Transcriptome Analysis Reveals That NEFA and β-Hydroxybutyrate Induce Oxidative Stress and Inflammatory Response in Bovine Mammary Epithelial Cells
, Junpeng Huang 1,†, Xiangxing Chen 2, Yexiao Yan 1, Lian Li 3 and Weiguo Zhao 1,*Round 1
Reviewer 1 Report
The manuscript entitled “Transcriptome analysis reveals that NEFA and β-hydroxybutyrate induce oxidative stress and inflammatory response in Bovine mammary epithelial cells” by Li et al. to evaluate the effect of NEFA and β-hydroxybutyrate on bovine mammary epithelial cells. Authors finds that NEFA and β-hydroxybutyrate can induce oxidative stress and inflammatory response probably via the MAPK signaling pathway in BMECs. The paper should become acceptable for publication pending minor revision.
1. Some spelling, spacing and editing issues need to be addressed. Pay attention to the spaces between the words.
2. The information about BMEC was very poor.
3. whether NEFA and β-hydroxybutyrate treatment will induce cells to produce different concentrations of intracellular NEFA and β-hydroxybutyrate?
4. Please add the evidence for the decision the concentrations and culture time of NEFA and β-hydroxybutyrate
5. Why did you choice GAPDH using single housekeeping gene, not multiple housekeeping genes?
6. Poor information about primary antibodies.
7. The description of result, “the standard errors of the mean (SEM)”, because you already defined SEM in the “Statistical analysis”.
8. The article is interesting, but the issues mentioned need to be adjusted.
Author Response
- Some spelling, spacing and editing issues need to be addressed. Pay attention to the spaces between the words.
Response: Thank you for pointing this out to us, we are awfully sorry for our mistakes, the manuscript has been carefully rechecked.
- The information about BMEC was very poor.
Response: Thank you for reminding us of this and sorry for the confusion caused by my inadequate expression. The BMECs were cell lines (MAC-T), which were a gift from Dr. Youping Sun, we have supplemented the information in our revised manuscript (please see line 71-72).
- whether NEFA and β-hydroxybutyrate treatment will induce cells to produce different concentrations of intracellular NEFA and β-hydroxybutyrate?
Response: Many thanks for this comment. Fatty acids (FAs) are essential constituents of all living cells and have significant roles as components of biomembranes, cell signaling, and energy substrates. Under the circumstance of starvation/fasting, de-esterification of FAs from stored lipids of the adipose tissue takes place by the action of a hormone-sensitive lipase, resulting in the temporary elevation of NEFAs in the circulation for coping with the body’s energy demands[1-3]. Therefore, different concentrations of NEFA will not induce cells to produce different concentrations of intracellular NEFA.
- Please add the evidence for the decision the concentrations and culture time of NEFA and β-hydroxybutyrate.
Response: Thank you for pointing this out to us. We have added supporting references in the Materials and Methods section (please see line 77).
- Why did you choice GAPDH using single housekeeping gene, not multiple housekeeping genes?
Response: Thank you for pointing this out to us. GAPDH was stably expressed in our study and was therefore chosen as a reference used for normalization, and we agree with the reviewer’s comment that multiple housekeeping genes would be better. However, in the present study, only single housekeeping gene was performed for the RT-qPCR analysis.
- Poor information about primary antibodies.
Response: Many thanks for this comment. Since bovine mammary epithelial cells (BMECs) were used as in vitro cell model in this study, and there are very few validated antibodies can be available in the experiment, thus only single antibody blotting was performed for the immunoblot analysis.
- The description of result, “the standard errors of the mean (SEM)”, because you already defined SEM in the “Statistical analysis”.
Response: Thank you very much for your comments and advices. Changes has been made in revised manuscript.
- The article is interesting, but the issues mentioned need to be adjusted.
Response: Thanks for your comments and patience. We have made revisions and modifications based on the comments which were marked in red highlighting.
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- Karpe, F.; Dickmann, J.R.;Frayn, K.N. Fatty acids, obesity, and insulin resistance: time for a reevaluation. Diabetes 2011, 60, 2441-2449.
- Adewuyi, A.A.; Gruys, E.;van Eerdenburg, F.J. Non esterified fatty acids (NEFA) in dairy cattle. A review. Vet Q 2005, 27, 117-126.
Reviewer 2 Report
1. The quality of Figure 1 is poor, please providing high quality figures.
2. In Figure 1G, H and I, the relative mRNA expression levels were analyzed by CON vs NEFA, CON vs BHBA or CON vs NEFA+ BHBA (t-test individually?), while in figure 3, 4 and 5, the data were analyzed by four groups together (one way ANOVA?). However, the experiment as described is a 2x2 design and should be analyzed as a 2-way analysis of variance. Presently all interactions means are being reported, and this would be inappropriate if treatment interactions are not significant, in which case only main effect means should be presented and discussed.
3. In Figure 3F, the detection item (SOD) should be provided and the quantitative result should be analyzed with DAPI or other internal reference. The same as Figure 4G, NO immunofluorescence staining.
4. The abbreviations should be defined, such as MDA and ROS in Abstract and Results.
5. In Figure 5D, the P-P38, P-JNK and P-ERK must be p-p38, p-JNK and p-ERK. Please provide the phosphorylation site.
6. Please provide the product code of the first antibody for western blotting analysis.
Author Response
Dear reviewer,
On behalf of my co-authors, we would like to express our great appreciation to you for the comments on our paper. We have studied comments carefully and done our best to make revision, which we hope meet with approval. Revised portion are marked in red in the paper and our responses to your comments as follows.
Responses
- The quality of Figure 1 is poor, please providing high quality figures.
Response: Many thanks for this comment. We have revised the Figure 1 in our revised manuscript, please check.
- In Figure 1G, H and I, the relative mRNA expression levels were analyzed by CON vs NEFA, CON vs BHBA or CON vs NEFA+ BHBA (t-test individually?), while in figure 3, 4 and 5, the data were analyzed by four groups together (one way ANOVA?). However, the experiment as described is a 2x2 design and should be analyzed as a 2-way analysis of variance. Presently all interactions means are being reported, and this would be inappropriate if treatment interactions are not significant, in which case only main effect means should be presented and discussed.
Response: Thank you very much for the comment. Dairy cows pass through a period of negative energy balance as they transition from late gestation to early lactation. Excessive concentrations of NEFAs or BHBA indicate an excess of NEB which is associated with detrimental health and production outcomes. In our experiment, we focused on exploring the biological roles of NEFA and BHBA on provoking oxidative stress and inflammatory responses in Bovine mammary epithelial cells (BMECs). Therefore, only control and treatment group were analyzed for comparison, the comparison between NEFA, BHBA and their combinative treatment groups was not analyzed. Figure 1G- I was the verification of the EDGs in BMECs treated with NEFA, BHBA and their combination, the EDGs among each group were not the same, thus, relative mRNA expression levels analyzed by CON vs NEFA, CON vs BHBA or CON vs NEFA+ BHBA need to be consistent with figure 1A-F.
- In Figure 3F, the detection item (SOD) should be provided and the quantitative result should be analyzed with DAPI or other internal reference. The same as Figure 4G, NO immunofluorescence staining.
Response: Thank you for pointing this out to us as would greatly improve the quality of this manuscript. Green fluorescence intensity of ROS and NO was quantified by Image J software, and the cell photos under DIC were supplemented in our revised manuscript (please see line208 and 221).
- The abbreviations should be defined, such as MDA and ROS in Abstract and Results.
Response: Many thanks for the comment. Manuscript was revised according to your comment (please see lines 21-23).
- In Figure 5D, the P-P38, P-JNK and P-ERK must be p-p38, p-JNK and p-ERK. Please provide the phosphorylation site.
Response: Thank you for reminding us of this. We have added the phosphorylation site in the Materials and Methods section (please see line 144-146).
- Please provide the product code of the first antibody for western blotting analysis.
Response: Thank you very much for your comments and patience. We have added the product code of the first antibody in the Materials and Methods section (please see line 143-151).
Reviewer 3 Report
This manuscript describes the induction of oxidative stress and inflammatory response by NEFA and BHBA in bovine mammary epithelial cells (BMEC), and I have one comment to the author.
Comment: In the Methods 5.1. Cell Culture and Treatments, ”BMECs were treated with NEFA (0.9 mM, 6h), BHBA (2.4 mM, 24h) and their combination. … The concentration of stock NEFA solution was 50 mM, containing 21.75 mM oleic acid, 15.95 mM palmitic acid, 7.2 mM stearic acid, 2.65 mM palmitoleic acid, and 2.45 mM linoleic acid”. Do these reflect actual circulating concentrations of NEFA and BHBA?
I think it would be better for the author to describe the rationale for the concentration of NEFA and BHBA used in this experiment.
Author Response
Comment: In the Methods 5.1. Cell Culture and Treatments, ” BMECs were treated with NEFA (0.9 mM, 6h), BHBA (2.4 mM, 24h) and their combination. … The concentration of stock NEFA solution was 50 mM, containing 21.75 mM oleic acid, 15.95 mM palmitic acid, 7.2 mM stearic acid, 2.65 mM palmitoleic acid, and 2.45 mM linoleic acid”. Do these reflect actual circulating concentrations of NEFA and BHBA?
I think it would be better for the author to describe the rationale for the concentration of NEFA and BHBA used in this experiment.
Response: Many thanks for the comment. a certain concentration of NEFAs and BHBA in the blood is part of a normal adaptation to NEB in early lactation. However, excessive concentrations of NEFAs or BHBA indicate an excess of NEB which is associated with detrimental health and production outcomes[1]. Elevated NEFA prepartum in the range of > 0.3 to 0.5 mmol/L within the last week of gestation are associated with greater risk for postpartum disease. Similarly, elevated NEFA (> 0.6 to 1 mmol/L) in individual cows within the first week postpartum would indicate higher disease risk. Concentrations of BHB have been used to clinically define a state of subclinical (> 1.4 mmol/L and < 2.6 mmol/L) and clinical (2.6 mmol/L) disease conditions[2, 3].And for the concentration of NEFA and BHBA used in this experiment, we have added supporting references in the Materials and Methods section (please see line 77).
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