Protection Impacts of Coix lachryma-jobi L. Seed Lactobacillus reuteri Fermentation Broth on Hydrogen Peroxide-Induced Oxidative Stress in Human Skin Fibroblasts
Round 1
Reviewer 1 Report
The manuscript entitled "Protection Impacts of Coix lachryma-jobi L. Seed Lactobacillus reuteri Fermentation Broth on Hydrogen Peroxide-induced Senescence in Human Skin Fibroblasts " by Fang et. al., projects the effectiveness of Coix seed Lactobacillus reuteri fermented extract (CLRF) in free radical scavenging and as a novel anti-ageing agent in the cosmetics industry.
Specific concerns:
1. Section 1: Line 61. Reference style should be checked and should be the same throughout the manuscript. Reference format should be followed according to author instructions.
2. Section 2.2: What was the absorbance (OD), or number of cells present in the starting cultures used for the fermentation?
3. Section 2.3: What are the differences in the absorbance measurement methods. A detailed description of Reagent III should be provided.
4. Section 2.4: Provide details of the enzyme marker?
5. Elaborate the factors that are responsible for the difference in the endpoint score (ES)? What are the results for the other fermentation methods? What was the ES for H2O2 only?
6. What by-products (such as ethanol etc.) are produced after fermentation? Do all the three fermentation methods produce similar by-products? Do the by-products have any impact on the observations?
7. Table S1: Should be transferred to the supplementary section.
8. Section 2.6: Authors should mention the value of the different concentrations used for the study.
9. Section 2.7: What enzyme was utilized as a standard?
10. Section 2.8: What fluorescent enzyme marker was utilized. Provide details?
11. Section 2.10: The model, make and specifications of the instrument should be mentioned.
12. Table number is missing for the details of the primer sequences.
13. Section 2.11: Cells from different colonies or same colonies had been used in case of the separate experiments that were conducted in the study?
14. Section 3.1: Representation of the results in a tabular format would be helpful.
15. Authors should check the value of p in line 272 as it does not match Figure 1.
16. Figures 1-5: Axis titles, axis labels, figure legends should be clearly represented.
17. Figure 3: What does the figure legend ’CLRY’ denote? Figure 3B: What was the viability with H2O2 only? Figure 3C: How CLRF is better compared to a common antioxidant such as vitamin C, vitamin E, lipoic acid, or flavonoid etc.?
18. Do CLRF/CW has any protective effect against reactive nitrogen species (RNS)?
19. How does CLRF/CW interfere with the apoptotic pathway?
20. The discussion section should explain and elaborate the findings of the current study in detail. Any background information should be transferred to the introduction section.
21. Elaborate the limitations of the study in more detail.
22. How does CLRF/CW protect against the DNA damaging effect of H2O2.
23. A detailed elaboration of the significance and novelty of the study in case of anti-aging should be beneficial.
Author Response
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Thank you for your review.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The authors intend to explore the anti-aging effect of Coix seed Lactobacillus reuteri fermented extract (CLRF) in human skin fibroblasts when challenged with H2O2. Overall, the study is promising. However, more in-depth experiments are warranted to establish the anti-aging effect of CLRF. Addressing the following comments may improve the manuscript:
· Authors mention about induction of senescence in the human skin fibroblasts in the title, but no test was conducted on senescence induction in HSF.
· Authors may need to use proper aging and senescence markers and evaluate telomerase activity to establish the anti-aging effect of CLRF for HSF.
· There is no mention of reference genes in real-time PCR.
· Authors measured mRNA expression of HAS. Which isoforms of HAS? Please note that different isoforms of HAS synthesize different molecular weight of HA and their biological functions vary.
· The referencing is inconsistent. Somewhere the references are denoted by a number, somewhere by name.
Author Response
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Thank you for your review.
Author Response File: 
Author Response.docx
Reviewer 3 Report
In the study, the authors show the fermentation of Coix seed using three different strains, examining the differences in the active ingredient content and in vitro free radical scavenging ability of the culture media. Fermentation with Lactobacillus reuteri was selected based on the results and it was able to increase the active ingredient content of the extract the best and had a stronger free radical scavenging ability. The results showed that human skin fibroblast cells viability, total antioxidant capacity, superoxide dismutase, catalase, collagen type I content and hyaluronic acid mRNA expression were significantly increased, while reactive oxygen species and the mRNA expression of matrix metalloproteinase-1 is reduced by Lactobacillus reuteri fermented extract-treated human skin fibroblast cells in comparison with other treatments and the control group. The results showed that Lactobacillus reuteri fermented extract can positively stimulate PI3K, AKT genes at the transcriptional level.
In my opinion is that the authors have presented an interesting and promising goal, and the research plan is thoughtful and well-structured. However, in several places in the introductory section, in the materials and methods section, they did not strive for concise wording and consistency. In the materials and methods section, several experiments are depicted accurately and in detail, however, there are parts that have gaps, so this also affects the understanding of the results. In the discussion, we can see well-outlined arguments, however, the conclusions drawn based on the results are exaggerated in some sentences.
Overall, I think that I would make the acceptance dependent on the revision of the article, so I recommend the article for a major revision. To help the revision, I summarize my observations in comments, furthermore there are some questions that were not clear when reading the article, so they will be found below.
Observations and Comments:
· A long introduction, with many sentences, does not focus on the succinct, short, to-the-point formulation. Over-explanatory descriptions are negligible.
For examples:
“Skin ageing features include loss of skin elasticity, dryness, hyperpigmentation, and wrinkles.”
“Microbial fermentation is a green and environmentally friendly method of biotechnological transformation which is capable of increasing the content of functional components such as polysaccharides, proteins and polyphenolics in the fermentation product.”
“Based on the above research gaps,”
· The link does not always follow the requested format, lines 41 “(Lephart, 2016)”and 61 (Xia et al., 2021; F. Yu et al., 2011). Please clarify your references.
· In section 2.1 Materials and Reagents, the list makes it difficult to follow in which experiments each material was used. I recommend the reagents, kits and their characteristics, mention which experiment you used, so that the reproducibility of the experiment can be examined more easily.
· An experimental description is incomplete, (2.3 Determination of Coix Seed Content (total protein), ELISA, qRT- PCR, Statistical Analysis), but instead of a description, it articulates evident characterizations. (“The ELISA method allows for the quick, easy and accurate detection of target enzyme activity or protein content”)
· Table S2. Primers: In addition to the forward and reverse orientation of primers, indicate whether you base the primer sequences in the 5'-3' or 3'-5' direction.
· “It is evident that CLRF and CW have some anti-ageing effects on HSF, and CLRF is more effective…” I would leave the drawing of conclusions from the results to the discussion section. The evident is a bit of a strong wording, rather suggested by the figures and results.
· Instead of listing the total protein, sugar and phenol content numerically in the results part, it would be much more transparent to display them in a table.
· In Figure 1, it is difficult to understand which significance values belong to which comparison, and which groups exactly were compared. Please describe it in detail in the text or indicate it in the table next to the figure.
· In the discussion, it is clear from the results that CW and CFLR have an effect on SOD, CAT, PI3P and AKT mRNA expression, ROS expression and proliferation. However, based on the results, the term anti-aging is still exaggerated, they should refine the wording. To think over the conclusion summarized from the results obtained.
Questions:
1. In in the qPCR section “Specific primers were designed using primer expression software” What software do you think? What did you use? What was used as a housekeeping gene and how were the qPCR results evaluated? What does relative expression mean in the graphs?
2. What do you mean by mapping analysis during statistical analysis? Exactly what statistical models were used in each experiment and how are the comparisons made?
3. What software was used to create Fig 6?
4. In section 1.7 Regulation of PI3K/AKT Signaling Pathway, would the blank group mean the control group, which is also shown in the figure? If so, please refer to it as you use it in the figure. How exactly did you perform the treatments before collecting the RNA sample, how was the treatment done for the control, M and CW, CLF?
5. In addition to measuring mRNA expression, was there any investigation into how this induction is experienced at the protein level?
Author Response
Please see the attachment.
Thank you for your review.
Author Response File: Author Response.docx
Reviewer 4 Report
The organization of introduction is good, but other chapters (Material and Methods, Results, and Discussion) need more work.
Here are my comments:
Materials and Methods
1) Materials and Reagents should be listed in the text. No need as a separate title.
2) Line 106-108: it should be in a sentence, combine this information into one sentence.
3) Line 109: please indicate g force value instead of rpm value.
4) Line 151-152: the information should be rewrite as a sentence.
5) Line 158-160: the information should be rewrite as a sentence.
6) Line 199-200: Please indicate six concentrations of sample solution in this section.
7) Line 206: Please add “%” symbol instead of “percent” word.
8) Line 237: Please briefly write the ELISA method used in this study.
Results
9) Line 269-272: Please expand this section. Differences between treatments should be indicated for each figure.
10) Figures: Please add A, B, C, and D the outside of each figure.
11) Line 293-294: Please list first treatments, then add controls.
12) Line 321-322: Please rewrite the sentence.
13) Line 328: No need separate paragraph. Please add after the previous sentence.
14) Please add an opening sentence in each section of the result chapter.
Discussion
This chapter needs more organization. Authors should indicate the main findings followed by their possible meaning. Then, this result and meaning should be supported by different studies. Moreover, this style should be applied for each main finding.
Future study should be separated from discussion chapter.
Author Response
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Thank you for your review.
Author Response File: Author Response.docx
Reviewer 5 Report
For a better reproductibility of this study, the informations concerning breed of chicken may be useful. Also, in which day of the incubation were the eggs open, in which day of the incubation were the CW and CLRF applied and in which quantity? If the applied quantity was 300 uL which were the criteria for this quantity was chosen?
Author Response
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Thank you for your review.
Author Response File: Author Response.docx
Reviewer 6 Report
The authors need to check the protein expression level of AKT and PI3K proteins.
The authors need to check the active levels of AKT and PI3K pathway by immunoblotting?
The authors need to test at-least either CLF/CSF for figure 4.
To prove their claims, the authors need to test at-least 2 independent skin cell lines and show the effect of CLRF.
In figure 4 why some of the bar graphs not having standard deviation bars (F, G, H, I, J)?
Figure 3, CLRY should CLRF
The quality of the figures can be improved, figure axis needs to be modified and large font size.
Written English need to be improved.
Author Response
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Thank you for your review.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
The authors modified the write-up. However, the following issues can further improve the manuscript.
Major:
1. Because the authors recognize that “the present study has limitations in the aging induction test of HSF” and “H2O2-induced oxidative stress causes DNA damage in HSF cells, telomere shortening and reduced telomerase activity in HSF cells. The fact that we did not conduct a detailed study for this issue is a limitation of our study.”: Authors are suggested to focus on the anti-oxidative property of CLRF but not on the anti-aging effect. They may highlight the application of the anti-oxidative properties of CLRF on skin aging.
2. Because the authors could not conduct any aging or senescence-related studies, I suggest replacing the word “senescence” with “oxidative stress” in the title and emphasizing the latter more than the former.
3. Line 19-20: “The protective effects of CLRF on hydrogen peroxide (H2O2)-induced senescence in human skin fibroblasts (HSF) are investigated” - Authors are suggested to substitute the word “senescence” with “oxidative stress”.
4. Line 25-27: “The results show that CLRF can stimulate the PI3K/AKT signaling pathway, inhibiting intracellular ROS levels, positively regulating COL-I genes and significantly reducing MMP-1 expression, with anti-oxidative damage and anti-aging effects.”- Authors are suggested to remove the phrase “anti-aging effects” as no study has been conducted in this direction. Instead, authors may highlight that increasing collagen synthesis and decreased MMP activity may slow the aging process.
5. Line 97-99: “the aim of this study was to investigate the action mechanism of fermented Coix seed on the regulation of cellular oxidative stress and skin anti-aging” – Authors are suggested to remove “skin anti-aging” as they did not perform any anti-aging experiment. Instead, they may like to highlight that the anti-oxidative properties of the fermented Coix seed might be explored to protect skin from aging.
6. Line 341: “HaCaT cells from the same colony were used for this experiment.”
Where the HaCaT cells came from?
7. Method: 2.7 Measurement of Intracellular ROS Levels – Authors measured fluorescence intensity. However, the cell number can not be the same in all wells. Therefore, to maintain consistency, authors are supposed to measure cell count or total protein or RNA concentration, and the fluorescence intensity should be normalized by cell count or total protein or RNA concentration.
8. Authors are encouraged to provide detailed explanatory legend in Figure 6.
9. In Figure 6, HAS (HAS1) is shown in the nucleus. However, all three isoforms of HAS are synthesized on the plasma membrane. Can the authors explain how HAS1 translocates to the nucleus?
10. What is the rationale to selecting HAS1 but not HAS2?
11. The Discussion section can be written better by explaining Figure 6 in detail.
Minor:
1. Line 39: “rad-icals” will be “radicals”. Remove hyphen.
2. Line 63-64: “Collagen, a glycosaminoglycan in the ECM,” – collagen is protein, but not glycosaminoglycan. Substitute the word “glycosaminoglycan” with “protein”.
3. Line 134, 138: “48h” should be “48 h”. The should be a gap between “48” and “h”.
4. Li.” 154: “600” should be “600 nm”.
5. Line 177: Authors are suggested to write absorbance sasame absorbance5” should be “765 nm”.
6. Line 260: “holes” should be “wells”.
7. Line 275-276: “the medium is poured out”. Tense should be consistent. Authors may write as: “the medium was discarded”.
8. Li.” 280-281: Active and passive voices mixed up. All sentences should be written consistently in the same voice.
9. Line 335: Sentence should be written in the past tense.
10. Page 9: Under “qRT-PCR”, authors are suggested to mention that β-actin was used as the reference gene.
Author Response
Please see the attachment.
Thank you very much for your review and suggestions.
Author Response File: Author Response.docx
Reviewer 3 Report
Dear Authors,
I would like to thank you for your answers, and I fully accept corrections. The changes greatly improved the text and the value of the article. With the proper description of the treatments, unfamiliar problems surfaced, which is why I still recommend your article for a major revision. My further comments and questions can be found below.
I consider one of the greatest values of the article, which you also mentioned several times, and the figures also showed, is the protective effect of CLRF and CW after the H2O2 treatment. In those figures, in the figure captions and in the description of the results, most of the comparison was made with a control with which no H2O2 treatment was performed. In these experiments, the comparison between the control and the marker group properly shows the effect of the H2O2 treatment, which is correct, but in the tests, if you want to highlight the protective effect of CW and CLRF, you should perform the comparison and statistical analysis between the marker group and samples groups, instead of comparison between control and samples groups. Here I am mainly referring to the figures and the description of chapters 3.4, 3.5, 3.6.
Furthermore, I would like to ask why the sentence “HaCaT cells from the same colony were used for this experiment.” was included in the statistical description and what do you think it means for the statistical analysis.
In addition, because of the sentence (“PI3K is a lipid kinase and AKT is a serine and threonine kinase which acts as the main downstream signal transducer of PI3K in various signaling cascades. PI3K/AKT is involved in regulating cell proliferation, apoptosis, and other physiological activities, and is a critical pathway associated with ageing and oxidative stress [26]”), I would indicate that the results are partially limited to exploring the results, such sentences should be in the introduction or the discussion section together with the reference.
In your letter, they carefully described the evaluation of qPCR and the calculation of relative expression, in my opinion, this could be presented in a compressed form in qPCR description.
Kind regards,
Author Response
Please see the attachment.
Thank you very much for your review and suggestions.
Author Response File: Author Response.docx
Reviewer 6 Report
No comments
Author Response
Thank you very much for your review and suggestions.
Round 3
Reviewer 2 Report
Thank you for the revision. The manuscript is improved. However, I would like to highlight that I disagree with the author's reply to Q.10. saying "HAS2 has a minor role in cell physiology". This is not correct, but rather just the opposite. Moreover, I did not find a detailed legend in Figure 6 as asked in Q.8.
Author Response
Please see the attachment.
Thank you very much for your review and suggestions.
Author Response File: Author Response.docx
Reviewer 3 Report
Dear Authors,
Thank you for the changes. I would like to mention that your article is getting better. Please would also like to mention that in the discussion he wrote: "In addition, CW and CLRF were able to inhibit the synthesis of MMP-1, protect COL-I and HA from catabolism." whereas, according to the results in Figure F4.E, it can be seen that CW has no significant effect on HA mRNA expression.
In the discussion, I'm glad that you mentioned the difference between CW and CLRF, which is clearly visible in the results.
Furthermore, reference 32 from line 492 should be placed at the end of the sentence because the rest of the sentence also presents this reference. You only need to put it behind the author if you combine several statements in one sentence. (but that's just a little thing)
My advise is that interpret your results more accurately and consistently next time.
Kind regards,
Author Response
Please see the attachment.
Thank you very much for your review and suggestions.
Author Response File: Author Response.pdf
