Therapeutic Potential of Deferiprone–Resveratrol Hybrid (DFP-RVT) Against Hepatic Iron Overload in β-Thalassemia Mice: A Proteomic Analysis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the study, the authors investigated the molecular impact of hepatic iron overload in a β-thalassemia mouse model and evaluated the therapeutic effects of a deferiprone-resveratrol hybrid (DFP-RVT) using a proteomics-based approach. They demonstrated that iron overload is associated with marked dysregulation of mitochondrial proteins, particularly those involved in oxidative phosphorylation and energy metabolism, while DFP-RVT treatment partially restored mitochondrial protein expression and attenuated inflammation- and injury-related pathways. These findings provide mechanistic insight into the mitochondrial basis of iron-induced liver injury and support the hepatoprotective role of DFP-RVT. However, several aspects would benefit from further clarification to strengthen the overall quality of the manuscript:

-The Introduction section would benefit from a more focused presentation to better highlight the scientific context and novelty of the present study. 

The last paragraph of the introduction section can be strengthened by adding information about the originality of the study and the research conducted. Also, several references must be added to lines 44-47, 52-58, and 69-75 on the introduction section.

-Although the authors refer to several studies related to the DFP-RVT hybrid, additional discussion is needed to help readers better understand its biological effects, mechanisms, and functional relevance.

The authors define the DFP-RVT hybrid in the Introduction (lines 69-98). However, the discussion of its biological effects is limited to a brief overview between lines 77 and 89 (references 13-16). While these references provide initial background, this section would benefit from the inclusion of additional relevant studies and/or a more detailed discussion to better explain the biological effects and underlying mechanisms of DFP-RVT. (e.g. doi: 10.3389/fmolb.2024.1364261, doi: 10.7717/peerj.15187)

-A different font size appears in Section 2.1. (Animal care). Please revise for consistency.

-Line 405, The correct annotation for “et al” is “et al.” Please revise accordingly in the manuscript. 

​​Latin phrases such as et al. should be consistently italicized throughout the manuscript and include a period after “al”. (See lines 77,85,209,405,413 and 416).

-It is recommended to update panels (a), (b) and (c) in Figures 3 and 5 with higher-resolution images to improve clarity and visualization.

The text within the figures is not easily readable and the current resolution limits clear interpretation. Therefore, updating these panels with higher-resolution images and larger more legible text is recommended.

-The Conclusion section remains insufficient in fully addressing the main findings and their broader implications. Please revise this section to more clearly reflect the study outcomes.

The current version of the conclusion section (in lines between 488-496) is very brief and does not adequately reflect the study outcomes or outline potential future directions. Revising this section to include a clearer summary of the results, their broader implications and possible future research directions will improve the quality of the manuscript.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Iron overload causes organ dysfunctions in liver, spleen and gonads. Among them, liver failure may be fetal in patients. In this manuscript, authors demonstrate that proteomic analyses of liver in mice. While authors previously report the efficiency of DEF-RVT in reference 16, they provide new findings. Some points should be clarified.

 

 

Major points

#1: When iron is overloaded in liver, ferritin protein level is increased. However, in table 2 and 3, there is no detection of modulated ferritin protein levels. How do authors think ?

 

#2: Proteomic analyses provide comprehensive data. However, quantification of each protein level is needed to measure conventional western blot.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

In this research article the authors generated iron overloaded beta thalassemic mice. Half of these treated mice received a special hybrid molecule of deferiprone-resveratrol (DFP-RVT) to reduce the iron deposition of the liver of the animals and to fight the oxidative stress of iron overloading. The authors proved the fact of iron overloading. Interestingly they used the liver tissues from a very similar previous experiment for proteomic analysis. The result of the latter one showed that the mitochondrial functions, especially ATP synthesis, heme and iron-sulfur cluster biogenesis suffered most from iron overload, and these functions were partially restored by the DFP-RVT administration.

I do not understand why the livers of the present experiment were not used or why additional experiments were not carried out. The authors should examine the hearts of the iron overloaded animals for example. Resveratrol is claimed to have an HbF expression level increase. Was this the case in the hybrid molecule treatment, too? I missed the control of the none-iron overloaded beta-thalassemic mice treated with the hybrid molecule. What was the reason for the 30 days interval between the iron treatment and the DFP-RVT administration?

The proteomic studies and their conclusions are correct. The figures and tables are informative. Statistical analysis is correct. Only the whole experimental setup is strange and unusual.

Author Response

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Author Response File: Author Response.pdf