Elicitation of Inhibitory Effects for AGE-Induced Oxidative Stress in Rotator Cuff-Derived Cells by Apocynin
, Takashi Kurosawa †, Yutaka Mifune *, Atsuyuki Inui, Hanako Nishimoto, Yasuhiro Ueda, Takeshi Kataoka, Kohei Yamaura, Shintaro Mukohara, Tomoya Yoshikawa, Issei Shinohara, Tatsuo Kato, Shuya Tanaka, Masaya Kusunose, Yuichi Hoshino, Takehiko Matsushita and Ryosuke KurodaRound 1
Reviewer 1 Report (Previous Reviewer 2)
ABSTRACT
The abstract must be rewritten to better introduce the subject, the methods used in a concise way
INTRODUCTION
The introduction is well written
MATERIAL AND METHODS
2.1 Human Rotator cuff-derived cells preparation and treatment
Why did you choose to treat your cells with 100 mg/ml AGEs?? Same question for 100M apocynin which is very important dose
2.2 Quantitative real-time polymerase chain reaction (PCR)
Using GAPDH as housekeeping gene is not appropriate because GAPDH is particularly sensitive to oxidative stress. Please normalize using another housekeeping gene.
2.3 Cell viability assay
Why did you choose to measure viability only at one point, 48 hours?
2.4 Apoptotic cells analysis
Why did you not complete the apoptotic analyses by exploration of molecular pathway related to apoptosis??
2.5 Detection of ROS production
Concerning DCFH-DA, please justify the concentration of 10 μM you used.
RESULTS
The results are not analyzed, please include value or percentage of your observations
Figure 3. include the merge panel. The number of apoptotic cells in AGE group seems to be relatively limited and does not seem to be conclusive Why did you not use flow cytometry to identify the percentage of apoptotic cells?
Figure 5. Please include the merge panel.
DISCUSSION
The discussion is too long and must be mainly focused on the interpretation of your results
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report (New Reviewer)
The manuscript drafted by Furukawa et al. reported the effects of apocynin on oxidative stress stimulated by AGEs in rotator cuff-derived cells. This study is preliminary and superficial. A more extensive future studies should be considered in future.
1. The ethical approval code should be mentioned in the Materials and Methods.
2. Line 85: The average age should be presented in mean ± standard deviation, followed by the unit (years).
3. Line 98: Please clarify the sentence “A - mM stock solution”.
4. How was the concentration of apocynin determined since only one concentration was used? I wonder why apocynin was not tested with a wide range of concentration for its effect on cell viability, which is commonly done in all in vitro studies.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report (New Reviewer)
Furukawa_Rotator cuff and apocynin_cimb_2023
I commend the authors on the completion of this manuscript. The aim is well defined, being clinically relevant. The presentation defines the research question.
But I have some minor concerns highlighted below.
Introduction
Line 58. Please explain the acronym ROS
2. Materials and Methods
Table 1. Please add a legend explaining data are mean ± SD or n(%)
Table 2. Please explain the acronym: qRT
Line 134. “Before the WST assay analysis, we cultured each 96-well plate at 5% CO2 and 37°C.” Please, remove it is repeated.
Line 144. Please explain the acronym: PBS
Discussion:
Line 344 “This result might be attributed to the inhibition of NOX activation by apocynin because glycation and oxidation influence each [38].” Please, syntax review.
Line 352. Please review the acronym: JNK
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report (Previous Reviewer 2)
the answers provided by the authors are conclusive
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
The detrimental effects of advanced glycation end-products (AGEs) on medical conditions are well explained in non-tendom tissues . This study focuses on the effects of AGEs at the cellular level and involves tendom extracellular matrix (ECM) in human patients. Manucript is well written. Materials and methods are adequate. However it is not clear whether twelve patients with rotator cuff tears (tenocytes donors) were diabetics or not. this is not explained in "material and methods" but it is mentioned at the begining of the discussion. Also large differences in age (32 vs72) are major limitations of this study. References are mostly from this century, however one reference concerning Maillard reaction is from the 1912. I think it can be removed. In conclusion, I recommend the paper to be published after minor revision.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 2 Report
1-Abstract
The abstract is well written
2-Introduction
The introduction is well written
3-Material and Methods
A table with characteristic of your patients must be included. The authorization number must be included.
RT-qPCR
GAPDH is strongly influence by oxidative stress and therefore is not a good housekeeping gene. Normalize your data with another housekeeping gene.
Cells viability
Indicate the concentration of apocynin and AGEs you used
Oxidative stress
Why you did not evaluated the antioxidant enzyme?
Apoptosis
Some molecular pathways must be completed to support the observations made by the TUNEL assay.
Figure
the size of the immunofluorescence pictures must be increased, they are too small
4-Discussion
The discussion must be rewritten. The discussion must be focused mainly on the data generated in this study. You mentioned several molecular pathways you did not explore in our study.
5-Conclusion
You must mentioned the limitations of our study and perspectives
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report
The manuscript, titled “Elicitation of Inhibitory Effects for AGEs-induced Oxidative Stress in Rotator Cuff-Derived Cells by Apocynin”, aims to evaluate in vitro apocynin effects on human rotator cuff-derived cells. This scientific work is very interesting. However, some problems, as indicated below, should be addressed before the document can be considered for the publication in this journal. This version of the manuscript is not enough complete. Here, I present all my comments in detail, but my global consideration is almost positive.
· Major revision:
-Recently, advanced glycation end-products (AGEs) have been gaining attention because the deposition of AGEs in organs and tissues can cause various diseases, such as arteriosclerosis, renal failure, and osteoporosis-
Aging is a multifactorial process developing through a complex net of interactions between biological and cellular mechanisms and it involves oxidative stress as well as protein glycation. Though many model systems are frequently used to study the biochemical alterations during aging as well as the condition of oxidative stress including the tissues from various parts of the body, erythrocytes or red blood cells get superiority amongst them. These cells are continually exposed to potential oxidative molecules in the blood flow. I suggest to add the following references, DOI: 10.3390/ijms231477.
-The accumulation of ROS under oxidative stress conditions results in the induction of lipid peroxidation and glycoxidation reactions, which leads to the elevated endogenous production of reactive aldehydes and their derivatives such as glyoxal, methylglyoxal (MG), malonic dialdehyde (MDA), and 4-hydroxy-2-nonenal (HNE) giving rise to advanced lipid-oxidation and glycation end products (ALEs and AGEs, respectively). Both ALEs and AGEs play key roles in cellular response to oxidative stress stimuli through the regulation of a variety of cell signaling pathways. Then, although this scientific work only summarizes the apocynin effects on advanced glycation end-products in the human rotator cuff-derived cells, I suggest to introduce in the "Introduction" some knowledges regarding ALEs. I suggest this reference, DOI: 10.1155/2019/3085756. Did authors investigate this aspect? They think they will be able to study it.
-The authors should add more knowledges about apocynin effects.
-IL-6 is a pleiotropic cytokine regulating different tissues and organs in diverse and sometimes discrepant ways (DOI: 10.1016/j.arr.2022.101697). Low information is reported in the text.
-Reactive oxygen species (ROS) are critical for senescence, and the relationship between AGEs and ROS has been recently reported-
-Therefore, the reduction of AGEs-induced oxidative stress might prevent rotator cuff degeneration due to senescence-
Aging is the progressive loss of tissue and organ function over time. The free radical theory of aging, later termed as oxidative stress theory of aging, is based on the structural damage-based hypothesis that age-associated functional losses are due to the accumulation of oxidative damage to macromolecules by ROS. The exact mechanism of oxidative stress-induced aging is still not clear, but probably increased ROS levels lead to cellular senescence, a physiological mechanism that stops cellular proliferation in response to damages that occur during replication. Senescent cells acquire an irreversible senescence-associated secretory phenotype involving secretion of soluble factors (interleukins, chemokines, and growth factors), degradative enzymes like matrix metalloproteases, and insoluble proteins/extracellular matrix components. I suggest to improve the introduction focuses, especially the key difference between aging and senescence, as they are different mechanism (DOI: 10.2147/CIA.S158513). In general, aging is the process of deterioration of cells with time, while senescence is a result of aging where the cells stop dividing and reach a state of arrest.
-CO2 is CO2 (the authors should modify it in all text).
-I suggest to uniform bar colors, like in Figure 3.
-I suggest to better organize the letter order in the Figure 2. In addition, I suggest to modify Figure 2 (b). This image is different respect with Bar of the Graph.
-ECM (Add the full sentence).
-Apocynin has been identified as a potent and selective inhibitor of NADPH oxidase. This enzyme is associated with oxidative stress. Why authors did not analyze its role in this context?
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
The answers provided by the authors are not fully satisfactory because molecular pathways concerning apoptosis should be added. From my point of view the justification of GAPDH as a housekeeping gene is not conclusive
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report
Aging is a multifactorial process developing through a complex net of interactions between biological and cellular mechanisms and it involves oxidative stress as well as protein glycation. Though many model systems are frequently used to study the biochemical alterations during aging as well as the condition of oxidative stress including the tissues from various parts of the body, erythrocytes or red blood cells get superiority amongst them. These cells are continually exposed to potential oxidative molecules in the blood flow. I suggest to add the following references, DOI: 10.3390/ijms23147781.
It is the correct reference: 10.3390/ijms23147781.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
