with activation of an Nrf2-associated antioxidant response. Likewise, modulation of the Wnt/β-catenin pathway was supported by the restoration of Wnt3A and LEF1 expression, nuclear localization of β-catenin, and the observation that the transcriptional responses induced by MAPE closely resembled those elicited by the canonical Wnt activator CHIR99021.

We agree with the reviewer that loss-of-function approaches (e.g., pharmacological inhibition or gene knockdown of Nrf2, β-catenin, or TERT) would provide stronger causal evidence to establish the necessity of these pathways. However, such mechanistic validation experiments were beyond the scope of the current study, which was designed to provide an initial characterization of the anti-senescent activity of MAPE in fibroblast models.

To address this point, we have carefully revised the manuscript to avoid overinterpretation and now describe the involvement of these pathways as associations consistent with pathway modulation rather than definitive causal mechanisms. We have also added statements in the Discussion section highlighting this limitation and emphasizing that future studies employing pathway inhibition or genetic perturbation will be necessary to establish causal relationships.

These clarifications have been incorporated into the revised Discussion section (Lines 639–649).

▶3.Because MAPE is a natural extract with potentially variable composition, the manuscript would benefit from a more rigorous description of extract standardization. Please clarify how batch consistency was ensured, provide the quantitative ranges (and preferably batch variability) of major constituents (e.g., chrysin, pinocembrin, galangin, total phenolics), and indicate whether the reported biological effects were reproduced across independent MAPE batches.

Reponse: We thank the reviewer for this important comment regarding extract standardization and batch consistency.

In the present study, the HPLC chromatogram shown in the manuscript corresponds to MAPE Lot 312241209, which was used for the biological experiments.

To further evaluate the reproducibility of the extraction process, two additional independent batches (Lot 232241206 and Lot 032241209) were prepared using the same microwave-assisted extraction conditions. The chemical consistency of these batches was assessed by HPLC using three representative propolis flavonoids—chrysin, pinocembrin, and galangin—as marker compounds.

The chromatographic profiles of the three batches showed highly similar peak patterns and retention times for the marker flavonoids. Quantitative analysis further confirmed comparable levels of these compounds across batches:

Chrysin: 33–37 ppm

Pinocembrin: 47–51 ppm

Galangin: 29–31 ppm

These results indicate good batch-to-batch consistency of the MAPE preparation based on marker flavonoid profiling. The chromatograms and quantitative comparison of the three independent batches are provided in Figure R1 for the reviewers’ reference.

Figure R1. Batch-to-batch consistency of MAPE evaluated by HPLC analysis.

Figure legend

Representative HPLC chromatograms of three independent MAPE batches are shown together with the chromatogram of the reference standards (STD). The chromatogram corresponding to the batch used in the manuscript is MAPE Lot 312241209, while two additional independent batches (Lot 232241206 and Lot 032241209) were analyzed to assess batch consistency. Quantitative analysis of the marker flavonoids chrysin, pinocembrin, and galangin demonstrated comparable levels across the three batches, confirming reproducible extract composition.

▶4.You mentioned that IL-6 mRNA and protein secretion, representative components of the senescence-associated secretory phenotype. Could you please provide supporting references for this statement? I was unable to identify an appropriate reference that substantiates this point.

Response: We thank the reviewer for this helpful comment. We agree that the statement describing IL-6 as a representative component of the senescence-associated secretory phenotype (SASP) should be supported by appropriate references. Accordingly, we have added references describing IL-6 as a prominent inflammatory cytokine secreted by senescent cells and a key component of the SASP (Coppe et al., 2008; Coppe et al., 2010). The relevant citation has now been included in the revised manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

The current study is quite interesting; however, the following should be addressed:

1- The authors mentioned in the title that MAPE is utilized in the study; however the introduction system did not include a section regarding the merits of the utilization of microwave-assisted extraction compared to conventional techniques.

2-Mention the temperature reached during microwave-assisted extraction.

3- No references were cited in the methodology section. 

4- The authors compared the MAPE with conventional extraction of propolis utilizing other solvents and conditions. Mention this in the methodological section in detail.

5- The authors reported the superiority of microwave-assisted extraction compared to other conventional methods and PG as a solvent, without a deep interpretation of the results, why PG revealed this superiority.

6- Figure 1 lack caption for figure F.

7-Sequence tests were performed to confirm the efficacy of MAPE as a Wnt/β-catenin signaling activator by upregulating Wnt3A and LEF1 while suppressing DKK1, and promoting TERT expression, however there was no deep interpretation of the results or correlation with the components of MAPE and the role of each component. 

8-For wound healing assay, report wound closure percent.

The methodological section needed to be revised to include all detailed experiment that their results appear in the manuscript.

Correlate between the components of propolis and reported activity.  

Author Response

Reviewer #3

The current study is quite interesting; however, the following should be addressed:

 

▶1. The authors mentioned in the title that MAPE is utilized in the study; however the introduction system did not include a section regarding the merits of the utilization of microwave-assisted extraction compared to conventional techniques.

Response: We thank the reviewer for this helpful comment. To clarify the rationale for using microwave-assisted extraction, we have added a paragraph in the Introduction describing the advantages of microwave-assisted extraction compared with conventional extraction techniques. The revised text highlights the improved extraction efficiency, reduced processing time, and compatibility of the butylene glycol extraction system for cosmetic applications (Introduction, Lines 85–95).

▶2.Mention the temperature reached during microwave-assisted extraction.

Response: We thank the reviewer for this helpful comment. The temperature reached during the microwave-assisted extraction process has now been clarified in the Methods section. During microwave irradiation, the extraction temperature was monitored and reached approximately 150 °C (Section 2.1, Lines 122–123).

▶3- No references were cited in the methodology section.

Response: We thank the reviewer for this helpful comment. In the revised manuscript, references supporting the experimental procedures have been added to the Materials and Methods section, including citations for the Folin–Ciocalteu assay for total phenolic content determination, SA-β-gal staining, and intracellular ROS measurement.

▶4- The authors compared the MAPE with conventional extraction of propolis utilizing other solvents and conditions. Mention this in the methodological section in detail.

Response: We thank the reviewer for this valuable comment. In response, we have expanded the Materials and Methods (Section 2.1) to provide a clearer description of the extraction procedures used for comparison with microwave-assisted extraction.

Specifically, we have added a detailed explanation indicating that propolis was extracted using several conventional solvent systems, including ethanol, dipropylene glycol, water, and butylene glycol under thermal extraction conditions (80 °C for 3 h, solvent-to-solid ratio 20:1, w/w). The extraction efficiency of these methods was initially evaluated by measuring total phenolic content (TPC), and microwave-assisted butylene glycol extraction (MAPE) showed the highest phenolic recovery. Based on this screening analysis, MAPE was selected for subsequent phytochemical characterization and biological evaluation.

This clarification has been incorporated into the revised manuscript in Section 2.1 (Lines 107-114).

▶5- The authors reported the superiority of microwave-assisted extraction compared to other conventional methods and PG as a solvent, without a deep interpretation of the results, why PG revealed this superiority.

Response: We thank the reviewer for this valuable comment. In response, we have expanded both the Introduction and Discussion sections to better explain the superiority of microwave-assisted extraction combined with butylene glycol (BG) as the extraction solvent. In the revised Introduction, we added background information describing the advantages of microwave-assisted extraction over conventional extraction methods, including enhanced mass transfer and reduced extraction time. In addition, the Discussion section has been expanded to provide a clearer interpretation of the observed extraction efficiency. Microwave irradiation promotes rapid dielectric heating, which enhances solvent penetration and facilitates the release of intracellular phenolic compounds from the propolis matrix. Furthermore, polyol solvents such as butylene glycol can improve the solubility and stability of phenolic compounds through hydrogen-bonding interactions. These factors likely contributed to the improved recovery of flavonoid markers observed in the MAPE preparation. The corresponding revisions have been incorporated into the manuscript (Introduction, Lines 85–95; Discussion, Lines 619–627)

▶6- Figure 1 lack caption for figure F.

Response: We thank the reviewer for this helpful comment. The caption for panel F in Figure 1 was inadvertently omitted in the original submission. The description for panel F, which presents the total phenolic content (TPC) analysis under different extraction conditions, has now been added to the revised Figure 1 legend.

▶7-Sequence tests were performed to confirm the efficacy of MAPE as a Wnt/β-catenin signaling activator by upregulating Wnt3A and LEF1 while suppressing DKK1, and promoting TERT expression, however there was no deep interpretation of the results or correlation with the components of MAPE and the role of each component.

Response: We thank the reviewer for this valuable comment. In response, we have expanded the Discussion to provide a clearer interpretation of the Wnt/β-catenin–related findings in relation to the phytochemical composition of MAPE. Specifically, we discussed the potential contribution of the major flavonoid constituents identified by HPLC analysis—chrysin, pinocembrin, and galangin—which have been reported to regulate antioxidant and signaling pathways such as Nrf2, NF-κB, and Wnt/β-catenin in various biological systems. Based on these reports, the revised Discussion now highlights that these flavonoid constituents may contribute, at least in part, to the coordinated antioxidant and signaling-modulating effects observed in this study. At the same time, we clarify that, because MAPE is a complex natural extract, the precise contribution of each individual component remains to be determined in future studies using purified compounds or fractionated extracts. These revisions have been incorporated into the Discussion section (Lines 616–627).

▶8-For wound healing assay, report wound closure percent.

Response: We thank the reviewer for this helpful suggestion. In response, we have clarified the Results section by explicitly describing the wound closure percentage obtained in the scratch-wound assay (Lines 396–399). The revised text now indicates that the reported wound closure values were calculated relative to the H₂O₂-treated group to reflect the recovery of fibroblast migration under oxidative stress conditions. The corresponding clarification has also been added to the figure legend

Detailed comments

▶The methodological section needed to be revised to include all detailed experiment that their results appear in the manuscript.

Response: We thank the reviewer for this comment. The Materials and Methods section has been revised and expanded to clearly describe the experimental procedures corresponding to the reported results. In particular, we added detailed descriptions of the comparative extraction procedures, microwave-assisted extraction protocol, and phytochemical analyses in Section 2.1 of the revised manuscript.

▶Correlate between the components of propolis and reported activity.

Response: We appreciate this suggestion. In the revised manuscript, the Discussion section has been expanded to better relate the phytochemical composition of MAPE with the observed biological activities. Specifically, we discussed the potential roles of chrysin, pinocembrin, and galangin, which were identified by HPLC analysis, and cited relevant literature describing their reported effects on antioxidant responses and signaling pathways such as Nrf2 and Wnt/β-catenin.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors clearly improved the quality of the manuscript

No comment

Author Response

Reviewer Comment

The authors clearly improved the quality of the manuscript.

Detailed comments: No comment.

 

Response:

We sincerely thank the reviewer for the positive evaluation of our revised manuscript. We appreciate the reviewer’s time and valuable feedback, which helped improve the quality and clarity of the manuscript.

Reviewer 2 Report

Although the authors have addressed many of the concerns, several issues still require further improvement.

1. The newly added introduction of Nrf2 signaling provides a standard overview of the pathway; however, in its current form, it reads more like a general textbook-style background section than a focused rationale for the present study. The description of the KEAP1–Nrf2–ARE axis and downstream antioxidant genes is broadly accurate, but its connection to dermal fibroblasts and the specific biological context of this manuscript remains insufficiently developed. In particular, the paragraph does not clearly explain why Nrf2 signaling is especially relevant to dermal fibroblast dysfunction, senescence, or the disease/aging phenotype under investigation.
In addition, the revised text still lacks a clear knowledge gap, unresolved question, or point of controversy that would justify the transition from general Nrf2 biology to the specific aims of this study. A stronger introduction should move beyond summarizing canonical pathway components and instead clarify what remains unknown about Nrf2 in dermal fibroblasts, why this gap is important, and how the present work addresses it. I therefore suggest that the authors substantially revise this section to make it more problem-driven, better integrated with the dermal fibroblast context, and more directly aligned with the central research question of the manuscript.

2. The additional batch comparison improves the response, but it still does not fully address the my original concern. The presented data only support partial chemical consistency based on three marker flavonoids, which is not equivalent to rigorous extract standardization for a complex natural product. More importantly, the authors have not provided evidence that the biological effects described in the manuscript are reproducible across independent MAPE batches. Since batch-to-batch variation is a major concern for natural extracts, chromatographic similarity alone is insufficient to support the robustness of the reported findings. The authors should clarify whether the key bioactivity assays were repeated with independent preparations and, if not, explicitly acknowledge this as a limitation.

3. The addition of Coppe et al. (2008, 2010) provides general support for the inclusion of IL-6 as a SASP factor. Because SASP composition is context-dependent, the term “representative component” may overstate the generality of this claim. "A well-established SASP-associated cytokine" may be more precise.

4.The nomenclature of genes and proteins should be revised throughout the manuscript to comply with internationally accepted conventions. Specifically, human gene symbols should appear in uppercase italics, whereas human protein symbols should appear in uppercase roman type (non-italic). Consistent formatting should be ensured throughout the text, figures, and tables.

Author Response

Reviewer Comment 1

The newly added introduction of Nrf2 signaling provides a standard overview of the pathway; however, in its current form, it reads more like a general textbook-style background section than a focused rationale for the present study. The description of the KEAP1–Nrf2–ARE axis and downstream antioxidant genes is broadly accurate, but its connection to dermal fibroblasts and the specific biological context of this manuscript remains insufficiently developed. In particular, the paragraph does not clearly explain why Nrf2 signaling is especially relevant to dermal fibroblast dysfunction, senescence, or the disease/aging phenotype under investigation.

In addition, the revised text still lacks a clear knowledge gap, unresolved question, or point of controversy that would justify the transition from general Nrf2 biology to the specific aims of this study.

 

Response

We thank the reviewer for this insightful comment. In response, the Nrf2-related section in the Introduction has been substantially revised to reduce the general textbook-style description of the KEAP1–Nrf2–ARE pathway and to better emphasize the biological relevance of Nrf2 signaling in dermal fibroblast aging.

Specifically, we clarified the role of oxidative stress in promoting fibroblast senescence and extracellular matrix deterioration in aging skin and highlighted the importance of Nrf2-mediated antioxidant defense in maintaining redox homeostasis in dermal fibroblasts. In addition, a clearer knowledge gap has been introduced regarding whether activation of the Nrf2 pathway can effectively counteract oxidative stress–induced fibroblast senescence and restore dermal ECM homeostasis.

These revisions establish a more problem-driven rationale linking Nrf2 signaling to dermal fibroblast dysfunction and better align the Introduction with the central objective of the present study. The revised text appears in the Introduction (Lines 52–65).

 

Reviewer Comment 2

The additional batch comparison improves the response, but it still does not fully address the original concern. The presented data only support partial chemical consistency based on three marker flavonoids, which is not equivalent to rigorous extract standardization for a complex natural product. More importantly, the authors have not provided evidence that the biological effects described in the manuscript are reproducible across independent MAPE batches.

 

Response

We appreciate the reviewer’s important comment regarding extract standardization and batch-to-batch reproducibility. In this study, HPLC analysis was used to identify three representative propolis flavonoids (chrysin, pinocembrin, and galangin), which are widely recognized marker compounds for propolis extracts. These analyses were used to confirm the characteristic chemical profile of the MAPE preparation.

We acknowledge that chromatographic similarity based on a limited number of marker compounds does not constitute full standardization of a complex natural extract. The biological assays in the present study were performed using a single well-characterized preparation of MAPE. To address the reviewer’s concern, this limitation has now been explicitly acknowledged in the Discussion section.

Future studies will aim to evaluate the reproducibility of biological activity across independent MAPE preparations and to further establish batch-to-batch consistency.

This clarification has been added to the Discussion section (Lines 635–639).

 

Reviewer Comment 3

Because SASP composition is context-dependent, the term “representative component” may overstate the generality of this claim. "A well-established SASP-associated cytokine" may be more precise.

Response

We thank the reviewer for this helpful suggestion. The terminology has been revised accordingly, and IL-6 is now described as “a well-established SASP-associated cytokine” rather than a “representative component” of SASP.

This change has been implemented in the Results section (Lines 303–305) and the Discussion section (Lines 591–594).

 

Reviewer Comment 4

The nomenclature of genes and proteins should be revised throughout the manuscript to comply with internationally accepted conventions.

 

Response

We thank the reviewer for pointing out this issue. The nomenclature of genes and proteins has been carefully revised throughout the manuscript to comply with internationally accepted conventions. Human gene symbols are now presented in uppercase italics, whereas protein symbols appear in uppercase roman type (non-italic). These formatting corrections have been applied consistently throughout the text, figures, and figure legends.

Author Response File: Author Response.docx

Reviewer 3 Report

The authors have performed the requested corrections, however minor changes should be addressed:

Remove line 112 to 114 to be after line 148

In photochemical profiling, mention that all Extract have been anzlyzed not only MAPE.

 

NA

Author Response

Reviewer Comment 1

The authors have performed the requested corrections, however minor changes should be addressed:

Remove line 112 to 114 to be after line 148.

 

Response

We thank the reviewer for this helpful suggestion. The paragraph describing the comparative extraction screening has been moved to the end of Section 2.1 (Line 145-154) to improve the logical flow of the extraction procedure and the subsequent evaluation of total phenolic content.

 

Reviewer Comment 2

In photochemical profiling, mention that all Extract have been analyzed not only MAPE.

 

Response

We thank the reviewer for this helpful comment. The manuscript has been revised to clarify that phytochemical profiling was performed for the different propolis extracts, not only MAPE. The relevant sentence has been modified accordingly in Section 2.1 (Lines 123–126).

Author Response File: Author Response.docx