Anti-Inflammatory Properties of Mineral-Balanced Deep Sea Water in In-Vitro and In-Vivo Models of Inflamed Intestinal Epithelium
Round 1
Reviewer 1 Report
This manuscript provided by Jain Nam and collaborators focuses on a study aimed to evaluate the effect of mineral controlled Deep Sea Water on intestinal health in enteritis models.
The Authors in this experimental study suggest that Deep Sea Water-derived mineral water intake can help reduce colitis and that supplementing drinking water with high magnesium and low salt content could have anti-inflammatory useful properties for the treatment of IBD.
The text is interesting and mostly clearly written except for the few minor points listed below:
- The manuscript contains multiple grammatical errors, typos and/or syntactic mistakes that need to be corrected. For example line 400 and TNF- ….add alpha.
- 2.5. Coculture for Inducing Inflammatory Reaction: are there other studies in the literature where this particular experimental model is used? Where did the authors start from? Please add a reference that supports the experimental scheme of in vitro testing.
- Line 399: A reference is needed
- The integration and synthesis in the conclusion section is fairly limited.
- Required English language modifications are not listed but would merit reading by a native English-speaker.
Author Response
Any recommended changes in the manuscript have been made in red font. We hope that the comments have been addressed adequately.
Referee: 1
Minor corrections:
- The manuscript contains multiple grammatical errors, typos and/or syntactic mistakes that need to be corrected. For example line 400 and TNF- ….add alpha.
: Thank you for providing these insights. These errors were revised accordingly.
- 2.5. Coculture for Inducing Inflammatory Reaction: are there other studies in the literature where this particular experimental model is used? Where did the authors start from? Please add a reference that supports the experimental scheme of in vitro testing.
: Thank you for your suggestion. We have clarified method section with a reference.
- Line 399: A reference is needed
: Thank you for bringing this to our attention. The references were revised in line 403.
- The integration and synthesis in the conclusion section is fairly limited.
: We agree that the integration and synthesis in the conclusion section is limited. However, we generated various water solutions to test various mineral compositions based on those present in DSW, we believe that we designed our study to determine if the efficacy of DSW is derived from the main minerals, calcium and magnesium, or whether there are other mineral effects.
- Required English language modifications are not listed but would merit reading by a native English-speaker.
Thank you for bringing this to our attention. The grammatical errors were corrected.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
In the manuscript entitled "Anti-inflammatory Properties of Mineral-balanced Deep Sea Water in In-vitro and In-vivo Models of Inflamed Intestinal Epithelium" by J. Nam et al., the authors present anti-inflammatory study of DSW vary depending on the proportion and amounts of magnesium and calcium, the main minerals in DSW. Especially, in an in vivo model, this DSW sample improved the pathological status in colons of BALB/c mice with DSS-induced colitis. The underlying mechanism was found to involve a reduction in the expression of pro-inflammatory genes, such as TNF-α and NOS2. Notwithstanding, I believe this work presents the overall quality and merit to be published in Applied Sciences in its present form.
Author Response
We appreciate the time and effort you have dedicated to reviewing our paper.
Reviewer 3 Report
Jain Nam et al. investigate the effects of DSW which has been altered to contain different ions. The different solutions are tested in vitro on such parameters as transepithelial electrical resistance, permeability and the gene expression of tight junction-related proteins; the solutions were tested as therapies for the DSS colitis model. The written expression of the manuscript is good. The bioinformatic approach is well explained; many papers gloss over this process.
General points
Generally speaking, “natural remedy" studies such as these are difficult to publish as the specific active component is not identified and the data tends to be observational. The study is not helped by the very modest effect of the treatments in DSS which would normally lead to a decision not to perform sequencing. The authors state in the abstract that HMLS does have a therapeutic effect, but the data really do not support this. Nevertheless, there are potential differences in the RNA sequencing data which may indicate changes to the pathophysiology. Looking across all the figures, the axis labels and figure legends are often too small to easily read. Duncan’s multiple range test is used throughout - this is regarded as less strict as tests such as ANOVA. It is not clear what the letters above bar graphs indicate in terms of statisitical differences - are they different if they share or don’t share a letter?
Specific points
Ln 174 What was the volume of the gavage of FD-4 given?
Ln194 What was the statistical test for the FDR?
Section 3.3 at Ln 244 Please explain and expand upon the reasons why these genes were chosen for measurement.
Figure 3 The methods sections says data are mean+/-SEM but there are no error bars on Figure 3a or 3b. It is not possible to tell if they are different or not without the variation. It may be helpful to calculate the area under the curve for each individual and plot that alongside the clinical data.
Ln 288 Figure 4 Either perform blinded histological severity scoring on the slides or define them as “representative samples”.
Figure 6 It is not clear how many colons from each group were analysed in this section (it is in the supplementary material, but could be mentioned here).
Figure 7 (a) and (b) are not defined in the figure legend.
Would the authors suggest treating DSS with laboratory-made combinations of salts to mimic DSW?
Author Response
Authors’ Responses to the Editor and Reviewers:
Any recommended changes in the manuscript have been made in red font. We hope that the comments have been addressed adequately.
Referee 3 :
General points
Generally speaking, “natural remedy" studies such as these are difficult to publish as the specific active component is not identified and the data tends to be observational. The study is not helped by the very modest effect of the treatments in DSS which would normally lead to a decision not to perform sequencing. The authors state in the abstract that HMLS does have a therapeutic effect, but the data really do not support this. Nevertheless, there are potential differences in the RNA sequencing data which may indicate changes to the pathophysiology. Looking across all the figures, the axis labels and figure legends are often too small to easily read. Duncan’s multiple range test is used throughout - this is regarded as less strict as tests such as ANOVA. It is not clear what the letters above bar graphs indicate in terms of statistical differences - are they different if they share or don’t share a letter?
You have raised an important question. We changed the figure alphabet to share same order. The alphabetical order is the order of values from small to large. This has been stated in the legends of all the figures.
Specific points
- Ln 174 What was the volume of the gavage of FD-4 given?
Thank you for bringing this to our attention. According to Animal Care & Use Standard, Oral gavage volume in the mice should be under 10ul/g. So, we followed the method. We have revised the text line 174 to reflect gavage volume.
- Ln194 What was the statistical test for the FDR?
Cuffdiff software is for multiple testing for p-value. These score statistics follow a chi-squared distribution, which is used to derive P values for DE.
- Section 3.3 at Ln 244 Please explain and expand upon the reasons why these genes were chosen for measurement.
Zona occludens-1 (ZO-1) localizes at the cytoplasmic surface of the cell membrane, close to the TJ strands. Occludin is a TJ protein consisting of four transmembrane domains altering epithelial permeability. Junctional adhesion molecules (JAMs) are contributing to the semi-permeable barrier, whereas cytosolic proteins link membrane components to the actin cytoskeleton. It is known that claudin-1, -3, and -4 are pore-sealing claudins. An increased expression of these proteins leads to a very tight epithelia, decreased solute permeability (mainly sodium ions) across the epithelial monolayer. To figure out permeability improvement of DSW derived mineral water function, we choosed those genes. We have rewritten in line 417-424 to be more in line with your comments.
- Figure 3 The methods sections says data are mean+/-SEM but there are no error bars on Figure 3a or 3b. It is not possible to tell if they are different or not without the variation. It may be helpful to calculate the area under the curve for each individual and plot that alongside the clinical data.
Thank you for raising this point. The Figure 3 was revised appropriately.
- Ln 288 Figure 4 Either perform blinded histological severity scoring on the slides or define them as “representative samples”.
Thank you for providing these insights. The Figure description was revised appropriately.
- Figure 6 It is not clear how many colons from each group were analysed in this section (it is in the supplementary material, but could be mentioned here).
Thank you for your suggestion. We have revised the figure 6 description to reflect the number of mice in this section.
- Figure 7 (a) and (b) are not defined in the figure legend.
Thank you for providing these insights. We defined in these figure legends.
- Would the authors suggest treating DSS with laboratory-made combinations of salts to mimic DSW?
Thank you for your suggestion, you have raised an important point; however, we believe that the data pertaining to the ratio of calcium to magnesium does not fit the purpose of this paper as this study is not focused solely on the ratio of calcium and magnesium. Rather, we generated various water solutions to test various mineral compositions based on those present in DSW. Although the health benefits of DSW are well known, many attempts have been made to determine its efficacy by focusing only on hardness. We, therefore, designed our study to determine if the efficacy of DSW is derived from the main minerals, calcium and magnesium, or whether there are other mineral effects.
Author Response File: Author Response.pdf
