Vitamin D in Irritable Bowel Syndrome: Exploring Its Role in Symptom Relief and Pathophysiology

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The review by Aggeletopoulou et al. addresses an interesting topic and is overall well written (apart from a few, somewhat inappropriate, terms reported as minor points below).

My main concern is with Paragraph 4.3. Vitamin D and Its Role in Gut Health and the Gut-Brain Axis in IBS and the accompanying Figure 2. They are based entirely on just two articles: refs. 81 and 82.

In particular, ref. 82 appears to be the only source of information on “vitamin D and the gut-brain axis in IBS”, which is instead a quite general and relevant issue. In my personal opinion, that paper (ref 82) is unclear about the in vitro cell models used to reproduce the gut–brain axes (the authors claim to model both the healthy gut/degenerative brain and the inflammatory bowel syndrome gut/degenerative brain axis) and it over-generalizes results obtained with cell lines. Using this only paper in a review to illustrate the role of vitamin D in the gut-brain axis in IBS leads to an excessive and misleading over-generalizing/over-interpretation of limited data obtained on specific cell lines.

Just an example to make the problem clearer: at lines 427-428, the authors state (citing ref 82) “The formulation (VitD3-NS) was also shown to cross the intestinal barrier and reach the brain, where it activated vitamin D signaling pathways by increasing the expression of the VDR [82]: how the formulation could have been shown to cross the intestinal barrier and reach the brain if the experiments have been performed in immortalized cell lines?

My suggestion is to revise the paragraph clearly describing - if the authors can get the information (I couldn't) -  the in vitro cell models used to modeling the gut–brain axes on which the cited data were obtained. If no other data (in animal model or in patients) are available on “vitamin D and the gut-brain axis in IBS” it should made it clear that, until new data are available – only speculations can be done.  I would also remove the bottom part of the figure, relative to the effect of VitD3-NS.

In summary: the paragraph should be revised either with the support of other data/citations, or by critically discussing the (limited) sources on which it is based and the general lack of data in the literature on the topic.

Other concerns:

• An important (and unresolved question) is whether the vitamin D deficiency observed in IBS patients is cause or consequence of the disease. The enhanced gut permeability/impaired intestinal function in patients suggests that vitamin D deficiency may be a consequence, and the enhanced Vitamin D levels observed after LFD are in line with this assumption. On the other hand, a strong biological plausibility also exists in favor of beneficial effects of vitamin D on the pathophysiology of the diseases and restoring vitamin D sufficiency could contribute in a positive loop to ameliorate IBS symptoms. A more explicit discussion of this controversial point might be useful to the reader.

Also, in line with this reasoning, I would be careful about the terms used when the cause-effect relationship is not demonstrated: e.g., at line 367-368, “while VDR ( or vitamin D?)  deficiency may downregulate TPH1 and disrupt serotonin balance” it would be better to use “is associated with down-modulation... disruption ” instead of may down-modulate ... and disrupt; similarly, in the title of figure 2, substitute “impact of vitamin D” with “relationship” or something similar, or at least “potential, proposed impact”.  

• The role of antimicrobial peptides is not clear: cited in the abstract as potential mediators of vitamin D effects on IBS pathophysiology, they are never discussed in the text. They are generically cited in the introduction of the paragraph Role of Vitamin D on IBS Pathophysiology as mechanisms regulated by vitamin D with potential impact on IBS, but never discussed in detail in the context of IBS.

Minor points:

• Scrolling with the mouse on figure 1 this greek writing appears:

Εικόνα που περιέχει κείμενο, διάγραμμα (according to Google translator: Image containing text, diagram)

Το περιεχόμενο που δημιουργείται από τεχνολογία ΑΙ ενδέχεται να είναι εσφαλμένο (according to Google translator: Content generated by AI technology may be incorrect).

Could the authors explain why the writing appears?

• Somewhat inappropriate terminology: 

Abstract, line 22-23: “The aim of this study is to investigate these mechanisms and assess the therapeutic potential of vitamin D supplementation in IBS patients”. Since this is a review, the terms “investigate” and “assess” do not seem appropriate: I would suggest rephrasing the sentence (e.g. “The aim of this review is to describe current knowledge/evidence on these mechanisms and on the therapeutic potential of vitamin D supplementation in IBS patients” or something similar)

Likewise, at the end of the introduction (line 81 – 84): This review aims to explore the potential role of vitamin D in the pathophysiology and management of IBS by examining .. (...). I would suggest to substitute “to explore” with “to illustrate/describe” or something similar.

Again, in a similar way, in Figure 1 legend (line 375) “the figure suggests” sounds strange: it is not the figure to suggest, but the authors that suggest in the figure.

• line 278-280 “However, while some studies report beneficial effects, others have found no significant impact, highlighting the need for further research to clarify the role of vitamin D in IBS pathophysiology”. This sentence seems out of place at the end of this introductory paragraph
• Ref 57 – Mistakenly cited at line 269? 
• Ref 65 – the name of the journal is missing
• Fig 1 legend:  The figure describes the serotonergic metabolism, not the signaling
• line 367-368 VDR deficiency or Vitamin D deficiency?
• Fig 2 legend: there is inconsistency about the relationship between vitamin D sufficiency/deficiency and ZO-1. Increased ZO-1 is reported in both sufficiency and deficiency.

Author Response

The review by Aggeletopoulou et al. addresses an interesting topic and is overall well written (apart from a few, somewhat inappropriate, terms reported as minor points below). 

 

Response to Reviewer 1:   Thank you very much for your positive feedback on our manuscript.  

 

Comment 1: My main concern is with Paragraph 4.3. Vitamin D and Its Role in Gut Health and the Gut-Brain Axis in IBS and the accompanying Figure 2. They are based entirely on just two articles: refs. 81 and 82. In particular, ref. 82 appears to be the only source of information on “vitamin D and the gut-brain axis in IBS”, which is instead a quite general and relevant issue. In my personal opinion, that paper (ref 82) is unclear about the in vitro cell models used to reproduce the gut–brain axes (the authors claim to model both the healthy gut/degenerative brain and the inflammatory bowel syndrome gut/degenerative brain axis) and it over-generalizes results obtained with cell lines. Using this only paper in a review to illustrate the role of vitamin D in the gut-brain axis in IBS leads to an excessive and misleading over-generalizing/over-interpretation of limited data obtained on specific cell lines.

Just an example to make the problem clearer: at lines 427-428, the authors state (citing ref 82) “The formulation (VitD3-NS) was also shown to cross the intestinal barrier and reach the brain, where it activated vitamin D signaling pathways by increasing the expression of the VDR [82]: how the formulation could have been shown to cross the intestinal barrier and reach the brain if the experiments have been performed in immortalized cell lines?

My suggestion is to revise the paragraph clearly describing - if the authors can get the information (I couldn't) -  the in vitro cell models used to modeling the gut–brain axes on which the cited data were obtained. If no other data (in animal model or in patients) are available on “vitamin D and the gut-brain axis in IBS” it should made it clear that, until new data are available – only speculations can be done.  I would also remove the bottom part of the figure, relative to the effect of VitD3-NS.

In summary: the paragraph should be revised either with the support of other data/citations, or by critically discussing the (limited) sources on which it is based and the general lack of data in the literature on the topic.

 

Response to comment 1: Thank you for your insightful comments regarding the section on vitamin D and the gut-brain axis in IBS. We acknowledge the over-reliance on ref. 82 and agree that its conclusions are based on in vitro models, which may not fully translate to in vivo conditions. To address this, we have revised the paragraph to explicitly clarify the limitations of the study and avoid over-generalization of its findings (pages 10-11, lines 426-446). Additionally, we have removed the bottom part of Figure 2 (Figure 3 in the revised manuscript), as suggested, to ensure the figure accurately reflects the available evidence.

Following your suggestion, we conducted a thorough literature search once again and confirm that there are currently no other published data on this specific topic. We understand that the available data are limited and underpowered; however, the purpose of this review is to present the existing literature and generate hypotheses rather than to critically evaluate individual studies. Nevertheless, we acknowledge that the reviewer’s concerns are valid, and we have adjusted the discussion accordingly to better reflect the strength and limitations of the evidence. Moreover, we have expanded the discussion to highlight the gap in literature and the need for further research in this area (page 11, lines 442-446).

 

Comment 2: An important (and unresolved question) is whether the vitamin D deficiency observed in IBS patients is cause or consequence of the disease. The enhanced gut permeability/impaired intestinal function in patients suggests that vitamin D deficiency may be a consequence, and the enhanced Vitamin D levels observed after LFD are in line with this assumption. On the other hand, a strong biological plausibility also exists in favor of beneficial effects of vitamin D on the pathophysiology of the diseases and restoring vitamin D sufficiency could contribute in a positive loop to ameliorate IBS symptoms. A more explicit discussion of this controversial point might be useful to the reader.

 

Response to comment 2: We thank the reviewer for highlighting the important question of whether vitamin D deficiency in IBS is a cause or consequence of the disease. In response, we have expanded the discussion in the conclusion section to address this issue and to provide a more balanced perspective emphasizing the need for future studies to explore whether vitamin D deficiency is a cause, consequence, or both in IBS (page 12, lines 492-506). 

 

Comment 3: Also, in line with this reasoning, I would be careful about the terms used when the cause-effect relationship is not demonstrated: e.g., at line 367-368, “while VDR ( or vitamin D?)  deficiency may downregulate TPH1 and disrupt serotonin balance” it would be better to use “is associated with down-modulation... disruption ” instead of may down-modulate ... and disrupt; similarly, in the title of figure 2, substitute “impact of vitamin D” with “relationship” or something similar, or at least “potential, proposed impact”. 

 

Response to comment 3: We have revised as suggested (page 10, lines 383-385) and page 11, line 453)

 

Comment 4: The role of antimicrobial peptides is not clear: cited in the abstract as potential mediators of vitamin D effects on IBS pathophysiology, they are never discussed in the text. They are generically cited in the introduction of the paragraph Role of Vitamin D on IBS Pathophysiology as mechanisms regulated by vitamin D with potential impact on IBS, but never discussed in detail in the context of IBS.

 

Response to comment 4: We acknowledge that the role of antimicrobial peptides was mentioned in the abstract but not sufficiently discussed in the main text. In the revised manuscript, we have expanded on the role of AMPs in IBS, particularly their regulation by vitamin D (page 7, lines 291-297). AMPs, including cathelicidins and defensins, play a key role in maintaining gut health by regulating the microbiota, preserving intestinal barrier integrity, and modulating immune responses. In IBS, impaired gut barrier function and dysbiosis contribute to inflammation, and AMPs help control microbial balance and reduce pathogenic overgrowth. Vitamin D, through its activation of the vitamin D receptor (VDR), enhances AMP production, potentially improving gut health. By restoring intestinal barrier function, reducing inflammation, and rebalancing microbiota, vitamin D may help alleviate IBS symptoms. 

 

Minor points:

Comment 1: Scrolling with the mouse on figure 1 this greek writing appears:

Εικόνα που περιέχει κείμενο, διάγραμμα (according to Google translator: Image containing text, diagram)

Το περιεχόμενο που δημιουργείται από τεχνολογία ΑΙ ενδέχεται να είναι εσφαλμένο (according to Google translator: Content generated by AI technology may be incorrect).

Could the authors explain why the writing appears?

 

Response to comment 1: We do not observe the issue mentioned. We believe the issue may be due to compatibility differences between software versions. The text you mentioned does not appear on our end and is not part of the figure we submitted. We trust that this will be addressed during the proofreading stage and will not appear in the final version. 

 

Comment 2: Somewhat inappropriate terminology: 

Abstract, line 22-23: “The aim of this study is to investigate these mechanisms and assess the therapeutic potential of vitamin D supplementation in IBS patients”. Since this is a review, the terms “investigate” and “assess” do not seem appropriate: I would suggest rephrasing the sentence (e.g. “The aim of this review is to describe current knowledge/evidence on these mechanisms and on the therapeutic potential of vitamin D supplementation in IBS patients” or something similar)

Response to comment 2: We have revised as suggested (page 1, lines 21-23).   

 

Comment 3: Likewise, at the end of the introduction (line 81 – 84): This review aims to explore the potential role of vitamin D in the pathophysiology and management of IBS by examining .. (...). I would suggest to substitute “to explore” with “to illustrate/describe” or something similar.

Response to comment 4: We have revised as suggested (page 3, line 86).  

 

Comment 4: Again, in a similar way, in Figure 1 legend (line 375) “the figure suggests” sounds strange: it is not the figure to suggest, but the authors that suggest in the figure.

Response to comment 4: We have revised as suggested (page 10, line 391).       

 

Comment 5: line 278-280 “However, while some studies report beneficial effects, others have found no significant impact, highlighting the need for further research to clarify the role of vitamin D in IBS pathophysiology”. This sentence seems out of place at the end of this introductory paragraph.

Response to comment 5: We have removed this sentence.     

 

Comment 6: Ref 57 – Mistakenly cited at line 269? 

Response to comment 6: Indeed, this reference is completely irrelevant and was placed there by mistake. Thank you very much for pointing it out.      

 

Comment 7: Ref 65 – the name of the journal is missing

Response to comment 7: We have revised. Thank you for pointing it out. 

 

Comment 8: Fig 1 legend:  The figure describes the serotonergic metabolism, not the signaling

Response to comment 8: We have revised as suggested, (page 9, line 372).      

 

Comment 9: line 367-368 VDR deficiency or Vitamin D deficiency?

Response to comment 9: Vitamin D deficiency is correct. We have revised as suggested, (page 10, line 384).        

 

Comment 10: Fig 2 legend: there is inconsistency about the relationship between vitamin D sufficiency/deficiency and ZO-1. Increased ZO-1 is reported in both sufficiency and deficiency.

Response to comment 10: In fact, ZO-1 levels are elevated in the presence of sufficient vitamin D, contributing to improved tight junction (TJ) integrity. However, during vitamin D deficiency, ZO-1 expression is reduced, leading to disrupted TJs and increased permeability. We have clarified this point in Figure Legend 2 (page 11, lines 456-457 and 460).

Reviewer 2 Report

Comments and Suggestions for Authors

Generally, this is a manuscript of good quality, reviewing the published data about possible mechanisms of vitamin D deficiency linked to IBS, as well as those of vitamin D supplementation. I like the way the Authors structured their whole paper, as well as the figures. The in-depth analysis of the existing meta-analyses, regarding the effects of vitamin D supplementation on quality of life and severity of symptoms in adult patients with IBS adds more value to the manuscript. References are generally good. I read attentively the iThenticate report and it looks fine. I have only some minor comments:

1. Title: Even though “Shedding Light on IBS:” sounds great, there are still many inconclusive and contradictory data. Maybe the Authors would agree to nuance the title.
2. In paragraph 3, Table 1 title refers to: “Summary of Meta-Analyses Assessing the Effects of Vitamin D Supplementation on IBS Symptom Severity and Quality of Life.”. I suggest the following text starting with effects on symptom severity and then on the quality of life, and not vice versa.
3. Effect of Vitamin D Supplementation on Quality of Life (QoL) – reference [54] is written in the line 166, while reference [53] in the line 185. Please correct and re-order.
4. Please delete redundant info throughout the whole manuscript.

Thank you

Author Response

Reviewer 2

Generally, this is a manuscript of good quality, reviewing the published data about possible mechanisms of vitamin D deficiency linked to IBS, as well as those of vitamin D supplementation. I like the way the Authors structured their whole paper, as well as the figures. The in-depth analysis of the existing meta-analyses, regarding the effects of vitamin D supplementation on quality of life and severity of symptoms in adult patients with IBS adds more value to the manuscript. References are generally good. I read attentively the iThenticate report and it looks fine. I have only some minor comments:

Response to Reviewer 2: Thank you very much for your positive feedback and for taking the time to thoroughly review the manuscript. Your comments are much appreciated, and we will address the minor points you've raised to further improve the manuscript.

 

Comment 1: Title: Even though “Shedding Light on IBS:” sounds great, there are still many inconclusive and contradictory data. Maybe the Authors would agree to nuance the title.

Response to comment 1:  We have revised to “Vitamin D in Irritable Bowel Syndrome: Exploring Its Role in Symptom Relief and Pathophysiology”. We hope this change meets your approval.

 

Comment 2: In paragraph 3, Table 1 title refers to: “Summary of Meta-Analyses Assessing the Effects of Vitamin D Supplementation on IBS Symptom Severity and Quality of Life.”. I suggest the following text starting with effects on symptom severity and then on the quality of life, and not vice versa.

Response to comment 2: We have re-arranged the text as suggested by the reviewer.

 

Comment 3: Effect of Vitamin D Supplementation on Quality of Life (QoL) – reference [54] is written in the line 166, while reference [53] in the line 185. Please correct and re-order.

Response to comment 3: This is because references 53 and 54 have already appeared in Table 1.

 

Comment 4: Please delete redundant info throughout the whole manuscript.

Response to comment 4: We have gone through the manuscript and removed any redundant information to make it more concise and clear.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for sending me a review of this publication.
In my opinion, the publication concerns an important problem, a significant medical issue (IBS) affecting an increasing part of the population.

In my opinion, the article is written correctly, but it does not bring new content beyond the known state of scientific knowledge. The authors have previously published important information on this topic, and they self-quote each other intensively, which is additionally emphasized by the high similarity values ​​(Percent match: 36%). As the authors themselves note very valuable in the Conclusion section, clinical trials on the subject of vitamin D and IBS are needed, not subsequent literature reviews: citation: "This variability underscores the need for further high-quality, randomized controlled trials to clarify the therapeutic potential of vitamin D in IBS, determine optimal dosing strategies, and identify specific patient subgroups that may benefit the most."

Kind regards,

reviewer

Author Response

Reviewer 3

Dear Authors,

Thank you for sending me a review of this publication. In my opinion, the publication concerns an important problem, a significant medical issue (IBS) affecting an increasing part of the population.

In my opinion, the article is written correctly, but it does not bring new content beyond the known state of scientific knowledge. The authors have previously published important information on this topic, and they self-quote each other intensively, which is additionally emphasized by the high similarity values ​​(Percent match: 36%). As the authors themselves note very valuable in the Conclusion section, clinical trials on the subject of vitamin D and IBS are needed, not subsequent literature reviews: citation: "This variability underscores the need for further high-quality, randomized controlled trials to clarify the therapeutic potential of vitamin D in IBS, determine optimal dosing strategies, and identify specific patient subgroups that may benefit the most."

 

Response to Reviewer 3  

Thank you for your thoughtful comments and for recognizing the significance of IBS as a growing medical issue. We acknowledge that there is already substantial evidence on this topic. However, to the best of our knowledge, there is no existing review that comprehensively summarizes the findings of meta-analyses while also providing a clear discussion of the pathophysiological mechanisms linking vitamin D to IBS. Additionally, we believe that Figures 2 and 3 (in the revised manuscript) add value by presenting a concise and visual overview of the existing literature. For these reasons, we are confident that our work contributes meaningfully to the current body of knowledge.

Reviewer 4 Report

Comments and Suggestions for Authors

Thank you for the review opportunity.

Here are my questions and suggestions if the authors choose to adhere to them:

Title: sounds a little too ambitious for my taste, i would reconsider, add the type of study conducted and so on, also, never use abbreviations such as "IBS" in the title.

Keywords: the role of the keywords is to enhance the search feature in order for more people to find your article and therefore, should not contain the same words as in the title, please reconsider.

Introduction: please add a section on the Vitamin D deficiency in adults in the worlds, it will enhance the need for studies in particular diseases

Please add a figure for the diagnosis criteria of IBS, since it's a critical part of this review, an "at-a-glance" figure would help alot in my pov.

Table 1: i would add the doses of vitamin D received and the assays used to determine Vitamin D, please keep in mind the assays used especially when conducting reviews:

Fraser WD, Milan AM. Vitamin D assays: past and present debates, difficulties, and developments. Calcif Tissue Int. 2013 Feb;92(2):118-27. doi: 10.1007/s00223-012-9693-3. Epub 2013 Jan 13. PMID: 23314742.

Figure 1 & 2. I really like them! but please specify the program used to create it, i'm guessing Biorender.

Please add the limitations of the review as well.

I would also take this opportunity to provide some suggestions for adequate vitamin D dosing and testing in IBS patients, this should been a separate section in your review.

References are ok, up to date.

 

Author Response

Reviewer 4

Thank you for the review opportunity. Here are my questions and suggestions if the authors choose to adhere to them.

 

Response to Reviewer 4: Thank you very much for your feedback and for taking the time to thoroughly review the manuscript.

 

Comment 1: Title: sounds a little too ambitious for my taste, i would reconsider, add the type of study conducted and so on, also, never use abbreviations such as "IBS" in the title.

 

Response to comment 1: We have revised to “Vitamin D in Irritable Bowel Syndrome: Exploring Its Role in Symptom Relief and Pathophysiology”. We hope this change meets your approval.

 

Comment 2: Keywords: the role of the keywords is to enhance the search feature in order for more people to find your article and therefore, should not contain the same words as in the title, please reconsider.

Response to comment 2: We respectfully disagree with this comment. Keywords are intended to enhance the discoverability of the article, and it is a common practice to include essential terms from the title to ensure that the study is easily found in relevant searches. The selected keywords (irritable bowel syndrome, IBS, vitamin D, gut microbiome, symptom severity, quality of life, pathophysiology) accurately represent the core aspects of our work and will help researchers locate our study more effectively. Therefore, we believe that our chosen keywords are appropriate and do not require modification.

 

Comment 3: Introduction: please add a section on the Vitamin D deficiency in adults in the worlds, it will enhance the need for studies in particular diseases.

 

Response to comment 3: We thank the reviewer for this comment. There is already a report in the manuscript saying that vitamin D deficiency represents a very common problem, affecting 30–50% of the global population across various demographic groups, and is linked to numerous chronic conditions (page 3, lines 77-80).      

 

 Comment 4: Please add a figure for the diagnosis criteria of IBS, since it's a critical part of this review, an "at-a-glance" figure would help alot in my pov.

 

Response to comment 4: Thank you for your suggestion. We agree that including a figure outlining the diagnostic criteria for IBS would be a valuable addition to this review. In response to your comment, we have created an "at-a-glance" figure summarizing the key diagnostic criteria for IBS based on the Rome IV criteria (figure 1 in the revises manuscript). We hope this addition enhances the clarity and utility of the review.  

 

Comment 5: Table 1: i would add the doses of vitamin D received and the assays used to determine Vitamin D, please keep in mind the assays used especially when conducting reviews: Fraser WD, Milan AM. Vitamin D assays: past and present debates, difficulties, and developments. Calcif Tissue Int. 2013 Feb;92(2):118-27. doi: 10.1007/s00223-012-9693-3. Epub 2013 Jan 13. PMID: 23314742.

 

Response to comment 5: Thank you for your suggestion. However, we would like to clarify that in Table 1, we have summarized data from meta-analyses rather than individual studies. Therefore, including specific doses of vitamin D and assays used in these meta-analyses is not feasible, as these details are often not consistent across the studies included in the analyses. While we understand the importance of such data, it is not applicable in this context due to the nature of the summarized data.

 

Comment 6: Figure 1 & 2. I really like them! but please specify the program used to create it, i'm guessing Biorender.

 

Response to comment 6: We appreciate your positive comments on Figures 2 and 3 (in the revised manuscript). As noted in the legends, we have already specified that they were created with BioRender.com (accessed on February 13, 2025). We have highlighted this for your attention.

 

Comment 7: Please add the limitations of the review as well.

 

Response to comment 7: In the revised manuscript we have added a section titled “Overcoming Methodological Challenges”, as suggested by the reviewer (page 12, lines 473-484).  

 

Comment 8: I would also take this opportunity to provide some suggestions for adequate vitamin D dosing and testing in IBS patients, this should been a separate section in your review.

 

Response to comment 8: We have provided some information in the revised manuscript (page 6, line 232-237).

 

Comment 9: References are ok, up to date.

 

Response to comment 9: Thank you for your feedback.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I thank the authors for accepting my suggestions

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for your response.

However, I stand by my opinion on the subject of the presented meta-analysis.

Reviewer 4 Report

Comments and Suggestions for Authors

Even if i don't agree with some replies from the authors, they adhered as much as they considered my suggestions fit. I recommend this article to be published.