Antifibrotic Effect of the TGF-β Type I Receptor Inhibitor EW-7197 on Anastomotic Healing in a Rat Choledochojejunostomy Model

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

I read your manuscript carefully. I have some comments that addressing them can increase the quality of your upcoming manuscript.

 

1- Your study exclusively reports histopathological outcomes, yet conclusions occasionally imply clinical implications. I recommend revising the conclusion, based on your findings.

2- The manuscript should report the clinical outcomes including anastomotic patency, biliary drainage, duct diameter, and cholestasis.

3- TGF-β–driven fibrosis is a dynamic, prolonged process. A single dose may not reflect realistic pharmacologic modulation. 

4- You mentioned that EW-7197 does not influence inflammation. As we know, the inflammation after surgery peaks between days 1–7 and Day-21 histology is too late to evaluate early inflammatory effects and leads to under-reported incidence.

5- Generally, the experimental model represents elective clean surgery, not emergency conditions, but the discussion part repeatedly emphasizes emergency biliary surgery.

6- Please describe more about the "Histopathological Scoring System".

 

Author Response

Response Letter – Reviewer 1

Dear Reviewer 1,

We sincerely thank you for your careful reading of our manuscript and for providing constructive and insightful comments. Your suggestions have greatly helped us to clarify the scope of our study, strengthen the interpretation of our findings, and improve the overall quality of the manuscript. Below, we provide point-by-point responses to your comments.

 

Comment 1: “Your study exclusively reports histopathological outcomes, yet conclusions occasionally imply clinical implications. I recommend revising the conclusion, based on your findings.”

Response 1:
We thank the reviewer for this important point. The Conclusion section has been revised to clearly reflect only the histopathological findings and to avoid overstating clinical implications. Clinical relevance is now presented cautiously, and the translational potential is suggested only for future studies. (Page 11 line 421-426)

 

Comment 2: “The manuscript should report the clinical outcomes including anastomotic patency, biliary drainage, duct diameter, and cholestasis.”

Response 2:
We acknowledge that functional clinical parameters were not assessed in this preclinical study. This limitation is now explicitly stated in the Limitations section, emphasizing that histopathological improvements do not necessarily indicate functional outcomes. (page 10 line 386-390)

 

Comment 3: “TGF-β–driven fibrosis is a dynamic, prolonged process. A single dose may not reflect realistic pharmacologic modulation.”

Response 3:
We agree and have added a discussion on the single-dose administration and the need for future studies to explore dose-response relationships, repeated or delayed dosing, and alternative administration routes. 8Page 4 line 153-158 ans page 10 line 391-395)

 

Comment 4: “You mentioned that EW-7197 does not influence inflammation. Inflammation after surgery peaks between days 1–7 and Day-21 histology is too late to evaluate early inflammatory effects.”

Response 4:
We have clarified that Day-21 evaluation captures late remodeling but may not reflect early peak inflammation (postoperative days 1–7). The Discussion and Limitations now highlight this temporal limitation of inflammatory assessment. (page 8 line 303-306 and page 10 line 378-379)

 

Comment 5: “Generally, the experimental model represents elective clean surgery, not emergency conditions, but the discussion repeatedly emphasizes emergency biliary surgery.”

Response 5:
We have revised the Discussion to temper repeated references to emergency surgery and clarified that the current model represents controlled elective surgery. Potential relevance to emergency conditions is now stated as speculative and for future investigation. (Page 8 line 307-322)

 

Comment 6: “Please describe more about the 'Histopathological Scoring System'.”

Response 6:
We have expanded the description of the semi-quantitative scoring system for edema, hyperemia, inflammation, and fibrosis in the Materials & Methods section, including grading criteria and references. (page 5,6 line 185-209)

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

• Apart from its effect of inhibition on hepatic, renal, and pulmonary fibrosis by blocking TGF-β and ROS signaling, which has been known for more than ten years, this study highlights the potential of EW-7197 as a therapeutic strategy to mitigate fibrosis-related complications in emergency biliary surgery, highlighting a novel field of action for Vactosertib. • What does it add to the subject area compared with other published material? To my knowledge, the effect of EW-7197 on choledocojejunostomy in emergency biliary surgery has not been evaluated to date. • The methodology of this study does not require improvement, but larger groups would be beneficial to enhance statistical power, as the authors already underlined. Even with the aforementioned limitations, the value of this study remains high. In the discussion chapter, it might be useful to make some remarks on the intraperitoneal delivery modality and on possible dosage variations in the study group. • Are the conclusions consistent with the evidence and arguments, and do they address the central question? The study's conclusions are consistent with the evidence and arguments presented.  

Author Response

Response Letter – Reviewer 2

Dear Reviewer 2,

We sincerely thank you for your thorough evaluation of our manuscript and for your constructive and scientifically valuable comments. Your suggestions have significantly contributed to improving the clarity, methodological transparency, and scientific rigor of our study. We have carefully revised the manuscript in accordance with your recommendations and provide detailed point-by-point responses below.

 

Comment 1:

“The novelty of the study should be clarified more explicitly.”

Response 1:
We appreciate this important suggestion. We have now explicitly clarified in the Discussion that, to the best of our knowledge, the effect of the selective ALK5 inhibitor EW-7197 on bilioenteric anastomotic healing has not previously been investigated. The novelty statement has been strengthened to emphasize the expansion of ALK5 inhibition research into gastrointestinal anastomotic fibrosis. (page 8 line 293-302)

 

Comment 2:

“The lack of molecular validation (e.g., Smad2/3 phosphorylation analysis) weakens the mechanistic interpretation.”

Response 2:
We agree that molecular confirmation would further strengthen the mechanistic interpretation. Accordingly, we have clearly acknowledged in both the Discussion and Limitations sections that Smad2/3 phosphorylation and other molecular markers were not assessed. We also clarified that the mechanistic interpretation is based on established literature regarding ALK5 inhibition and TGF-β signaling. (page 8,9 line 323-339 and page 10 line 386-390)

 

Comment 3:

“The discussion should better contextualize the timing of inflammatory assessment.”

Response 3:
We have revised the Discussion to explicitly state that inflammation after gastrointestinal surgery typically peaks within postoperative days 1–7 and that assessment at day 21 reflects the remodeling phase rather than early inflammatory activity. This clarification prevents overinterpretation of the inflammatory findings. (page 8 line 303-306)

Comment 4:

“The clinical implications appear somewhat overstated given that only histopathological parameters were assessed.”

Response 4:
We thank the reviewer for this observation. The Conclusion and Discussion have been revised to limit interpretation strictly to histopathological outcomes. We now clearly state that functional parameters such as anastomotic patency, biliary drainage, luminal diameter, and cholestasis markers were not evaluated, and therefore clinical relevance remains to be established. (page 9,10 line 369-376 and page 11 line 424-426)

 

Comment 5:

“The dosing strategy (single intraperitoneal administration) requires further justification.”

Response 5:
We have expanded the Discussion and Limitations sections to explain the rationale for single-dose intraperitoneal administration and to acknowledge that repeated dosing, delayed treatment protocols, or alternative routes (e.g., oral delivery) may produce different outcomes. We also emphasized the need for future dose–response investigations. (page 8 line 293-302 and page 10 line 391-395)

Final Statement to Reviewer 2

We again thank the reviewer for the constructive feedback, which has substantially improved the scientific clarity and balance of our manuscript. We believe that the revised version addresses all concerns and presents the findings in a more rigorous and appropriately cautious manner.

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for addressing my comments.