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  • Isabella Morais Tavares Huber 1,2,3,*,
  • Emerson Luiz Botelho Lourenço 2,4 and
  • Leonardo Garcia Velasquez 8
  • et al.

Reviewer 1: Anonymous Reviewer 2: Vijai Kumar Reddy Tangadanchu Reviewer 3: Laxman Subedi

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for inviting me to review this manuscript. The study appears to investigate the potential cytoprotective effects of Curcuma longa against chemotherapy-induced hematological toxicity, specifically in relation to docetaxel-induced bone marrow suppression and splenic alterations. The study's design, which includes varying doses of Curcuma longa in combination with docetaxel, appears relevant for understanding the phytochemical’s protective role in this context. Below, I provide suggestions to improve clarity, conciseness, and overall content quality.

ABSTRACT

  1. The information is clear, but could benefit from a brief mention of why docetaxel is commonly used despite its toxicity, to provide context for the reader. Also, a stronger connection between Curcuma longa's antioxidant properties and its potential protective effects would strengthen the rationale for its use in this study. Consider rephrasing to highlight the potential clinical relevance more explicitly.
  2. The methodology is well-described, but the term "subgroups" could be more specific, such as "treatment groups," to avoid confusion with statistical terms. Also, providing more details on how the rats were allocated (e.g., randomly, by specific criteria) would enhance the transparency of the methodology. Additionally, specifying whether the hematological parameters measured were baseline or post-treatment could clarify the results section.
  3. The Abstract presents the findings clearly. Still, it could be improved by emphasizing the statistical significance of the results, especially in the high-dose group, where a "non-significant increase in relative spleen weight" is mentioned. This could be clearer if stated as "no statistically significant change" to avoid ambiguity. Additionally, the term "attenuated these alterations" could be rephrased to specify whether the effect was a complete reversal or a partial improvement, which would help the reader gauge the effectiveness of Curcuma longa more accurately.
  4. The conclusion effectively summarizes the study’s findings, but could emphasize more clearly the implications for further research or clinical use. A more direct link between the results and future translational studies would strengthen the closing statement. Consider adding a note on what specific aspects of docetaxel toxicity future studies should focus on to improve the relevance of Curcuma longa as a potential adjuvant strategy.

INTRODUCTION

  1. The opening paragraph effectively establishes the global burden of cancer and the continued relevance of cytotoxic chemotherapy. To further strengthen the impact, the authors could briefly contextualize hematological toxicity as a clinical limitation and a biological bottleneck that affects long-term treatment efficacy and patient survival, thereby reinforcing the urgency of the problem.
  2. The Introduction could benefit from a more explicit mechanistic link between docetaxel’s mode of action (e.g., microtubule stabilization) and the downstream hematopoietic and splenic effects, which would help bridge pharmacology with observed toxicities.
  3. The section on natural compounds and Curcuma longa is well-positioned and relevant. That said, the authors may briefly distinguish between evidence derived from curcumin alone and that from whole Curcuma longa preparations, as this distinction is essential for interpreting translational relevance and justifying the intervention used in the present study.
  4. While the rationale for using phytochemicals as adjuvants is convincing, the Introduction could be strengthened by acknowledging potential limitations or controversies surrounding curcumin/Curcuma bioavailability and dose translation. Addressing these points preemptively would demonstrate critical engagement with the existing literature.
  5. To further enhance clarity, the authors could explicitly state why the spleen was selected as a target organ alongside hematological parameters, emphasizing its role in extramedullary hematopoiesis and immune regulation under chemotherapeutic stress.

MATERIALS AND METHODS

  1. The experimental design is generally well described; however, the term “controlled case experimental study” is unconventional in animal research. The authors may consider adopting more standard terminology, such as “controlled experimental animal study” or “randomized preclinical study,” to improve clarity and alignment with reporting standards (e.g., ARRIVE guidelines).
  2. The grouping strategy based on treatment duration (7, 14, and 21 days) is logical and clearly structured. Nevertheless, the rationale for selecting these specific time points should be briefly justified, particularly in relation to docetaxel pharmacodynamics, bone marrow recovery kinetics, or prior literature on cumulative toxicity.
  3. The extensive listing of subgroups (G1A–G5C) ensures transparency but results in some redundancy. The authors could summarize this design in a table and use a concise narrative description in the text, thereby improving readability without compromising methodological rigor.
  4. Stating that no inclusion or exclusion criteria were defined a priori may raise concerns for some readers; the authors might clarify that all healthy animals meeting baseline age and weight criteria were included, to reinforce methodological robustness.
  5. The sample size justification relies on precedent rather than formal power analysis. While this is common in exploratory animal studies, the authors may wish to acknowledge this as a limitation or briefly discuss the expected effect size that informed the chosen group size.
  6. The allometric scaling approach used to determine the docetaxel dose is described in detail and enhances transparency. That said, the authors could strengthen this section by briefly explaining why a single-dose docetaxel model was selected instead of repeated dosing, given that clinical regimens often involve multiple cycles.
  7. The Curcuma longa dosing range is broad and potentially informative. The rationale for including a very high dose (500 mg/kg/day) should be more explicitly justified, particularly with respect to translational relevance, safety considerations, or prior toxicological data.
  8. The absence of environmental enrichment may warrant brief justification, especially given the increasing emphasis on refinement in animal welfare. Additionally, the behavioral observation protocol could be clarified by specifying which parameters (e.g., locomotion, grooming, feeding) were systematically assessed.
  9. Blood collection via the retro-orbital plexus is a standard technique but remains controversial. The authors may briefly justify this choice and note any measures taken to minimize pain or tissue damage, thereby strengthening the ethical transparency of the study.
  10. The Methods section would benefit from a brief explanation of why relative spleen weight was selected as the sole splenic endpoint, especially since morphological or histological assessments are mentioned elsewhere in the manuscript.
  11. The statistical analysis plan is sound and clearly reported. One point for clarification is whether ANOVA was performed separately for each time point or across pooled data; explicitly stating this would help readers better interpret the results.

RESULTS

  1. The opening paragraph appropriately outlines the time-dependent analytical framework. Still, it would benefit from a more explicit statement of the primary comparative objectives (e.g., docetaxel vs. control, docetaxel vs. Curcuma-treated groups) to guide the reader through the complex results that follow.
  2. The global ANOVA analysis across time points is a valuable overview; however, the biological interpretation of these pooled temporal effects is limited. The authors may consider explicitly stating that these results reflect time-driven trends rather than treatment-specific effects to avoid potential misinterpretation.
  3. Several parameters are reported as non-significant (e.g., platelets, spleen weight), yet these outcomes are central to the study hypothesis. A brief acknowledgment that the lack of significance may reflect high variability or limited power would provide important context.
  4. The observation that erythrocytes and hemoglobin peak at 14 days and decline at 21 days is potentially meaningful, but the manuscript would benefit from a short mechanistic interpretation (e.g., transient compensatory hematopoiesis followed by cumulative toxicity).
  5. The progressive increase in RDW is a notable finding that aligns with chemotherapy-induced dyserythropoiesis. Highlighting this parameter more explicitly as an indicator of red cell heterogeneity and marrow stress would strengthen the narrative.
  6. The reference to spleen body weight as non-significant in the general analysis contrasts with later subgroup variability. The authors may wish to clarify that this global analysis may mask subgroup-specific effects explored in Section 3.2.
  7. The nomenclature used for groups (e.g., AG50, BG25, CGC, CGQ) is not intuitive and should be clearly defined at the beginning of this subsection or in a legend to avoid reader confusion.
  8. At 7 days, the isolated difference in MCHC (lower in the 50 mg/kg group) is reported without interpretation. The authors should comment on whether this represents an early transient effect, biological variability, or a potential dose-specific response.
  9. The 14-day findings suggest that intermediate doses of Curcuma longa may modulate leukocyte and neutrophil counts. This is a key result supporting the study hypothesis and should be emphasized more explicitly, ideally with a concise summary statement highlighting the apparent hematoprotective window.
  10. At 21 days, the divergent patterns observed in MCV, MCHC, and RDW across doses are complex but potentially significant. The authors may improve clarity by explicitly contrasting docetaxel-only groups with Curcuma-treated groups, rather than listing intergroup differences sequentially.
  11. The significant variability observed in spleen weight at 21 days (particularly the extreme standard deviation in the G500 group) raises concerns about outliers or measurement consistency. The authors should address this variability explicitly and clarify whether any exploratory analyses were conducted to assess its impact on statistical outcomes.
  12. Across all periods, leukocyte and platelet data are reported comprehensively. Still, the absence of consistent significant differences should be briefly contextualized, especially given the known sensitivity of these parameters to docetaxel toxicity.

DISCUSSION

  1. It would benefit from substantial condensation. Several mechanistic explanations (e.g., NF-κB inhibition, Nrf2 activation, cytokine modulation, extramedullary hematopoiesis) are reiterated across multiple subsections. Streamlining these recurring concepts and cross-referencing them rather than re-explaining them would markedly improve coherence and reader engagement.
  2. In multiple instances, the narrative appears to overinterpret trends that did not reach statistical significance, particularly regarding platelet preservation and splenic enlargement at high Curcuma longa The authors are encouraged to adopt more consistently cautious language (e.g., “suggests,” “may indicate”) and to distinguish statistically supported findings from hypothesis-generating observations clearly.
  3. The discussion of splenic responses, while biologically plausible and richly supported by literature, is disproportionately long relative to the strength of the data (i.e., relative spleen weight only, with high variability and no significance). This section would be strengthened by consolidation and by clearly framing splenomegaly as a secondary, exploratory finding that requires histological and functional validation.
  4. The emphasis on intermediate Curcuma longa doses (25–50 mg/kg) as the most consistently beneficial is one of the strongest and most original contributions of the study. This point should be more clearly highlighted as a central conclusion of the Discussion, potentially serving as a unifying theme that contrasts protective hematological effects with the potential for adverse immune overstimulation at higher doses.

CONCLUSIONS

  1. The statement that the study provides “robust preclinical evidence” may benefit from slight moderation. While the data are comprehensive, the absence of bone marrow histopathology and functional immune assays suggests that terms such as “strong” or “substantial” evidence might be more appropriate and scientifically conservative.
  2. The interpretation of RDW stabilization and splenic modulation as indicators of broader hematopoietic homeostasis is biologically plausible; however, this inference should be more explicitly framed as mechanistic speculation rather than a demonstrated outcome, given the limited structural and functional analyses performed.

Thank you again for the opportunity to review this manuscript. The study presents valuable insights into the potential of Curcuma longa as a therapeutic adjunct to mitigate chemotherapy-induced toxicity. With minor refinements, the manuscript can become even more effective at conveying the research's significance.

Comments on the Quality of English Language

 The English could be improved to more clearly express the research.

Author Response

Comment 1: The information is clear, but could benefit from a brief mention of why docetaxel is commonly used despite its toxicity, to provide context for the reader. Also, a stronger connection between Curcuma longa's antioxidant properties and its potential protective effects would strengthen the rationale for its use in this study. Consider rephrasing to highlight the potential clinical relevance more explicitly.

Response 1: We agree with this comment. We added a brief contextual justification for docetaxel’s widespread use despite cumulative toxicity and strengthened the link between the antioxidant/anti-inflammatory properties of Curcuma longa and the study rationale, highlighting potential translational relevance.
Location in revised manuscript: Abstract, Background (p. 2, para. 1, lines 2-6, 8, 11, 13, 15 and 16).

Comment 2: The methodology is well-described, but the term "subgroups" could be more specific, such as "treatment groups," to avoid confusion with statistical terms. Also, providing more details on how the rats were allocated (e.g., randomly, by specific criteria) would enhance the transparency of the methodology. Additionally, specifying whether the hematological parameters measured were baseline or post-treatment could clarify the results section.

Response 2: We agree. We replaced “subgroups” with “treatment groups” in the Abstract, explicitly stated random allocation, and clarified that hematological parameters were assessed post-treatment.
Location: Abstract(p. 2, para. 1, lines 2-6,).

Comment 3: The Abstract presents the findings clearly. Still, it could be improved by emphasizing the statistical significance of the results, especially in the high-dose group, where a "non-significant increase in relative spleen weight" is mentioned. This could be clearer if stated as "no statistically significant change" to avoid ambiguity. Additionally, the term "attenuated these alterations" could be rephrased to specify whether the effect was a complete reversal or a partial improvement, which would help the reader gauge the effectiveness of Curcuma longa more accurately.

Response 3: We agree. We rephrased “non-significant increase” to “no statistically significant change” and replaced “attenuated” with “partially mitigated” to avoid overstating the effect.
Location: Abstract, Results (p. 2, para. 1, line 11).

Comment 4: The conclusion effectively summarizes the study’s findings, but could emphasize more clearly the implications for further research or clinical use. A more direct link between the results and future translational studies would strengthen the closing statement. Consider adding a note on what specific aspects of docetaxel toxicity future studies should focus on to improve the relevance of Curcuma longa as a potential adjuvant strategy.

Response 4: We agree. We strengthened the final statement by explicitly indicating that future translational studies should focus on hematological toxicity, bone marrow suppression, and immune-related adverse effects to clarify the potential role of Curcuma longa as a low-toxicity adjuvant.
Location: Abstract (p. 2, para. 1, lines 15 and 16).


Comment 5: The opening paragraph effectively establishes the global burden of cancer and the continued relevance of cytotoxic chemotherapy. To further strengthen the impact, the authors could briefly contextualize hematological toxicity as a clinical limitation and a biological bottleneck that affects long-term treatment efficacy and patient survival, thereby reinforcing the urgency of the problem.

Response 5: We agree. We expanded the opening rationale to explicitly frame hematological toxicity as a dose-limiting barrier leading to dose reductions/delays and potentially affecting outcomes.
Location: Introduction (p. 3, para. 1, lines 6-9).

Comment 6: The Introduction could benefit from a more explicit mechanistic link between docetaxel’s mode of action (e.g., microtubule stabilization) and the downstream hematopoietic and splenic effects, which would help bridge pharmacology with observed toxicities.

Response 6: We agree. We added a mechanistic bridge connecting docetaxel’s microtubule stabilization/antimitotic activity with downstream myelosuppression and potential splenic responses under hematopoietic stress.
Location: Introduction (p. 3, para. 2, lines 12-14).

Comment 7: The section on natural compounds and Curcuma longa is well-positioned and relevant. That said, the authors may briefly distinguish between evidence derived from curcumin alone and that from whole Curcuma longa preparations, as this distinction is essential for interpreting translational relevance and justifying the intervention used in the present study.

Response 7: We agree. We added a clarifying distinction between literature on curcumin and on whole Curcuma longa preparations/extracts, reinforcing why the present intervention (extract) is relevant.
Location: Introduction, Curcuma paragraph (p. 3, para. 3, lines 23-26).

Comment 8: While the rationale for using phytochemicals as adjuvants is convincing, the Introduction could be strengthened by acknowledging potential limitations or controversies surrounding curcumin/Curcuma bioavailability and dose translation. Addressing these points preemptively would demonstrate critical engagement with the existing literature.

Response 8: We agree. We added a concise limitation statement acknowledging ongoing debate on bioavailability, pharmacokinetics, and dose translation from animal models to humans, framed as rationale for cautious interpretation.
Location: Introduction (p. 3, para. 3, lines 30-32).

Comment 9: To further enhance clarity, the authors could explicitly state why the spleen was selected as a target organ alongside hematological parameters, emphasizing its role in extramedullary hematopoiesis and immune regulation under chemotherapeutic stress.

Response 9: We agree. We added an explicit justification that the spleen was included due to its role in extramedullary hematopoiesis and immune regulation under chemotherapeutic stress.
Location: Introduction, final paragraph (p. 3, para. 4, lines 34-37).

Comment 10: The experimental design is generally well described; however, the term “controlled case experimental study” is unconventional in animal research. The authors may consider adopting more standard terminology, such as “controlled experimental animal study” or “randomized preclinical study,” to improve clarity and alignment with reporting standards (e.g., ARRIVE guidelines).

Response 10: We agree. We replaced the term with “controlled experimental animal study” / “randomized preclinical animal study” to align with standard reporting language.
Location: Materials and Methods, opening sentence (p. 3, para. 5, line 48).

Comment 11: The grouping strategy based on treatment duration (7, 14, and 21 days) is logical and clearly structured. Nevertheless, the rationale for selecting these specific time points should be briefly justified, particularly in relation to docetaxel pharmacodynamics, bone marrow recovery kinetics, or prior literature on cumulative toxicity.

Response 11: We agree. We added a brief rationale linking the chosen time points to expected time-dependent marrow suppression/recovery kinetics and cumulative toxicity patterns under docetaxel exposure.
Location: Experimental design / grouping rationale (p. 4, para. 1, lines 1-4).

Comment 12: The extensive listing of subgroups (G1A–G5C) ensures transparency but results in some redundancy. The authors could summarize this design in a table and use a concise narrative description in the text, thereby improving readability without compromising methodological rigor.

Response 12: We agree. We kept transparency but condensed the narrative, and we prepared a group-design table summarizing treatment arms across time points (control, docetaxel-only, Curcuma 25/50/500).
Location: Materials and Methods, new Table (p. 4, para.1, line 5, and Table 1).

Comment 13: Stating that no inclusion or exclusion criteria were defined a priori may raise concerns for some readers; the authors might clarify that all healthy animals meeting baseline age and weight criteria were included, to reinforce methodological robustness.

Response 13: We agree. We clarified that all healthy animals meeting baseline age and weight criteria were included, while maintaining that no animals or datapoints were excluded.
Location: Materials and Methods (p. 3, para. 5, lines 49-51).

Comment 14: The sample size justification relies on precedent rather than formal power analysis. While this is common in exploratory animal studies, the authors may wish to acknowledge this as a limitation or briefly discuss the expected effect size that informed the chosen group size.

Response 14: We agree. We acknowledged that formal power analysis was not performed and framed sample size as consistent with exploratory preclinical precedent, noting this as a limitation of inference.
Location: Materials and Methods (p. 4, para. 2, lines 43-45).

Comment 15: The allometric scaling approach used to determine the docetaxel dose is described in detail and enhances transparency. That said, the authors could strengthen this section by briefly explaining why a single-dose docetaxel model was selected instead of repeated dosing, given that clinical regimens often involve multiple cycles.

Response 15: We agree. We added a brief justification that the single-dose model was selected to isolate time-dependent hematotoxic effects while limiting repeated-procedure burden, acknowledging clinical regimens use cycles.
Location: Materials and Methods (p. 5, para. 3, lines 6-9).

Comment 16: The Curcuma longa dosing range is broad and potentially informative. The rationale for including a very high dose (500 mg/kg/day) should be more explicitly justified, particularly with respect to translational relevance, safety considerations, or prior toxicological data.

Response 16: We agree. We added explicit rationale that 500 mg/kg/day was included as a high-dose exploratory arm to evaluate a broad dose range and potential biphasic effects, with cautious interpretation regarding translational relevance.
Location: Materials and Methods (p. 5, para. 5, lines 23-25).

Comment 17: The absence of environmental enrichment may warrant brief justification, especially given the increasing emphasis on refinement in animal welfare. Additionally, the behavioral observation protocol could be clarified by specifying which parameters (e.g., locomotion, grooming, feeding) were systematically assessed.

Response 17: We agree. We added a brief justification for standard housing (and lack of enrichment) and specified behavioral monitoring parameters (e.g., locomotion, grooming, feeding, posture/alertness).
Location: Materials and Methods (p. 5, para. 5, lines 34-36).

Comment 18: Blood collection via the retro-orbital plexus is a standard technique but remains controversial. The authors may briefly justify this choice and note any measures taken to minimize pain or tissue damage, thereby strengthening the ethical transparency of the study.

Response 18: We agree. We added ethical clarification describing anesthesia/handling measures aimed at minimizing pain/distress and reaffirmed compliance with approved protocols.
Location: Materials and Methods (p. 5, para. 6, lines 38-40).

Comment 19: The Methods section would benefit from a brief explanation of why relative spleen weight was selected as the sole splenic endpoint, especially since morphological or histological assessments are mentioned elsewhere in the manuscript.

Response 19: We agree. We added a brief explanation that relative spleen weight was used as a quantitative surrogate marker of splenic response to hematopoietic stress / potential extramedullary hematopoiesis, while noting that histology would be required for validation.
Location: Materials and Methods (p. 5, para. 6, lines 44-46).

Comment 20: The statistical analysis plan is sound and clearly reported. One point for clarification is whether ANOVA was performed separately for each time point or across pooled data; explicitly stating this would help readers better interpret the results.

Response 20: We agree. We explicitly clarified that analyses were performed (I) across time points for the general temporal analysis and (II) separately within each time point for group-wise comparisons, to prevent misinterpretation.
Location: Materials and Methods (p. 6, para. 4, lines 13-17).

Comment 21: The opening paragraph appropriately outlines the time-dependent analytical framework. Still, it would benefit from a more explicit statement of the primary comparative objectives (e.g., docetaxel vs. control, docetaxel vs. Curcuma-treated groups) to guide the reader through the complex results that follow.

Response 21: We agree. We added a clear opening sentence stating that the primary comparisons were docetaxel vs control and docetaxel-only vs Curcuma-treated groups at each time point (7/14/21 days).
Location: Results (p. 6, para. 6, lines 24-28)

Comment 22: The global ANOVA analysis across time points is a valuable overview; however, the biological interpretation of these pooled temporal effects is limited. The authors may consider explicitly stating that these results reflect time-driven trends rather than treatment-specific effects to avoid potential misinterpretation.

Response 22: We agree. We explicitly stated that the general ANOVA across time points primarily captures time-dependent trends and should not be overinterpreted as treatment-specific.
Location: Results (p. 6, para. 7, lines 33-35).

Comment 23: Several parameters are reported as non-significant (e.g., platelets, spleen weight), yet these outcomes are central to the study hypothesis. A brief acknowledgment that the lack of significance may reflect high variability or limited power would provide important context.

Response 23: We agree. We added a brief contextual sentence noting that non-significant results (e.g., platelets, spleen mass) may reflect biological variability and limited power in an exploratory preclinical design.
Location: Results (p. 6, para. 9, lines 46-48).

Comment 24: The observation that erythrocytes and hemoglobin peak at 14 days and decline at 21 days is potentially meaningful, but the manuscript would benefit from a short mechanistic interpretation (e.g., transient compensatory hematopoiesis followed by cumulative toxicity).

Response 24: We agree. We added a short interpretive clause indicating this pattern may reflect transient compensatory hematopoiesis followed by cumulative toxicity.
Location: Results (p. 6, para. 5, lines 39-41).

Comment 25: The progressive increase in RDW is a notable finding that aligns with chemotherapy-induced dyserythropoiesis. Highlighting this parameter more explicitly as an indicator of red cell heterogeneity and marrow stress would strengthen the narrative.

Response 25: We agree. We added a sentence emphasizing RDW as an indicator of erythrocyte heterogeneity and marrow stress, consistent with chemotherapy-induced dyserythropoiesis.
Location: Results (p. 6, para. 5, lines 42-43).

Comment 26: The reference to spleen body weight as non-significant in the general analysis contrasts with later subgroup variability. The authors may wish to clarify that this global analysis may mask subgroup-specific effects explored in Section 3.2.

Response 26: We agree. We clarified that the global analysis may mask subgroup-specific variability, which is explored in period-specific analyses.
Location: Results, Section 3.1 → 3.2 transition (p. 6, para. 9, lines 46-48).

Comment 27: The nomenclature used for groups (e.g., AG50, BG25, CGC, CGQ) is not intuitive and should be clearly defined at the beginning of this subsection or in a legend to avoid reader confusion.

Response 27: We agree. We added a clear definition of group nomenclature at the start of Section 3.2 and in the Table 3 legend (e.g., A/B/C denote time; 25/50/500 denote dose; GC control; GQ docetaxel-only).
Location: Results (p. 7, para. 1, lines 32-34).

Comment 28: At 7 days, the isolated difference in MCHC (lower in the 50 mg/kg group) is reported without interpretation. The authors should comment on whether this represents an early transient effect, biological variability, or a potential dose-specific response.

Response 28: We agree. We added a brief interpretation stating this isolated finding likely represents an early transient effect or biological variability, and it was not sustained across time points.
Location: Results, Section 3.2 (p. 7, para. 2, lines 36-39).

Comment 29: The 14-day findings suggest that intermediate doses of Curcuma longa may modulate leukocyte and neutrophil counts. This is a key result supporting the study hypothesis and should be emphasized more explicitly, ideally with a concise summary statement highlighting the apparent hematoprotective window.

Response 29: We agree. We added a concise summary sentence emphasizing that intermediate doses (25–50 mg/kg) appear to represent a hematoprotective window, particularly for leukocyte/neutrophil preservation at day 14.
Location: Results, Section 3.2 (p. 7, para. 3, lines 43-45).

Comment 30: At 21 days, the divergent patterns observed in MCV, MCHC, and RDW across doses are complex but potentially significant. The authors may improve clarity by explicitly contrasting docetaxel-only groups with Curcuma-treated groups, rather than listing intergroup differences sequentially.

Response 30: We agree. We revised phrasing to more explicitly contrast docetaxel-only versus Curcuma-treated groups when describing erythrocyte index patterns (MCV/MCHC/RDW), improving readability without altering results.
Location: Results, Section 3.2 (p. 7, para. 4, lines 50-51 and p. 8, para. 1, lines 1-2).

Comment 31: The significant variability observed in spleen weight at 21 days (particularly the extreme standard deviation in the G500 group) raises concerns about outliers or measurement consistency. The authors should address this variability explicitly and clarify whether any exploratory analyses were conducted to assess its impact on statistical outcomes.

Response 31: We agree. We added a statement acknowledging the marked variability at 21 days (especially G500), indicating potential outliers/heterogeneity. We also clarified that no post hoc outlier exclusion/removal was performed, consistent with the predefined analysis approach.
Location: Results, Section 3.2 (p. 8, para. 2, lines 3-7).

Comment 32: Across all periods, leukocyte and platelet data are reported comprehensively. Still, the absence of consistent significant differences should be briefly contextualized, especially given the known sensitivity of these parameters to docetaxel toxicity.

Response 32: We agree. We added a brief caution that the absence of consistent significance should be interpreted carefully due to intergroup variability and the exploratory nature of the model.
Location: Results, Section 3.2 (p. 8, para. 3, lines 8-11).

Comment 33: It would benefit from substantial condensation. Several mechanistic explanations (e.g., NF-κB inhibition, Nrf2 activation, cytokine modulation, extramedullary hematopoiesis) are reiterated across multiple subsections. Streamlining these recurring concepts and cross-referencing them rather than re-explaining them would markedly improve coherence and reader engagement.

Response 33: We agree. We condensed repeated mechanistic explanations by replacing repeated NF-κB/Nrf2/cytokine descriptions with short synthesis sentences and cross-referenced earlier mechanistic mentions to improve coherence and reduce redundancy.
Location: Discussion, Sections 4.6–4.8 (p. 14, paras. 1 and 2, lines 1-10, p. 14, paras. 5-7, lines 24-41, p. 14, para. 9, line 51 and p. 15, para. 1, lines 1-2).

Comment 34: In multiple instances, the narrative appears to overinterpret trends that did not reach statistical significance, particularly regarding platelet preservation and splenic enlargement at high Curcuma longa The authors are encouraged to adopt more consistently cautious language (e.g., “suggests,” “may indicate”) and to distinguish statistically supported findings from hypothesis-generating observations clearly.

Response 34: We agree. We revised wording to use more conservative language (“suggests”, “may indicate”, “numerical trend”) and explicitly distinguished statistically supported findings from exploratory observations, particularly for platelet and splenic outcomes.
Location: Discussion, Section 4.5 (p. 13, para. 5, lines 30-35).

Comment 35: The discussion of splenic responses, while biologically plausible and richly supported by literature, is disproportionately long relative to the strength of the data (i.e., relative spleen weight only, with high variability and no significance). This section would be strengthened by consolidation and by clearly framing splenomegaly as a secondary, exploratory finding that requires histological and functional validation.

Response 35: We agree. We substantially shortened the splenic discussion and framed splenomegaly as a secondary/exploratory observation requiring histological and functional validation (histomorphology, immunophenotyping, cytokines).
Location: Discussion, Section 4.5 (p. 13, para. 5, lines 30-35).

Comment 36: The emphasis on intermediate Curcuma longa doses (25–50 mg/kg) as the most consistently beneficial is one of the strongest and most original contributions of the study. This point should be more clearly highlighted as a central conclusion of the Discussion, potentially serving as a unifying theme that contrasts protective hematological effects with the potential for adverse immune overstimulation at higher doses.

Response 36: We agree. We strengthened the Discussion’s unifying message by emphasizing intermediate doses (25–50 mg/kg) as the most consistent hematoprotective window, while noting that high-dose exposure was associated with greater variability and potential immune overstimulation signals.
Location: Discussion + Translational implications (Sections 4.4 and 4.10) (p. 13, para. 2, lines 9-12 and p. 15, para. 8, lines 41-47).

Comment 37: The statement that the study provides “robust preclinical evidence” may benefit from slight moderation. While the data are comprehensive, the absence of bone marrow histopathology and functional immune assays suggests that terms such as “strong” or “substantial” evidence might be more appropriate and scientifically conservative.

Response 37: We agree. We replaced “robust preclinical evidence” with more conservative wording (“strong preclinical evidence”) and explicitly acknowledged that the absence of bone marrow histopathology and functional immune assays limits mechanistic inference.
Location: Conclusion (p. 16, para. 6, line 33).

Comment 38: The interpretation of RDW stabilization and splenic modulation as indicators of broader hematopoietic homeostasis is biologically plausible; however, this inference should be more explicitly framed as mechanistic speculation rather than a demonstrated outcome, given the limited structural and functional analyses performed.

Response 38: We agree. We revised the conclusion to state that RDW stabilization and splenic changes suggest a possible modulation and are hypothesis-generating, explicitly identifying mechanistic interpretations as speculative given limited structural/functional analyses.
Location: Conclusion, mid-paragraph (p. 16, para. 7, lines 37-41).

Please see the attachment with the specific alterations. Thank you very much for your support.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript titled “Impact of Curcuma longa on hematopoiesis and splenic mass in an animal model undergoing docetaxel chemotherapy” presents a preclinical evaluation of Curcuma longa as a potential adjuvant to reduce docetaxel-induced hematologic toxicity. The overall rationale is well developed, and the study design includes several strengths. However, a number of design, reporting, and interpretative issues currently limit the translational relevance and mechanistic rigor of the work.

The introduction, particularly in its discussion of curcumin biology, is overly detailed. A more concise framing focused on hematopoiesis, myelosuppression, and splenic responses would improve clarity and alignment with the study objectives.

In the Introduction, the authors should consider citing a recent review (https://aces.onlinelibrary.wiley.com/doi/full/10.1002/asia.202400380) on diarylheptanoids that detail the structural classes of curcuminoids and closely related analogues, along with their reported biological activities. Such a citation would help place the present work on Curcuma longa derived curcuminoids within the broader chemical and functional landscape of diarylheptanoids and provide additional context for the diverse hematologic and immunomodulatory effects discussed.

The in vivo design, including multiple doses and time points, is a notable strength. However, the absence of a Curcuma longa–only control group limits interpretation. Without this arm, it is difficult to distinguish true protective effects from intrinsic hematologic stimulation, especially given the splenic enlargement observed at higher doses.

The description of dose extrapolation from human to rats using the SMR approach is detailed but would benefit from clearer separation between conceptual explanation and the final working dose. A brief justification that 2.5 mg/kg docetaxel adequately models clinically relevant myelosuppression would also strengthen the methods section.

Preparation and administration of Curcuma longa are described adequately. However, characterization of the extract is limited to “95% curcuminoids.” Information on batch identity, certificate of analysis, and stability would improve reproducibility and help address potential sources of biological variability.

Outcome measures focus primarily on CBC parameters and relative spleen weight. While informative, this narrow scope constrains mechanistic interpretation. The absence of histopathology, bone marrow analyses, or cytokine and oxidative stress markers limits support for claims of hematoprotective or immunomodulatory effects.

Interpretation of the splenic findings is particularly problematic. Although spleen weight differences are reported as statistically non-significant, the discussion later emphasizes a large numerical increase at the highest dose and extrapolates to hypersplenism, fibrosis, or immune overstimulation. These interpretations are not supported by histological data and create internal inconsistency between the results and discussion.

The discussion of erythroid indices and RDW is conceptually interesting. However, the modest changes observed, often within physiological ranges, do not fully justify extensive mechanistic speculation involving Nrf2 signaling, iron metabolism, or preserved erythropoietic efficiency. Similar concerns apply to interpretations of leukocyte and platelet preservation, which rely heavily on prior literature rather than strong effects demonstrated in the current dataset.

The translational discussion appropriately raises hypotheses regarding maintenance of dose intensity and reduction of supportive interventions. However, the level of certainty exceeds what can be supported by a single hematologic rat model without tumor burden, pharmacokinetics, or efficacy endpoints.

The limitations section acknowledges the absence of histopathology and cytokine profiling, which is appropriate. Additional limitations should also be discussed, including the lack of Curcuma longa–only groups, use of a single sex, and potential confounding from repeated gavage and handling stress.

The manuscript is generally well written. Terminology is occasionally inconsistent, with Curcuma longa and curcumin used interchangeably, which should be corrected given the use of a multicomponent extract. In the mechanistic discussion, a tighter linkage to the specific findings of this study, with clearer distinction between demonstrated effects and literature-based inference, would improve focus.

In summary, the study addresses an important clinical question and uses a reasonably rigorous in vivo approach. The data appears internally consistent and support a more modest conclusion that intermediate doses of Curcuma longa are associated with partial attenuation of docetaxel-induced cytopenias in rats. With more cautious interpretation, improved statistical handling, and additional mechanistic data in future studies, this work could make a useful contribution to supportive care literature. At present, however, the mechanistic and translational conclusions should be substantially moderated to align with the evidence presented.

 

Author Response

1. The manuscript titled “Impact of Curcuma longa on hematopoiesis and splenic mass in an animal model undergoing docetaxel chemotherapy” presents a preclinical evaluation of Curcuma longa as a potential adjuvant to reduce docetaxel-induced hematologic toxicity. The overall rationale is well developed, and the study design includes several strengths. However, a number of design, reporting, and interpretative issues currently limit the translational relevance and mechanistic rigor of the work. The introduction, particularly in its discussion of curcumin biology, is overly detailed. A more concise framing focused on hematopoiesis, myelosuppression, and splenic responses would improve clarity and alignment with the study objectives. 

Comments: We thank the reviewer for this valuable suggestion. The Introduction has been carefully revised and condensed to improve focus and clarity. Redundant mechanistic details were removed, and the framing was refocused on hematopoiesis, chemotherapy-induced myelosuppression, and splenic responses, in closer alignment with the study objectives. This restructuring improves coherence while preserving essential background information relevant to the experimental design

2. In the Introduction, the authors should consider citing a recent review (https://aces.onlinelibrary.wiley.com/doi/full/10.1002/asia.202400380) on diarylheptanoids that detail the structural classes of curcuminoids and closely related analogues, along with their reported biological activities. Such a citation would help place the present work on Curcuma longa derived curcuminoids within the broader chemical and functional landscape of diarylheptanoids and provide additional context for the diverse hematologic and immunomodulatory effects discussed.

Comments: We appreciate this helpful recommendation. The suggested review on diarylheptanoids has now been cited in the Introduction. This reference was incorporated to contextualize curcuminoids within the broader chemical class of diarylheptanoids and to acknowledge the diversity of related compounds with reported biological and immunomodulatory activities. The text was adjusted to ensure that this addition complements, rather than expands, the Introduction.

3. The in vivo design, including multiple doses and time points, is a notable strength. However, the absence of a Curcuma longa–only control group limits interpretation. Without this arm, it is difficult to distinguish true protective effects from intrinsic hematologic stimulation, especially given the splenic enlargement observed at higher doses.

Comments: We agree with the reviewer that the absence of a Curcuma longa–only group represents a limitation. This point has now been explicitly addressed in the Limitations section. The original experimental design focused on evaluating Curcuma longa as a potential modulator of docetaxel-induced hematological toxicity rather than as a standalone hematopoietic stimulant. Nevertheless, we acknowledge that the inclusion of such a control group would have allowed clearer discrimination between intrinsic hematologic effects and protective modulation, particularly in relation to splenic outcomes.

4. The description of dose extrapolation from human to rats using the SMR approach is detailed but would benefit from clearer separation between conceptual explanation and the final working dose. A brief justification that 2.5 mg/kg docetaxel adequately models clinically relevant myelosuppression would also strengthen the methods section.

Comments: Thank you for this important methodological comment. The Methods section has been revised to clearly separate the conceptual explanation of the SMR-based dose extrapolation from the final working doses used in the study. In addition, a concise justification has been added explaining that the selected docetaxel dose (2.5 mg/kg) is widely used to reproducibly induce clinically relevant myelosuppression, thereby supporting its suitability for the present investigation.

5. Preparation and administration of Curcuma longa are described adequately. However, characterization of the extract is limited to “95% curcuminoids.” Information on batch identity, certificate of analysis, and stability would improve reproducibility and help address potential sources of biological variability.

Comments: We acknowledge this point. Additional information has been added to the Methods section specifying the batch identity, supplier-provided certificate of analysis, and declared curcuminoid content. While extended phytochemical profiling and stability testing were beyond the scope of the current study, this information improves reproducibility and transparency and is now clearly reported.

6. Outcome measures focus primarily on CBC parameters and relative spleen weight. While informative, this narrow scope constrains mechanistic interpretation. The absence of histopathology, bone marrow analyses, or cytokine and oxidative stress markers limits support for claims of hematoprotective or immunomodulatory effects.

Comments: We agree that the outcome measures are primarily functional and hematological in nature. This was an intentional design choice aligned with the study’s aim to evaluate systemic hematological patterns rather than molecular mechanisms. To address this limitation, we have further moderated mechanistic language throughout the Discussion and added an in silico pathway- and target-based integrative subsection. This analysis is explicitly presented as supportive and hypothesis-generating, without implying direct mechanistic validation. The need for future studies incorporating histopathology, bone marrow analyses, and molecular markers is now more clearly emphasized.

7. Interpretation of the splenic findings is particularly problematic. Although spleen weight differences are reported as statistically non-significant, the discussion later emphasizes a large numerical increase at the highest dose and extrapolates to hypersplenism, fibrosis, or immune overstimulation. These interpretations are not supported by histological data and create internal inconsistency between the results and discussion.

Comments: We appreciate this observation. The Discussion of splenic outcomes has been substantially revised. Interpretations suggesting hypersplenism, fibrosis, or immune overstimulation have been removed or clearly reframed as exploratory hypotheses. The revised text now emphasizes that spleen weight differences were not statistically significant and that numerical trends should be interpreted cautiously in the absence of histological validation, thereby resolving the previously noted internal inconsistency.

8. The discussion of erythroid indices and RDW is conceptually interesting. However, the modest changes observed, often within physiological ranges, do not fully justify extensive mechanistic speculation involving Nrf2 signaling, iron metabolism, or preserved erythropoietic efficiency. Similar concerns apply to interpretations of leukocyte and platelet preservation, which rely heavily on prior literature rather than strong effects demonstrated in the current dataset.

Comments: We thank the reviewer for highlighting this issue. The Discussion has been carefully revised to reduce speculative mechanistic extrapolation. Interpretations related to Nrf2 signaling, iron metabolism, and preserved erythropoietic efficiency are now framed more conservatively and explicitly linked to functional patterns rather than causal mechanisms. Greater emphasis has been placed on the modest magnitude of changes and their consistency with partial attenuation rather than robust preservation.

9. The translational discussion appropriately raises hypotheses regarding maintenance of dose intensity and reduction of supportive interventions. However, the level of certainty exceeds what can be supported by a single hematologic rat model without tumor burden, pharmacokinetics, or efficacy endpoints.

Comments: We agree and have substantially moderated the translational discussion. Statements implying clinical impact, dose intensity maintenance, or reduced supportive interventions have been revised to reflect a hypothesis-generating perspective. The revised section now clearly states that translational implications are speculative and require validation in tumor-bearing models and pharmacokinetic and efficacy studies.

10. The limitations section acknowledges the absence of histopathology and cytokine profiling, which is appropriate. Additional limitations should also be discussed, including the lack of Curcuma longa–only groups, use of a single sex, and potential confounding from repeated gavage and handling stress.

Comments: We thank the reviewer for this constructive suggestion. The Limitations section has been expanded to include absence of a Curcuma longa–only group, use of a single sex and potential confounding effects related to repeated gavage and handling stress. 

11. The manuscript is generally well written. Terminology is occasionally inconsistent, with Curcuma longa and curcumin used interchangeably, which should be corrected given the use of a multicomponent extract. In the mechanistic discussion, a tighter linkage to the specific findings of this study, with clearer distinction between demonstrated effects and literature-based inference, would improve focus.

Comments: We appreciate this important clarification. Terminology has been standardized throughout the manuscript. Curcuma longa is now consistently used when referring to the administered extract, while curcumin is used only when discussing literature-based molecular mechanisms.

12. In summary, the study addresses an important clinical question and uses a reasonably rigorous in vivo approach. The data appears internally consistent and support a more modest conclusion that intermediate doses of Curcuma longa are associated with partial attenuation of docetaxel-induced cytopenias in rats. With more cautious interpretation, improved statistical handling, and additional mechanistic data in future studies, this work could make a useful contribution to supportive care literature. At present, however, the mechanistic and translational conclusions should be substantially moderated to align with the evidence presented.

Comments: We fully agree with this recommendation. The Conclusions section has been revised to reflect a more modest and evidence-aligned interpretation. The revised conclusion now emphasizes that intermediate doses of Curcuma longa were associated with partial attenuation of docetaxel-induced cytopenias in rats, without overstating mechanistic or translational certainty.

 

We are very grateful to you for the insightful and rigorous critique, which substantially improved the focus, balance, and scientific precision of the manuscript. We believe that the revisions undertaken have strengthened the work. Thank you very much.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript addresses a relevant and timely topic, namely the mitigation of chemotherapy-induced hematological toxicity using Curcuma longa as an adjuvant. However, the study remains largely descriptive, with limited mechanistic validation and several issues related to statistical analysis, interpretation, and translational relevance. Thus, revision based on following suggestion could strengthen its quality:

Q1. The novelty of the study is limited, as hematoprotective effects of curcumin/Curcuma longa in chemotherapy models have been widely reported. The authors should clearly articulate the specific knowledge gap addressed by this work, particularly in relation to docetaxel-specific toxicity, dose–time interactions, or splenic responses.

Q2. The study includes two independent variables (treatment/dose and time), yet statistical analysis relies primarily on multiple one-way ANOVA tests. A two-way ANOVA or mixed-effects model would be more appropriate to evaluate treatment × time interactions and reduce the risk of type-I error. If the current approach is retained, a clear methodological justification is required.

Q3. The manuscript places strong emphasis on splenic enlargement, particularly at the highest dose and longest treatment duration. However, these effects are highly variable and often not statistically significant. Without histopathological or immunological validation, conclusions regarding extramedullary hematopoiesis or immune overstimulation remain speculative and should be toned down.

Q4. Extensive mechanistic discussion is provided (e.g., NF-κB, Nrf2, PI3K/Akt pathways), but no molecular, cytokine, oxidative stress, or histological data are presented. The authors should clearly frame these interpretations as hypothetical or provide supporting experimental data or in silico data.

Q5. The highest dose of Curcuma longa (500 mg/kg/day) raises concerns regarding physiological relevance and clinical translatability. Discussion of human-equivalent dosing, bioavailability limitations of curcumin, and potential interactions with docetaxel is insufficient and should be expanded.

Q6. The authors should consider focusing the main text on key hematological endpoints and moving secondary data to supplementary material.

 

Author Response

Coment 1: Q1. The novelty of the study is limited, as hematoprotective effects of curcumin/Curcuma longa in chemotherapy models have been widely reported. The authors should clearly articulate the specific knowledge gap addressed by this work, particularly in relation to docetaxel-specific toxicity, dose–time interactions, or splenic responses.

Response 1: We thank the reviewer for this important comment. We agree that the general hematoprotective properties of curcumin/Curcuma longa have been previously described. However, the present study addresses a more specific and underexplored knowledge gap by focusing on docetaxel-induced hematological toxicity, incorporating a time-resolved experimental design (7, 14, and 21 days) and a broad Curcuma longa dose range, allowing the identification of dose–time–dependent hematological patterns. In the revised manuscript, we have clarified that the primary novelty lies in the identification of an intermediate-dose therapeutic window (25–50 mg/kg) associated with more consistent hematological preservation, contrasted with increased variability at higher doses. We have also explicitly framed splenic findings as exploratory and secondary outcomes, rather than central mechanistic conclusions. These clarifications have been incorporated in the Introduction (page 3, paragraph 4, line 33), Discussion Sections 4.5 (page 13, paragraph 5, line 30) and 4.10 (page 15, paragraph 8, line 41), and Conclusion (page 16, paragraph 7, line 38), where the study’s contribution is now more clearly positioned within the context of docetaxel-specific toxicity and translational relevance.

Coment 2: Q2. The study includes two independent variables (treatment/dose and time), yet statistical analysis relies primarily on multiple one-way ANOVA tests. A two-way ANOVA or mixed-effects model would be more appropriate to evaluate treatment × time interactions and reduce the risk of type-I error. If the current approach is retained, a clear methodological justification is required.

Response 2: We appreciate the reviewer’s statistical insight and acknowledge that a two-way ANOVA could formally assess treatment × time interaction effects. However, given the exploratory nature of the study, the limited sample size per subgroup (n = 7), and the primary objective of identifying time-specific hematological patterns, we intentionally applied separate one-way ANOVA analyses at each time point. This methodological rationale has now been explicitly clarified in the Statistical Analysis subsection of Materials and Methods (page 6, paragraph 4, line 14). This approach was chosen to reduce model overfitting and maintain interpretability within the constraints of the experimental design.

Coment 3: Q3. The manuscript places strong emphasis on splenic enlargement, particularly at the highest dose and longest treatment duration. However, these effects are highly variable and often not statistically significant. Without histopathological or immunological validation, conclusions regarding extramedullary hematopoiesis or immune overstimulation remain speculative and should be toned down.

Response 3: We fully agree with the reviewer’s concern. In response, we have substantially condensed the discussion of splenic findings and reframed splenic enlargement as a secondary, exploratory observation, rather than a definitive outcome. In the revised manuscript interpretations related to extramedullary hematopoiesis and immune overstimulation are now explicitly described as hypothesis-generating; emphasis on splenomegaly has been reduced, and the high variability and lack of statistical significance are clearly acknowledged; and the need for histopathological and functional validation has been explicitly stated. These changes were implemented primarily in Discussion Section 4.5 (page 13, paragraph 3, line 16), as well as reflected in the Conclusion (page 16, paragraph 7, line 41).

Coment 4: Q4. Extensive mechanistic discussion is provided (e.g., NF-κB, Nrf2, PI3K/Akt pathways), but no molecular, cytokine, oxidative stress, or histological data are presented. The authors should clearly frame these interpretations as hypothetical or provide supporting experimental data or in silico data.

Response 4: We thank the reviewer for highlighting this point. In the revised manuscript, all mechanistic interpretations involving NF-κB, Nrf2, PI3K/Akt, cytokine modulation, and antioxidant pathways have been consistently reframed as biologically plausible hypotheses, rather than demonstrated mechanisms. Specifically repetitive mechanistic explanations were condensed into concise synthesis statements, language was modified to use cautious terminology (e.g., “may,” “suggests,” “is consistent with”), and the absence of molecular and histological validation is now explicitly acknowledged as a limitation. These revisions were applied throughout Discussion Sections 4.4 (page 13, paragraph 1, line 3 and page 13, paragraph 2, line 9), 4.6 (page 13, paragraph 8, line 51) 4.7 (page 14, paragraph 5, line 24), with additional clarification in Section 4.11 (Limitations - page 16, paragraph 5, line 25) .

Coment 5: Q5. The highest dose of Curcuma longa (500 mg/kg/day) raises concerns regarding physiological relevance and clinical translatability. Discussion of human-equivalent dosing, bioavailability limitations of curcumin, and potential interactions with docetaxel is insufficient and should be expanded.

Response 5: We agree with the reviewer and have addressed this concern by reframing the high-dose Curcuma longa group (500 mg/kg) as an upper-bound exploratory condition, rather than a clinically translatable dose. In the revised Discussion, we emphasize that intermediate doses (25–50 mg/kg) demonstrated the most consistent and favorable hematological profile; the increased variability and potential immune overstimulation at high doses are highlighted as cautionary findings< and limitations related to bioavailability, dose translation, and safety margins are now explicitly discussed. These clarifications are presented in Discussion Sections 4.5 (page 13, paragraph 3, line 16) and 4.10 (page 15, paragraph 8, line 41).

Coment 6: Q6. The authors should consider focusing the main text on key hematological endpoints and moving secondary data to supplementary material.

Response 6: We appreciate this suggestion. In the revised manuscript, we have streamlined the main text to emphasize key hematological endpoints, particularly erythroid and leukocyte parameters, which represent the primary outcomes of the study. Secondary observations, including splenic findings and exploratory trends, are now clearly framed as supportive or hypothesis-generating, with reduced narrative weight in the main text. While all data remain presented for transparency, the discussion focus has been adjusted in accordance with the reviewer’s recommendation.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for your work.

Author Response

We sincerely thank you for the careful evaluation of our manuscript and for the constructive comments provided throughout the review process. We greatly appreciate the time and effort dedicated to assessing our work. Your suggestions were highly valuable and contributed significantly to improving the clarity, structure, and scientific robustness of the manuscript. We are grateful for the positive reassessment following the revisions and for the recognition that the implemented changes strengthened the overall quality of the study.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have satisfactorily addressed all comments from the previous reviewers, reflecting careful attention to detail and a strong commitment to manuscript improvement. The work now meets the expected standards of scientific quality and rigor for publication.

Author Response

We sincerely thank the reviewer for this very positive and encouraging assessment. We are grateful for the careful evaluation and thoughtful feedback provided throughout the review process, which substantially contributed to improving the clarity, rigor, and overall quality of the manuscript. We are pleased that the revisions have met the expected standards of scientific quality and rigor, and we truly appreciate the reviewer’s time and expertise.

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for the revision as per the suggestion, which has strengthened the quality of the manuscript.

Q1. Add a schematic graphical abstract.

Q2. In Table 2, what do "a" and "b" mean? Please highlight it in a note.

Q3. The data are not sufficient; please either include histology results (H&E) or support your results using in silico docking.

Q4. This is the original article; could you please mention why the author is using other data?

Author Response

Q1. Add a schematic graphical abstract.

Response:  We thank the reviewer for this suggestion. A schematic graphical abstract has now been added to the manuscript. The graphical abstract clearly illustrates the experimental design, highlighting the chemotherapy-induced hematological injury, the Curcuma longa intervention (25, 50, and 500 mg/kg, oral gavage).

Q2. In Table 2, what do "a" and "b" mean? Please highlight it in a note.

Response: We thank the reviewer for this comment. A clarifying note has been added to Table 2 stating that different lowercase letters (a, b) within the same row indicate statistically significant differences between groups according to Tukey’s post-hoc test (p < 0.05), whereas values sharing the same letter do not differ significantly.

Q3. The data are not sufficient; please either include histology results (H&E) or support your results using in silico docking.

Response: We acknowledge the reviewer’s concern. Unfortunately, H&E analysis could not be performed due to the unavailability of preserved tissue from the original experiment. To address this limitation (as discussed in the Limitations section) and strengthen the biological interpretation of the findings, we have incorporated an in silico pathway- and target-based analysis, based on curated biological databases and peer-reviewed literature, to provide mechanistic context for the observed hematological outcomes. This new subsection has been added to the Discussion, clearly framed as supportive and hypothesis-generating, without implying direct molecular validation.

Q4. This is the original article; could you please mention why the author is using other data?

Response: We thank the reviewer for this important point. All hematological and splenic data presented as Results were generated exclusively from the experimental model described in this manuscript. Additional figures and analyses based on previously published studies or curated biological databases (e.g., Figure 1 and the in silico pathway-based analysis) were included solely for contextualization and hypothesis generation. These data are clearly identified as literature-derived or database-based and are not presented as original experimental results.

 

We sincerely thank you for the careful evaluation of our manuscript and for the constructive comments provided. Your insights were highly valuable and contributed substantially to improving the clarity, methodological transparency, and overall scientific rigor of the study. We believe that the revisions implemented in response to these comments have strengthened the manuscript and enhanced its relevance within the field. Thank you again.

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for the revision as per the suggestion. Please update the schematic image in the manuscript. In Table 4, please incorporate the binding affinity of curcuma longa with reported molecular targets (based on in silico docking) and add a 2D representative in silico image also in the original manuscript instead of supplementary data.

Author Response

Comments: Please update the schematic image in the manuscript. 

Response: We thank the reviewer for this valuable and constructive suggestion.The schematic image has been updated and a conceptual figure was added to the manuscript (Figure 1), summarizing a proposed, literature-supported biological framework integrating docetaxel-induced hematological toxicity and the modulatory effects of Curcuma longa.

Comments: In Table 4, please incorporate the binding affinity of curcuma longa with reported molecular targets (based on in silico docking) and add a 2D representative in silico image also in the original manuscript instead of supplementary data.

Response: The table 4 was expanded to incorporate literature-reported in silico docking binding affinity ranges between curcumin and the indicated molecular targets, while fully preserving the original pathway-based biological interpretation and its relation to the observed hematological outcomes. A representative 2D schematic illustration of curcumin interaction with the COX-2 binding pocket, newly created for this manuscript and based on literature-reported molecular docking studies, has been incorporated into the main manuscript for illustrative purposes, replacing the supplementary material as requested.

We believe these revisions strengthen the mechanistic clarity and visual integration of the in silico findings with the experimental results. Thank you again for your valuable support.