Nicotinamide Supplementation Mitigates Oxidative Injury of Bovine Intestinal Epithelial Cells through Autophagy Modulation
and Xiaoshi Wei 1,2,*Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
Well done on a nice manuscript. It is good to see work supporting the positive effects of Niacinamide.
Title Suggestion; Nicotinamide supplementation alleviates oxidative injury in bovine intestinal epithelial cells and the autophagic process plays a crucial role in the alleviation.
L12 undegradable? while -> and comma after damage
L18 The small
L20 delete the
L21 while -> but
L23 H2O2
L25 delete comma
L26 H2O2
L28 And -> Additionally, delete the first the
L34 Injury
L38 ruminants
L39 it also
L40 barriers,
L42 delete target
L43 the weaning
L44 the small
L48 Previously, we
L51 are few -> is little
L62 maintain
L65 injury
L72 delete And
L89 delete was
L92 replace 、with ,
L94 injury
L97 delete the to-> for
L101 delete the
L106 protocol of the manufacturer -> manufacturer’s protocol.
L109 , culturing for -> and incubated for hole -> well
L114 the Nanjing (and same for all other companies unless in brackets)
L121 a Total
L123 by -> with a or using a
L129 Amplification -> Denaturation
L130 cycles of amplification at
L144 electrophoretically
L146 to the laboratory formulas. -> standard methods
L147 purchased from
L149 was rocking or agitation used? (and for other incubations
L148 what was the concentration of BSA and what was the dilution buffer?
L161 by the -> with a
L165 potentials
L166 instructions of the commercial kits - kit instructions.
L173 a fluorescence microscope (manufacturer)
L178 then washed
L182 microscopy
L184 can be mediated
L186 delete the
L187 an autophagy
L194 differences
L200 delete first The
L201 The H2O2 treatment decreased the cell viability of BIECs, and NAM at 2 mM increased it (Figure
L202 P < 0.05) but not at 4 and 8 mM.
L203 H2O2, and decreased with NAM (1, 2, 4, and 8 mM) (Figure
L204 when challenged
L205 delete first the T-AOC (Figure 1C).
L232 expression
L240 delete the to -> as
L241 protein expressions or expression (optional)
L243 protein expressions or expression (optional)
L248 H202
L249 H202
L250 H202 H202
L253 Inconsistency throughout between spacings in (P≤0.05) and (P < 0.05)
L256 < 0.05), and decreased with NAM at 4 mM the total 256 autophagy (P < 0.05)
L262 to simcompared to a similar level as CON
L301 per -> post
L348 produce -> exhibit
Figure appears a bit fuzzy and can be sharpened up using methods in the attachment.
Comments for author File: 
Comments.pdf
Comments on the Quality of English Language
There are a lot of editorial corrections suggested in the non-discussion parts of the manuscript and a way of improving the sharpness of the figures is offered.
Author Response
Please see the attachment.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments and Suggestions for Authors
Comments and Suggestions for Authors
After reviewing the manuscript entitled “The autophagy plays vital roles in alleviating oxidative injure of bovine intestinal epithelial cells by nicotinamide supplementation”, the following suggestions were made it. The manuscript is very interesting and provides novel information on the effects of nicotinamide on autophagy and oxidative damage of intestinal epithelial cells. The study uses scientifically valid methods; however, several details must be corrected before the manuscript can be considered for publication in this prestigious journal. Specific comments are listed below.
Simple Summary
Line 13: Please specify what type of disorders.
Line 13, 14, and 15: Abbreviations should not be used in the simple summary. Please replace the abbreviations “NAM” with the full name.
Abstract
Line 20: Please specify what type of symptoms and nutritional consequences.
Line 20: Delete “Our”.
Line 23: Please change “(H2O2)” to “(H2O2)”.
Line 24: Delete “The results of this study”. This phrase is obvious and should be avoided in the abstract.
Lines 24-30: The results shown in the abstract should be rewritten since they are very ambiguous in their current form. In these lines, the response variables (or at least the most important ones) that increased or decreased should be specified. Likewise, the significance level at which the changes were detected or at which no changes were detected must be specified.
Keywords: Oxidative injure; bovine intestinal epithelial cell; nicotinamide; autophagy. These words used as keywords are the same as those previously used in the title of the manuscript. Keywords should be different from those in the title (but related to the topic) to broaden the reach of academic search engines in case the manuscript is later published.
Introduction
Lines 41-43: Please add at least one sentence explaining how oxidative stress affects the migration and proliferation of continuously self-renewing epithelial cells.
Line 45: The authors should mention the main strategies that have been used to mitigate oxidative stress and prevent its effects on intestinal damage.
Line 49: The abbreviation “ROS” has not been previously specified.
Line 64: Please delete “[1,4,15]”.
Material and methods
Lines 75-86: Authors must justify why they used these cultivation methods, and this justification must be supported by relevant scientific references.
Line 99: The authors should clearly indicate which methods were used to measure changes in gene expression since several methods are currently available. After specifying this information, it must also be justified with relevant references explaining why those methods were used.
Lines 175-182: Authors must justify why they used these immunofluorescence methods, and this justification must be supported by relevant scientific references.
Lines 192-197: The authors must indicate which statistical tests they used to evaluate the data's normality and homogeneity of variance. This is very important to know if the correct tests were used. Also, could you explain why you used Duncan's test to compare the differences between treatments?
Results
Line 199: Please do not use abbreviations in the subheadings of any section.
Lines 201-288: The entire wording of the results section needs to be improved. The significance value obtained must be specified immediately after mentioning the direction of the effect. For example, the sentence “The H2O2 treatment decreased the cell viability, and NAM at 2 mM increased that of 201 BIECs (Figure 1B, P < 0.05)” should be changed to “The H2O2 treatment decreased (p < 0.05) the cell viability, and NAM at 2 mM increased (p < 0.05) that of 201 BIECs (Figure 1B)”.
Lines 201-288: The authors must modify the format used to indicate significance levels throughout the results section. The instructions for authors indicate that the p should be lowercase and italics. However, the authors used capital P.
Figure 1 A: Please change “Cell viability(%)” to “Cell viability (%)”.
Lines 248-250: Please change “H2O2” to “H2O2”.
Lines 296-299: The paragraph on lines 296-299 should be changed immediately after paragraph 284-288.
Discussion
Lines 307-309: These lines must be removed. In the discussion section, the results found should be explained and contrasted with those obtained from similar studies. However, lines 307-309 only mention what was evaluated in the present study. That information was previously added to the end of the introduction section.
Line 318: Please delete “And”.
Lines 318-321: This sentence needs to be clarified; the authors should modify it and clearly indicate who found results consistent with those obtained in the present study. Likewise, the reference should be added immediately after mentioning the study with which the results are being contrasted and should not be placed until the end of the sentence (as it currently stands).
Line 322: It should be specified in what type of tissue from dairy cows the authors of the study reviewed and cited found an increase in antioxidant activity. This specification is important since intestinal cells were used in the present study.
Line 336: What antioxidants were used by Li et al.? Were they NAM? Otherwise, the authors should change the reference to another study where the same antioxidant (NAM) as in the current study was used.
Conclusion
Line 403: Please delete “Our data suggested that”.
Comments on the Quality of English Language
The manuscript needs a moderate grammar check as there are several details and some sentences are not clear.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for Authors
General Comment:
The manuscript titled "The Autophagy Plays Vital Roles in Alleviating Oxidative Injury of Bovine Intestinal Epithelial Cells by Nicotinamide Supplementation" submitted to Animals Journal explores the impact of Nicotinamide on oxidative stress induced by H2O2 in intestinal epithelial cells. I have several concerns regarding the manuscript which need to be addressed before I can recommend this article for publication.
Specific Comments:
1. The clarity of language in the manuscript is inadequate, which significantly hinders comprehension. The title itself lacks clarity and impact, suggesting the need for a thorough language review throughout the manuscript.
2. Correct the statement “For ruminant, the small intestine is an important site for the digestion and absorption of non-degradable starches and proteins from the rumen [1], also plays a vital role in physical and biochemical barrier and immune homeostasis [2,3].
3. Please revise the sentence for clarity and grammatical accuracy: "For ruminants, the small intestine is an important site for the digestion and absorption of non-degradable starches and proteins from the rumen [1], and it also plays a vital role in physical and biochemical barrier functions and immune homeostasis [2,3]."
4. Revised the sentence for clarity “Line 69-70, cow small intestinal epithelial cells (taken from jejunal segments, Bovine intestinal epithelial cells, BIECs),
5. Could you provide details on how the bovine intestinal epithelial cells (BIECs) were stored and transported?
6. The exclusion of Nrf2 and MDA as markers in the study is not justified. Please explain why these were not considered.
7. On line 186, a reference to previous work is mentioned but not cited. Please provide the appropriate citation.
8. Replace "oxidative injure" with "oxidative injury" throughout the manuscript
9. The manuscript focuses exclusively on oxidative stress. Please explain the rationale behind not assessing inflammatory responses alongside oxidative stress.
10. The statement on the effects of Nicotinamide is unclear. Do you mean to describe "Effect of Nicotinamide on H2O2-mediated oxidative stress in BIECs?"
11. Discussion and Conclusion: The conclusion and discussion sections require a comprehensive re-drafting to better reflect the findings and logically conclude the study.
12. The manuscript must discuss the limitations of the study explicitly in the conclusion section.
Comments on the Quality of English Language
Extensive editing of English language required
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for Authors
The authors have responded satisfactorily to each of my suggestions. Therefore, I recommend acceptance of the manuscript.
Comments on the Quality of English Language
The quality of English is good
Author Response
Dear reviewer,
Thanks for your suggestion. We have revised the English language throughout the manuscript. Hoping it is better now.
Reviewer 3 Report
Comments and Suggestions for Authors
The title, abstract, discussion, and conclusion sections are notably weak particularly from a scientific language perspective, and still require significant revision to clearly convey the implications and the scope of the research.
Comments on the Quality of English Language
Extensive editing of English language required
Author Response
Dear reviewer,
Thanks for your suggestion. We have revised the English language throughout the manuscript. Hoping it could be better and get your approval.
Round 3
Reviewer 3 Report
Comments and Suggestions for Authors
Title is not ok.
Suggested title: Nicotinamide Supplementation Mitigates Oxidative Injury of Bovine Intestinal Epithelial Cells through Autophagy Modulation
The last three lines of summary (16-19) and abstract (31-34) are duplicated.
Discussion and conclusion still need revision
Line 390-391 replace the sentence with "In addition, the NAM supplementation modulated the autophagy pathway, which played a key role in the mitigation of oxidative injury of BIECs".
Comments on the Quality of English Language
Moderate editing of English language required
Author Response
Dear reviewer,
Thanks for your suggestion. We have revised the manuscript as you suggested. Please see the revised vision. Hoping it could get your approval.
