Cardiovascular Protection with Sulfasalazine During Doxorubicin Challenge: Evidence from Oxidative and Histologic Endpoints

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled Cardiovascular Protection with Sulfasalazine During Doxorubicin Challenge: Evidence from Oxidative and Histologic Endpoints presents an interesting and relevant study investigating the potential cardioprotective effects of sulfasalazine against early doxorubicin-induced myocardial and vascular injury in a rat model. The authors address an important clinical problem, considering the well-known cardiotoxic limitations of anthracycline therapy and the need for novel supportive strategies. The experimental design combines hemodynamic, electrocardiographic, histological, and oxidative stress assessments, providing a comprehensive approach to evaluate the protective effects of sulfasalazine.

The study provides valuable data suggesting that short-term sulfasalazine pretreatment may attenuate oxidative stress, tissue injury, and functional alterations associated with doxorubicin exposure. The manuscript is generally well organized and the findings are of potential interest to the field. However, several minor issues regarding clarification of methodology, data presentation, and interpretation should be addressed to further improve the quality and scientific rigor of the manuscript.

Therefore, I recommend minor revision before further consideration for publication:

1. The Introduction is well written and provides sufficient background for the research question. The authors may consider briefly introducing the concept of cancer therapy-related cardiac dysfunction (CTRCD) to place their study within the current cardio-oncology context and strengthen its clinical relevance.
2. The body weight range (294-467 g) is quite broad. Did baseline body weights differed between groups?
3. Randomization is mentioned, but describe in more detail “simple randomization”.
4. Please clarify whether any animals developed severe adverse effects or died before completion of the study.
5. Please explain better this statement “All procedures, treatments, and subsequent measurements were conducted in a standardized, sequential series between groups, rather than processing the entire cohort of one group before the other; this prevented the time of day and treatment order from affecting the results.“.
6. In the Study Design section, there is no need to list all procedures performed (e.g., BP measurement, ECG, histological and biochemical analyses), as these methods are described in detail in the following sections. The Study Design section could be more focused on the overall experimental protocol and group allocation.
7. For improved clarity and readability, the authors may consider numbering the main sections and subsections throughout the manuscript in accordance with the journal's formatting guidelines.
8. Avoid presenting the same data in both figures and tables, so consider using either a figure or a table depending on which format best highlights the findings. Also, p-values can be only presented in figure or table. There is no need for repetitiveness.
9. Please avoid starting sentences with numerals ("28 male Wistar albino rats...") and revise the wording to standard scientific writing style.
10. The Discussion section is comprehensive, however, some conclusions regarding the mechanisms of SSZ-mediated cardioprotection appear stronger than supported by the presented data. Since pathways such as Nrf2 activation, NF-κB inhibition, and ROS-related signaling were not directly measured, these mechanisms should be presented as possible explanations rather than confirmed findings.

Author Response

C1. The Introduction is well written and provides sufficient background for the research question. The authors may consider briefly introducing the concept of cancer therapy-related cardiac dysfunction (CTRCD) to place their study within the current cardio-oncology context and strengthen its clinical relevance.

R1. Thank you for this valuable suggestion. We agree that introducing the concept of cancer therapy-related cardiac dysfunction (CTRCD) strengthens the clinical context of our study. Accordingly, we have revised the Introduction to briefly describe CTRCD as a clinically important complication of anticancer therapy and highlighted that anthracycline-induced cardiotoxicity represents one of its best-recognized forms. This addition better places our experimental findings within the contemporary cardio-oncology framework and emphasizes their potential translational relevance. (P:2 L:44-49).

Reference:Contaldi, C., D'Aniello, C., Panico, D., Zito, A., Calabro', P., Di Lorenzo, E., Golino, P., & Montesarchio, V. (2025). Cancer-Therapy-Related Cardiac Dysfunction: Latest Advances in Prevention and Treatment. Life, 15. https://doi.org/10.3390/life15030471

 

C2. The body weight range (294-467 g) is quite broad. Did baseline body weights differed between groups?

R2. Thank you for this important observation. Although the overall body weight range of the animals was relatively broad, randomization resulted in comparable baseline body weights across the experimental groups. As shown in Table 1, no statistically significant difference was observed in baseline body weight among the groups (p = 0.293). To avoid any ambiguity, we have revised the Methods section to explicitly state that baseline body weights were comparable between groups before treatment initiation. (P:6 L: 275-279).

 

C3. Randomization is mentioned, but describe in more detail “simple randomization”.

R3. Thank you for this valuable suggestion. We agree that the randomization procedure should be described more explicitly. Therefore, the Methods section has been revised to clarify that each rat was assigned a unique identification number and allocated to the experimental groups using computer-generated random numbers before the initiation of the experimental procedures. This additional information improves the transparency and reproducibility of the study. (P:3 L:127-131).

 

C4. Please clarify whether any animals developed severe adverse effects or died before completion of the study.

R4. Thank you for this important comment. No animals died or developed severe adverse effects before completion of the study. As expected following doxorubicin administration, transient red discoloration of the urine was observed in some animals, which is a well-recognized pharmacological effect of doxorubicin rather than an indicator of severe toxicity. To clarify this point, we have added this information to the Methods section. (P:3 L:139-143).

 

C5. Please explain better this statement “All procedures, treatments, and subsequent measurements were conducted in a standardized, sequential series between groups, rather than processing the entire cohort of one group before the other; this prevented the time of day and treatment order from affecting the results.“.

R5. Thank you for this helpful comment. We agree that the original wording was not sufficiently clear. The Methods section has been revised to better explain that animals from different experimental groups were processed in an alternating sequence throughout each experimental session rather than completing all procedures for one group before proceeding to the next. This approach minimized potential confounding effects related to the time of day and procedural order. (P: L:107-112).

 

C6. In the Study Design section, there is no need to list all procedures performed (e.g., BP measurement, ECG, histological and biochemical analyses), as these methods are described in detail in the following sections. The Study Design section could be more focused on the overall experimental protocol and group allocation.

R6. Thank you for this helpful suggestion. We agree that the Study Design section should focus primarily on the overall experimental protocol rather than detailed descriptions of the outcome assessments. Accordingly, we have revised this section to emphasize the experimental groups, treatment allocation, timeline, and tissue collection, while removing detailed descriptions of blood pressure measurements, ECG recordings, and histopathological and biochemical analyses, which are presented in their respective subsections. This revision improves the organization of the Methods section and avoids unnecessary repetition. (P:4 L:144; P:4 L:155-158).

 

C7. For improved clarity and readability, the authors may consider numbering the main sections and subsections throughout the manuscript in accordance with the journal's formatting guidelines.

R7. Thank you for this helpful suggestion. We have revised the manuscript by numbering the main sections and subsections in accordance with the journal's formatting guidelines, which we believe improves the overall organization and readability of the manuscript. (P:2-15).

 

C8. Avoid presenting the same data in both figures and tables, so consider using either a figure or a table depending on which format best highlights the findings. Also, p-values can be only presented in figure or table. There is no need for repetitiveness.

R8. Thank you for this valuable suggestion. We agree that presenting the same data in multiple formats may result in unnecessary repetition. Accordingly, Tables 2, 4, and 5 have been removed from the revised manuscript, and the corresponding data are presented only in the figures. In addition, the Results section has been revised to avoid redundant reporting of numerical values and p-values, which are now presented only in the relevant figures. These revisions improve the clarity and readability of the manuscript while preserving all essential findings.

 

C9. Please avoid starting sentences with numerals ("28 male Wistar albino rats...") and revise the wording to standard scientific writing style.

R9. Thank you for this helpful suggestion. We have revised the manuscript to avoid starting sentences with numerals in accordance with standard scientific writing conventions. The relevant sentences have been rephrased throughout the manuscript to improve readability and comply with the journal’s style. (P:3 L:99,127).

 

C10. The Discussion section is comprehensive, however, some conclusions regarding the mechanisms of SSZ-mediated cardioprotection appear stronger than supported by the presented data. Since pathways such as Nrf2 activation, NF-κB inhibition, and ROS-related signaling were not directly measured, these mechanisms should be presented as possible explanations rather than confirmed findings.

R10. Thank you for this insightful comment. We agree that the molecular mechanisms underlying the cardioprotective effects of SSZ were not directly investigated in the present study. Accordingly, we have revised the Discussion to avoid definitive mechanistic statements and instead present the involvement of pathways such as NF-κB inhibition, Nrf2 activation, and ROS-related signaling as plausible mechanisms supported by previous literature. (P:13 L:452-459; P:13 L:466-474; P:14 L:475; P:14 L:498-504; P:15 L:526-532).

Reviewer 2 Report

Comments and Suggestions for Authors

Dr. Ozhan et al. studied the effect of a sulfasalazine intervention on cardiotoxicity induced by doxorubicin treatment. They found that a short-course sulfasalazine pretreatment mitigates doxorubicin-induced myocardial and aortic injury in rats, as shown by reduced blood pressure, arrhythmogenic effects, and tissue damage. The mechanism of protection is related to decreased oxidative stress along with restored antioxidant levels and aortic thickness. The authors concluded that sulfasalazine treatment has the potential for preventing early oxidative and structural cardiovascular damage during doxorubicin treatment.

A previous study demonstrated that sulfasalazine treatment decreases oxidative stress during doxorubicin exposure in cultured H9C2 cells (PMID: 39963866). In the current study, authors show that sulfasalazine treatment in vivo also provides beneficial cardioprotective effects during doxorubicin exposure in rats. Thus, there is some novelty in the current study.

Comments:

Line 121-122: “28 rats were divided into three groups.”

As the authors discussed in the limitations section, it would have been beneficial to include a sulfasalazine-only control group in the current study. This is a critical cohort for evaluating the effects of sulfasalazine treatment alone on blood pressure and heart rate. Data received from this group would clarify several issues in the current study.

 

Line 241 Statistical Analyses

Line 274-280 ECG changes

Please use the proper statistical analysis in this section. The authors should use a chi-square test or Fisher's exact test to perform the analysis, rather than a t-test or one-way ANOVA.

 

Table 1 and other table

Please double-check to ensure consistent reporting of decimal places.

 

Figure 1 and other figures

Please add lines to indicate specific statistical differences between the experimental groups. Additionally, please expand the figure legend to include more experiment-related information.

Author Response

C1. A previous study demonstrated that sulfasalazine treatment decreases oxidative stress during doxorubicin exposure in cultured H9C2 cells (PMID: 39963866). In the current study, authors show that sulfasalazine treatment in vivo also provides beneficial cardioprotective effects during doxorubicin exposure in rats. Thus, there is some novelty in the current study.

R1. Thank you for your positive evaluation of our study. We appreciate your recognition that, although the antioxidant effects of sulfasalazine against doxorubicin-induced injury have previously been demonstrated in H9C2 cells, our study extends these findings by providing in vivo evidence of its cardioprotective effects in a rat model of doxorubicin-induced cardiotoxicity. We believe that the integration of functional, biochemical, histopathological, and vascular assessments enhances the translational relevance and novelty of our findings. No changes to the manuscript were considered necessary in response to this comment.

 

C2. Line 121-122: “28 rats were divided into three groups.”

As the authors discussed in the limitations section, it would have been beneficial to include a sulfasalazine-only control group in the current study. This is a critical cohort for evaluating the effects of sulfasalazine treatment alone on blood pressure and heart rate. Data received from this group would clarify several issues in the current study.

R2. Thank you for this important comment. We agree that the inclusion of a sulfasalazine-only control group would have provided additional information regarding the independent effects of SSZ on cardiovascular and biochemical parameters under physiological conditions. As appropriately noted, this represents a limitation of the present study. Accordingly, we have emphasized this point in the Limitations section and acknowledged that future studies incorporating an SSZ-only group would help distinguish the intrinsic effects of SSZ from its protective effects against DOX-induced cardiotoxicity. (P:19 L:542-549).

 

C3.  Line 241 Statistical Analyses

Line 274-280 ECG changes

Please use the proper statistical analysis in this section. The authors should use a chi-square test or Fisher's exact test to perform the analysis, rather than a t-test or one-way ANOVA.

R3. Thank you for this valuable comment. We agree that the ECG findings represent categorical data and therefore require an appropriate statistical approach. Accordingly, the statistical analysis of the ECG findings has been revised using Fisher's exact test, as recommended. The Methods section and the corresponding Results have been updated to reflect this change. (P:6 L:269-270; P:7 L:295-296; P:8 L:307).

 

C4. Table 1 and other table

Please double-check to ensure consistent reporting of decimal places.

R4. Thank you for this careful observation. We have thoroughly reviewed Table 1 and all remaining tables and standardized the reporting of decimal places throughout the manuscript. Values within each parameter are now presented using a consistent number of decimal places, while maintaining an appropriate level of precision for the corresponding measurements. The reporting format of the p-values has also been checked and standardized. (P:7 L:281)

 

C5. Figure 1 and other figures

Please add lines to indicate specific statistical differences between the experimental groups. Additionally, please expand the figure legend to include more experiment-related information.

R5. Thank you for this helpful suggestion. We have revised Figure 1 and the other quantitative figures by adding comparison lines to indicate the specific statistically significant differences between the experimental groups. The figure legends have also been expanded to include the experimental groups, sample sizes, data presentation format, statistical tests, and definitions of the significance symbols. These revisions allow the statistical comparisons to be interpreted directly from the figures and improve their overall clarity. (P:7 L:287-293; P:9 L:320-328; P:10 L:337-344).

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

My concerns have been properly addressed in the revised manuscript.