Immunomodulatory Effects of Epilobium angustifolium Extract in DSS-Induced Colitis: Attenuation of Inflammatory and Metabolic Markers in Mice

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript investigates the immunomodulatory effects of Epilobium angustifolium extract (EAE) in a DSS-induced colitis mouse model. The authors build upon previous work (Ref. 33), using plasma samples from the same experimental cohort to perform proteomic analysis and explore molecular mechanisms. The therapeutic potential of EAE is promising, especially as an alternative to glucocorticoid treatment. However, several concerns must be addressed before the manuscript can be considered for publication.

 

Major Points

1. Clarify the Relationship with Ref. 33

   The manuscript heavily relies on data previously published in Ref. 33. To avoid concerns of salami publication, the authors should clearly explain the scope and novelty of the current study in the Introduction, especially how it differs from and builds upon Ref. 33.

2. Excessive Referencing

   The number of references is unusually high for an original article, giving the impression of a review. This may reflect a lack of novel data and an overreliance on speculative interpretation. The authors should consider reducing and focusing the references to support key findings.

3. Redundancy and Focus

   The Introduction includes excessive background on ulcerative colitis. In the Results and Discussion, descriptions of cytokines and chemokines are overly detailed and heavily sourced from literature, which detracts from the core findings. The manuscript should focus more on the specific molecular mechanisms by which EAE exerts its anti-inflammatory effects.

 

Minor Points

1. The x-axis labels in Figures 3–5 are overly complex. Simplifying them and including a legend for the bar colors would improve clarity.
2. Figures 2, 3–5, and 6 appear to present overlapping results in different formats. This may be perceived as data inflation. Figures 2 and 6 alone may suffice.
3. Lines 173–186 contain repetitive descriptions. Similar redundancy is found in lines 162–168 and 223–228. These should be consolidated.
4. Line 204 refers to Figure 5, which may be incorrect. Please verify.
5. Line 206: The numbering of quercetin may be incorrect. And, it appears that other compounds (e.g., neochlorogenic acid) have higher content levels.
6. In Table 1, units such as µg/mg and mg/g should be standardized for clarity.
7. Please verify the content value for compound No. 12 in Table 1.

Author Response

Responses to Reviewer 1

This manuscript investigates the immunomodulatory effects of Epilobium angustifolium extract (EAE) in a DSS-induced colitis mouse model. The authors build upon previous work (Ref. 33), using plasma samples from the same experimental cohort to perform proteomic analysis and explore molecular mechanisms. The therapeutic potential of EAE is promising, especially as an alternative to glucocorticoid treatment. However, several concerns must be addressed before the manuscript can be considered for publication.

 

Major Points

 

Comment 1: Clarify the Relationship with Ref. 33

The manuscript heavily relies on data previously published in Ref. 33. To avoid concerns of salami publication, the authors should clearly explain the scope and novelty of the current study in the Introduction, especially how it differs from and builds upon Ref. 33.
Response: We appreciate the reviewer’s comment and fully agree with the importance of clarifying the relationship between the present study and Ref. 33. Our previous work (Ref. 33) was primarily a clinical and histological evaluation, providing detailed analysis of disease activity, tissue pathology, and overall therapeutic outcomes of the Epilobium extract (EAE) in a DSS-induced colitis model. In contrast, the scope of the current manuscript is centered on elucidating the molecular immunological mechanisms underlying EAE’s ulceroprotective and immunomodulatory effects, using advanced proteomic analysis of plasma from the same experimental cohort. While both studies address complementary facets of EAE’s therapeutic potential, their focus, methodology, and scientific contributions are distinct.

We considered publishing these findings jointly, but the resulting manuscript would be excessively lengthy, heterogeneous in its results, and difficult to follow, potentially obscuring the mechanistic insights that are the focus here. Thus, in line with best publishing practices, we opted to publish the molecular and clinical findings separately to maintain clarity and depth. To further clarify the novelty and scope of the present study, we have explicitly described how it builds upon and differentiates itself from the previous report (Ref. 33) in the Introduction section. 

 

Comment 2: Excessive Referencing: The number of references is unusually high for an original article, giving the impression of a review. This may reflect a lack of novel data and an overreliance on speculative interpretation. The authors should consider reducing and focusing the references to support key findings.
Response: We appreciate the reviewer’s concern regarding the number of references in the Results section. To clarify, the extensive referencing was not due to a lack of novel data; rather, it reflects our commitment to annotating the function and biological significance of the numerous proteins found to be differentially expressed in our study. The results reported are entirely original, generated from a systematic proteomic mapping of over 30 inflammatory, immune, and metabolic markers in a clinically relevant DSS-induced colitis model. Given the multifactorial nature of IBD and the intricate immune-metabolic interplay involved, we considered functional annotation of each marker essential for scientific transparency and interpretability.

Importantly, to address the reviewer’s recommendation, we have removed non-essential references from the Results section, focusing only on those directly supporting our key findings in the Discussion section. We stress that no cited reference was used to substitute for original results: all data are derived from our experiments, and, to the best of our knowledge, no prior study has characterized the DSS colitis model at this molecular depth or explored the therapeutic effects of E. angustifolium in such a broad proteomic context.

 

Comment 3: Redundancy and Focus

The Introduction includes excessive background on ulcerative colitis. In the Results and Discussion, descriptions of cytokines and chemokines are overly detailed and heavily sourced from literature, which detracts from the core findings. The manuscript should focus more on the specific molecular mechanisms by which EAE exerts its anti-inflammatory effects.

Response: We thank the reviewer for raising this point. We agree that a focused description of the molecular mechanisms by which EAE exerts its anti-inflammatory and ulceroprotective effects is essential for clarity and impact. However, we believe that including background on colitis, with emphasis on its molecular complexity and immune-metabolic crosstalk, is highly relevant for contextualizing our findings and illustrating how the observed changes in multiple proteins contribute to EAE’s therapeutic action.

Because our study systematically profiled over 30 inflammatory, immune, and metabolic proteins, a network-level analysis was required to accurately interpret their functions in the context of our findings (i.e. the molecular mechanisms of EAE) and within the multifactorial pathology of DSS-induced colitis. Detailing the roles of these proteins in inflammation and immune regulation is critical to understanding how EAE mediates its effects beyond just describing a change in the measured biomarkers.

 

Minor Points

 

Comment 4: The x-axis labels in Figures 3–5 are overly complex. Simplifying them and including a legend for the bar colors would improve clarity.

Figures 2, 3–5, and 6 appear to present overlapping results in different formats. This may be perceived as data inflation. Figures 2 and 6 alone may suffice.
Response: We thank the reviewer for their constructive input. We fully agree that simplifying the presentation and eliminating overlapping formats would enhance interpretability for readers. In response, we have removed Figures 3-5 and now present a single, consolidated heatmap (formerly Figure 6, now Figure 3) alongside Figure 2 for the final submission. We thank you for highlighting this issue and believe these changes result in a clearer, more focused presentation of our findings.

 

Comment 5: Lines 173–186 contain repetitive descriptions. Similar redundancy is found in lines 162–168 and 223–228. These should be consolidated.
Response: Thank you for bringing this to our attention. We have revised the relevant sections to eliminate duplications and repetitive descriptions in the Materials and Methods and Results Sections, ensuring that each methodological aspect is presented only once and in appropriate detail.

 

Comment 6: Line 204 refers to Figure 5, which may be incorrect. Please verify.
Response: Thank you for noticing. We have reviewed and corrected the Figure reference in line 204 (citing Figure 1, line 198 in the revised manuscript) to ensure accuracy and consistency with the revised manuscript.

 

Comment 7: Line 206: The numbering of quercetin may be incorrect. And, it appears that other compounds (e.g., neochlorogenic acid) have higher content levels.

In Table 1, units such as µg/mg and mg/g should be standardized for clarity.
Response: Thank you for noticing. Accordingly, the correction has been done.

 

Comment 8: Please verify the content value for compound No. 12 in Table 1.
Response: Thank you for noticing. Accordingly, the correction has been done.

Reviewer 2 Report

Comments and Suggestions for Authors

Why is there no description of the colon at necropsy?  Did the mice receiving diss show signs of colitis such as bloody feces or loss of weight?   A vehicle with diss should havbeen done.  what was the effect o treatments without diss?  Diss model is not specific for us.  What was the molecular weight of diss.   Many of the figures were difficult to interpret.  Why was there no statistical evaluation. I could not access your paper showing eff ectiveness. Could you at least give some data from this study?

Author Response

Responses to Reviewer 2

 

Comment 1: Why is there no description of the colon at necropsy?  Did the mice receiving diss show signs of colitis such as bloody feces or loss of weight? 
Response: Thank you for your question. The development of clinical signs, such as bloody feces, weight loss, and changes in stool consistency, was systematically assessed using the Disease Activity Index (DAI), a validated scoring approach for DSS-induced ulcerative colitis. These clinical observations confirmed successful colitis induction in the DSS-treated groups. In addition, we performed histological analysis of colon samples, with detailed micrographs. Both the DAI scoring and histological assessment are referenced and described in the manuscript:

Simeonova et al., Ulceroprotective Effects of Epilobium angustifolium Extract in DSS-Induced Colitis in Mice, Current Issues in Molecular Biology, 2025, 47, 444 (https://doi.org/10.3390/cimb47060444).
MDPI link: https://www.mdpi.com/1467-3045/47/6/444

We kindly refer the reviewer to that publication for comprehensive information.

 

Comment 2: A vehicle with diss should havbeen done.  what was the effect o treatments without diss?
Response: If we understand your question correctly, you are asking whether a group treated with EAE or other interventions in the absence of DSS-induced colitis was included for comparison. In our study, the primary aim was to evaluate the ulceroprotective effects of EAE specifically under conditions of experimentally induced mucosal injury. Therefore, we did not assess the effects of EAE in a healthy, DSS-non-treated group, since no ulcerative damage is present in such animals and ulceroprotection cannot be meaningfully evaluated. Nonetheless, a naïve untreated control group was included to serve as a reference for physiological and baseline parameters. This approach is standard in preclinical IBD research, where treatment effects are principally interpreted in the context of inflammation and tissue injury.

Comment 3: Diss model is not specific for us.  What was the molecular weight of diss.
Response: The DSS-induced colitis is a widely accepted and validated experimental approach, extensively used animal model for studying the pathogenesis and therapy of human ulcerative colitis, as it closely recapitulates key clinical and histopathological features of the disease(https://doi.org/10.1093/ibd/izad312; https://doi.org/10.3390/foods12051073; https://doi.org/10.1186/s12866-021-02342-8; https://doi.org/10.1038/s41598-021-84761-1; https://doi.org/10.3390/nu15133029). The DSS used in our study was obtained from MP Biomedicals and is a standardized “Colitis grade” formulation, specifically developed for reliable induction of ulcerative colitis in preclinical models. As specified by the manufacturer, the DSS had a molecular weight range of 36,000–50,000 g/mol. This information is clearly indicated in the Materials and Methods section.

 

Comment 4: Many of the figures were difficult to interpret. 
Response: Thank you for pointing this out. We fully agree that simplifying the presentation and eliminating overlapping formats would enhance interpretability for readers. According to both reviewers’ recommendations, we have removed Figures 3-5 and now present a single, consolidated heatmap (formerly Figure 6, now Figure 3) alongside Figure 2 for the final submission. We thank you for highlighting this issue and believe these changes result in a clearer, more focused presentation of our findings.

 

Comment 5: Why was there no statistical evaluation.
Response: Thank you for raising this point. Due to our use of pooled serum samples, statistical evaluation was not technically feasible: within each group, only a single pooled sample was analyzed, meaning that the duplicate signals for each protein on the array originate from the same pooled specimen. Standard statistical tests require independent biological replicates; performing statistics on two technical signals per protein would not provide meaningful or interpretable results. This limitation is inherent to the semi-quantitative approach of membrane-based proteome profiling, and aligns with published methodologies for high-throughput cytokine and protein arrays using pooled samples. In many peer-reviewed studies employing this design, statistical analysis is omitted, as biological averaging and population-level trends are the main focus:
1. Li J, Lin B, Li X, Tang X, He Z, Zhou K. Biomarkers for predicting response to tyrosine kinase inhibitors in drug-sensitive and drug-resistant human bladder cancer cells. Oncol Rep. 2015 Feb;33(2):951-7. doi: 10.3892/or.2014.3639; https://www.spandidos-publications.com/10.3892/or.2014.3639

2. Berestjuk I, Lecacheur M, Carminati A, Diazzi S, Rovera C, Prod'homme V, Ohanna M, Popovic A, Mallavialle A, Larbret F, Pisano S, Audebert S, Passeron T, Gaggioli C, Girard CA, Deckert M, Tartare-Deckert S. Targeting Discoidin Domain Receptors DDR1 and DDR2 overcomes matrix-mediated tumor cell adaptation and tolerance to BRAF-targeted therapy in melanoma. EMBO Mol Med. 2022 Feb 7;14(2):e11814. doi: 10.15252/emmm.201911814
   https://pmc.ncbi.nlm.nih.gov/articles/PMC8819497/

For further clarification, please refer to the data analysis guidelines provided by the array manufacturer: https://www.rndsystems.com/products/proteome-profiler-mouse-xl-cytokine-array_ary028.

 

Comment 6: I could not access your paper showing eff ectiveness. Could you at least give some data from this study?
Response: Thank you for your comment. To facilitate access, we have provided a direct link to our published paper demonstrating the effectiveness of Epilobium angustifolium extract in DSS-induced colitis: https://doi.org/10.3390/cimb47060444. Key findings from this study show that EAE significantly reduced colonic inflammation, improved antioxidant defenses, and attenuated histopathological injury in the DSS model, demonstrating efficacy comparable to dexamethasone

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I have confirmed that the author has proactively addressed all points raised and made revisions to the paper. The revised paper is acceptable for publication.

Reviewer 2 Report

Comments and Suggestions for Authors

Why were figures showing effects o markers removed?