Zearalenone-Induced Mechanical Damage of Intestinal Barrier via the RhoA/ROCK Signaling Pathway in IPEC-J2 Cells
Round 1
Reviewer 1 Report
Major
This manuscript entitled “Zearalenone induced mechanical damage of intestinal barrier via RhoA/ROCK Signaling Pathway in IPEC-J2 Cells” demonstrate the zeralenone-induced damage of the intestinal barrier via RhoA/ROCK signaling pathway by utilizing the monolayer culture of procine epithelial cell line, IPEC-J2. Results and conclusion of this study is suitable for publishing on the IJMS after the revisions according to follows.
page 4, Figure 4
This figure seems to have shown that the TEER of the 8 day-cultured cell layer decreases by ZEN, but not of the 6 day-cultured cell layer. However, there is no description of that in the text of Results. Moreover, the author should discuss the reason of different results between 6-day and 8-day culture monolayers.
page 14, line14
LSD test as post-hoc test after ANOVA is inadequate in more than four groups, because multiplicity issue is not considered in LSD. As multiple comparisons, Tukey-Kramer, Dunnett, or Williams method should be used according to the purpose.
Minor
page 2, line 32
“signalling” to “signaling”
page 3, line 8
Abbreviation CCK-8 is the first appearance here, so that the long form should be described here.
page 3, line 11
“Cell” to “cell”
page 3, line 17 “The functional integrity … activity values.”,
The author should refer the papers indicating the grounds. Reference no. 27 referred by page 16, line 24 “AP is found in many tissues and cells … membrane integrity [27].” should be referred here.
page 4, line 1 Figure 3
Is not common title of Figure necessary before (A) and (B)?
page 4, line 13 and/or page 6, line 1 (Figure 5 legend)
Please describe how long cotreatment with ZEN and Y-27632.
page 4, line 13
Please describe the explanation for Y-27632 here like “an ATP-competitive inhibitor of ROCK, Y-27632”, because Y-27632 is the first appearance.
page 5, line 5
“binded” to “bound”
page 5, line 11
“unthickened” to “unthicken”
page 5, line 11
“amount” is “number”, right?
page 5, line 14
“shown” is better than “depicted”, I guess.
page 6, line 11
“protein” to “mRNA”
page 8, line 9
“treament” to “treatment”
page 10, line 24 “AP is found in many tissues and cells … membrane integrity [27].”
This sentence should place page 3, line 17 as described above comment.
page 10, line 34
What is “DON”?
page 10, line 39
“ocludin” to “occluding”
page 10, line 39, 45
“Claudin” to “claudin”
page 10, line 45
“Occludin” to “occluding”
page 11, line 6
“Disruption” to “disruption”
page 11, line 11
“As shown, ZEN …” to “As shown in Fig 6B, Fig 9C and Fig 9E, ZEN …”
The end of the sentence, (Fig 6B, Fig 9C and Fig 9E) will be deleted.
page 13, line 5 “After fixed”
Method of fixation should be described.
page 14, line 19
“remodelde” to “remodeled”
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
IN the introduction, a strange sentence is used: "The purpose of this paper is to elucidate the mechanical damage caused by ZEN to the intestine and its related mechanism". How can a drug induce "mechanical damage", remains to be explained. I mean, compounds can have an impact on cell morphology and function, structural proteins, and biophysical properties - but it is all via regulation of biochemical and/or transcriptional changes. That should be in the focus (and it is not always).
What bothers me more, however, are the dramatic concentrations of ZEN compound used in this study - up to 180 µM. Effects, for example on TEER, become visible only at 120 µM, still a very high concentration. How is this high dose physiologically relevant for human nutrition and toxicology? What are the effective concentrations achieved from the uptake of ZEN by food or drink? That should be clarified, and placed in relation to the data shown.
The same concentrations also feature in other assays, shown in Fig. 2 and 3. and 4. Fig. 4 doesn't show the ZEN concentration, I assume its 160 µM as indicated in the text (please add). 160 µM are then used throughout the text, is there a reason why? Does this have anything to do with concentrations observed in nature?
Concerning Fig. 6: its not unclear why the authors use an almost confluent cell culture here, instead of showing just a few cells, with clear staining. The quality of the images (resolution, signal/noise ratio) isn't impressive either.
Similar quality issues apply also for Fig. 8, which lacks focus and is rather blurry; higher magnification (and better quality) would be really beneficial. Try again! (Nevertheless, the morphological differences are obvious). Howeve, I am surproidsed to see ZO-1 expression at all. IN our own experience, Z=-1 and other cell adhesion proteins only become critical and highly (and functionally) expressed in 3D cultures; when cells are embedded in a matrix. Maybe comment...
Otherwise, the discussion doesn't add much to the picture. It summarizes more or less the downstream signaling initiated by or with ROCK kinases (and Y-inhibitors), a field very well investigated and understood. But if its really the main downstream mechanisms and pathways affected by the compound ZEN, that currently remains unclear (due to the dosage issues).
In conclusion, while the paper is reasonably written, the experiments (and controls) all appear relevant, I simply question the relevance of the findings. Please clarify how you justify using such high concentrations of a compound that is trace contamination in foods. What's a realistic concentration in food, and what's the likely uptake? Apparently, the compound is almost insoluble in water (and in cells, probably), how do you even get these concentrations in experiments without precipitation? Are there any papers you could refer to that justify this experimental strategy?
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Author Response
please see Attachment
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The authors well revised the manuscript, and it has become suitable for publishing. However, there are minor corrections because of my wrong comments as follows,
Comments 16, 18
I’m sorry, “occluding” was wrong.
“occludin” is right.
