Quercetin Alleviates Lipopolysaccharide-Induced Cell Damage and Inflammation via Regulation of the TLR4/NF-κB Pathway in Bovine Intestinal Epithelial Cells
, Yinghao Huang 1,†, Qianbo Ma 1, Maocheng Jiang 1, Kang Zhan 1,2,3 and Guoqi Zhao 1,2,3,*
Round 1
Reviewer 1 Report
The authors described the influence of quercetin in modulating the inflammatory reaction induced by LPS on bovine intestinal epithelial cells (BIECs). The study is very simple and easy to follow. The experiments are essential and appropriate to the study. Although too much mRNA expression much (Real time PCR) is described not followed by investigation regarding the corresponding proteins.
However, before the article can be accepted for a publication some questions need to be solve:
- Quercetin is considered a senolytic agent responsible in inducing the death of senescent cells by apoptosis. How can you combine the senolytic proprieties of Quercetin with your data regarding the viability of BIEC after quercetin and quercetin /LPS treatment?
- How do you explain the upregulation effect on antioxidant enzymes in BIECs treated with only quercetin (paragraph 3.2)?
- Paragraph 3.7: the magnification of the fluorescence images is too low to demonstrate the translocation of p65. Please include/substitute the images with other at higher magnification where it is possible to well detect the nucleus and its fluorescent signal.
- Discuss the data also taking in consideration the senolytic effect of quercetin
Author Response
Response to Reviewer 1 Comments
The authors described the influence of quercetin in modulating the inflammatory reaction induced by LPS on bovine intestinal epithelial cells (BIECs). The study is very simple and easy to follow. The experiments are essential and appropriate to the study. Although too much mRNA expression much (Real time PCR) is described not followed by investigation regarding the corresponding proteins.However, before the article can be accepted for a publication some questions need to be solve:
1、Quercetin is considered a senolytic agent responsible in inducing the death of senescent cells by apoptosis. How can you combine the senolytic proprieties of Quercetin with your data regarding the viability of BIEC after quercetin and quercetin/LPS treatment?
Response for point 1: Thank you very much for your valuable question. We are sorry that we did not conduct cell senescence experiments, but through the current cell viability experiments, the results show that high concentrations of quercetin can inhibit cell viability, and the higher the concentration, the more obvious the effect; but in low doses of quercetin From the results of quercetin treatment, the use of quercetin did not affect the viability of cells; and in the subsequent inflammatory response experiments, the treatment time of cells was 6 hours, the stimulation time was shorter, and the experimental results also showed that low doses The quercetin is also beneficial to cell activity, so we choose a low dose of quercetin to treat cells in a shorter time without affecting the active physiological state of cells; Time treatment, the aging properties of quercetin had no major effect on the results of this experiment.
2、How do you explain the upregulation effect on antioxidant enzymes in BIECs treated with only quercetin (paragraph 3.2)?
Response for point 2: Thank you very much for your question. Our previous research results showed that quercetin is rich in flavonoids, which have anti-inflammatory, antibacterial and antioxidant effects, so adding quercetin plays an important role in the antioxidant aspect of cells.
3、Paragraph 3.7: the magnification of the fluorescence images is too low to demonstrate the translocation of p65. Please include/substitute the images with other at higher magnification where it is possible to well detect the nucleus and its fluorescent signal.
Response for point 3: Thank you very much for your valuable question. In response to your question of P65 transfer, we have added immunofluorescence experiments. The experimental results show that when LPS treatment, the level of intracellular phosphorylated P65 increases, and the corresponding transfer of p65 occurs after phosphorylation. Bit, from the membrane to the nucleus, see Figure 8. From L 268 to L 269.
4、Discuss the data also taking in consideration the senolytic effect of quercetin.
Response for point 4: Thank you very much for your valuable suggestions. We checked relevant information, and there are many studies showing that quercetin has anti-aging effects, but the focus of this paper is to study the anti-inflammatory and antioxidant properties of quercetin, especially the protective effect on inflammation caused by LPS . Therefore, no apoptosis-related indicators were detected during the experiment, so the data on apoptosis was not involved in the discussion. But your suggestion has inspired us a lot and is of great significance to our future research. Thank you again for your suggestion.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The authors reviewed “Quercetin Alleviates Lipopolysaccharide-induced Cell Damage and Inflammation via Regulation of the TLR4/NF-κB Pathway in Bovine Intestinal Epithelial Cells”. Overall, the work is interesting and will be beneficial for the fellow researchers. I will recommend it after some minor revisions.
- The abstract needs revision and restructuring of many sentences for better understanding.
- We will suggest to improve the presentation of all the images.
- The introduction needs to be revised and modified to provide a structured context.
- Methods need to be described properly.
- The result and discussion part have not been supported by references. Cite the latest references that support the study.
- The conclusion is overly simple-similar to other parts, there is no useful information that can be obtained after reading the perspective. The conclusion should provide some expert opinion too.
- Critical proofreading of the whole manuscript is needed. There are plenty of typos, grammatical mistakes, scientific inaccuracies, etc. inside the manuscript, which are too many to be pointed out in detail
Author Response
Response to Reviewer 2 Comments
The authors reviewed “Quercetin Alleviates Lipopolysaccharide-induced Cell Damage and Inflammation via Regulation of the TLR4/NF-κB Pathway in Bovine Intestinal Epithelial Cells”. Overall, the work is interesting and will be beneficial for the fellow researchers. I will recommend it after some minor revisions.
1、The abstract needs revision and restructuring of many sentences for better understanding.
Response for point 1: Thank you very much for your question. We have revised and restructured the sentences in the abstract of the paper. From L 14 to L 40.
2、We will suggest to improve the presentation of all the images.
Response for point 2: Thank you very much for your question. We have supplemented and replaced the pictures in the article accordingly. (Figure 8). From L 268 to L 269.
3、The introduction needs to be revised and modified to provide a structured context.
Response for point 3: Thank you very much for your suggestion. We have carefully revised the introduction of the paper. From L 44 to L 85.
4、Methods need to be described properly.
Response for point 4: Thank you very much for your suggestion, we have made serious revisions to the materials methods. From L 86 to L 155.
5、The result and discussion part have not been supported by references. Cite the latest references that support the study.
Response for point 5: Thank you very much for your suggestion. We have updated the latest literature in the Results and Discussion section of the paper. From L 468 to L 491.
6、The conclusion is overly simple-similar to other parts, there is no useful information that can be obtained after reading the perspective. The conclusion should provide some expert opinion too.
Response for point 6: Thank you very much for your question. We have reworked the conclusions of the paper. Based on your suggestion, we have corrected it as follows:
The present study has proved that quercetin can effectively alleviate oxidative stress and damage of barrier function of BIEC induced by LPS in vitro. It can be seen that quercetin may be a promising bioactive extract for protecting the natural biological performance of BIECs and alleviating the negative effects of LPS. Therefor, in the production we can improve the health status and performance of dairy cows by adding quercetin to the diet to reduce the effects of intestinal inflammation. From L 362 to L 368.
7、Critical proofreading of the whole manuscript is needed. There are plenty of typos, grammatical mistakes, scientific inaccuracies, etc. inside the manuscript, which are too many to be pointed out in detail
Response for point 7: Thank you for your kindly suggestions. We have examined our manuscript in detail and made careful revisions.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The authors satisfactory answered to the questions. Anyway, I still find the quality of immunofluorescence images poor. I strongly suggest them to change immunofluorescence low magnification images with high magnification images (at least 600X magnification) to better appreciate the translocation of NFkB in the nucleus after LPS treatment.
Author Response
List of Responses
Dear Editors and Reviewers:
Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled “Quercetin Alleviates Lipopolysaccharide-induced Cell Damage and Inflammation via Regulation of the TLR4/NF-κB Pathway in Bovine Intestinal Epithelial cells” (ID: cimb-1896151). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction in detail one by one which we hope meet with approval. Revised portion are highlighted in yellow in the paper. The main corrections in the paper and the responds to the reviewer’s comments are as flowing:
Response to Reviewer 1 Comments
1、The authors satisfactory answered to the questions. Anyway, I still find the quality of immunofluorescence images poor. I strongly suggest them to change immunofluorescence low magnification images with high magnification images (at least 600X magnification) to better appreciate the translocation of NF-kB in the nucleus after LPS treatment.
Response for point 1: We are very grateful for your valuable suggestions, which are very helpful to improve the quality of our manuscript. We have revised the pictures according to your suggestions. We hope this picture can meet the requirements, and look forward to your valuable comments. From L268-L269.
Author Response File: Author Response.pdf
Round 3
Reviewer 1 Report
The authors improved the quality of figure 8. The manuscript can be accepted for a publication.
