Protective Effects of Astaxanthin against Oxidative Stress: Attenuation of TNF-α-Induced Oxidative Damage in SW480 Cells and Azoxymethane/Dextran Sulfate Sodium-Induced Colitis-Associated Cancer in C57BL/6 Mice
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
The manuscript entitled “Protective Effects of Astaxanthin against Oxidative Stress: Attenuation of TNF-α-Induced Oxidative Damage in SW480 Cells and AOM/DSS-Induced Colitis Associated Cancer in C57BL/6 Mice” submitted to Marine drugs by Dr. Zhang and co-workers presents some interesting findings on the effects of astaxanthin on oxidative stress/cytokine synthesis/inflammation but needs a profound review.
Line 32. “Among these molecules, reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced endogenously at the highest concentrations and have major biological effects [3]. Reactive oxygen species (ROS) are essential….” Abbreviations should be presented the first time.
Line 35. “in the production and regulation of ROS are superoxide dismutase (SOD), acyl-CoA oxidase (ACOX), xanthine oxidase (XO), d-amino acid oxidase (DAO) and NADPH oxidase (NOX)…” This sentence is too much ambiguous. Please clarified the enzymes involved in ROS production and ROS metabolism.
Line 38. “Low levels of ROS” or physiological levels of ROS?
Line 70. “AST is the most potent antioxidant active substance currently available in nature”. This statement needs to be supported by bibliography.
Introduction sections should present astaxanthin. What is astaxanthin dietary source? Astaxanthin dietary concentrations? What was astaxanthin concentrations and astaxanthin doses chosen?
Line 88. “Interestingly, ROS levels and SOD activity recovered in a dose-dependent manner after AST treatment”. In my opinion, concentration-dependent is more suitable than dose-dependent in this in vitro context. Please checking along the manuscript.
Line 122. “The colon length was lower in the model group than in the normal group…” Model group, normal group……what means??? In figures I see control group and AOM/DSS group. Please checking along the manuscript.
I can not understand a lot of sentences along the manuscript. Per example “Similarly, the expression of p-p65 was increased and suppressed by AST treatment (Figure 5 163 C-D) (line 165)” What means??? English should be reviewed.
The title of the figure legends should be improved. “Figure 5. Effects of AST on major proteins and inflammatory factors” This title is too much ambiguous.
“The contents of proinflammatory cytokines IL-6, IL-1β and TNF-α. CTR was the control group, - was the AOM/DSS model group, and 50–200 mg/kg was the AST dose group. The presentation of AOM/DSS groups without/with treatment should be improved in the figures.
Discussion section should be improved. Authors should mention the novelty of these findings considering the important bibliography on astaxanthin-inflammation. In this way, several references could be considered (doi: 10.1080/10408398.2021.1983766; doi: 10.1186/s12876-014-0212-z).
Materials section should describe astaxanthin source.
Line 278. “All the data are expressed as the mean ± standard deviation (mean ± sem)” What happen with the number of experiments/determinations? This important aspects is not described in the manuscript.
Comments on the Quality of English Language
I can not understand a lot of sentences along the manuscript. Per example “Similarly, the expression of p-p65 was increased and suppressed by AST treatment (Figure 5 163 C-D) (line 165)” What means??? English should be reviewed.
Author Response
Question 1: Line 32. “Among these molecules, reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced endogenously at the highest concentrations and have major biological effects [3]. Reactive oxygen species (ROS) are essential….” Abbreviations should be presented the first time.
Response 1: Thank you for your suggestion. We revised abbreviations as you suggested (lines 39-41).
Question 2: Line 35. “in the production and regulation of ROS are superoxide dismutase (SOD), acyl-CoA oxidase (ACOX), xanthine oxidase (XO), d-amino acid oxidase (DAO) and NADPH oxidase (NOX)…” This sentence is too much ambiguous. Please clarified the enzymes involved in ROS production and ROS metabolism.
Response 2: Thank you for your suggestion. We have clarified the enzymes involved in ROS production (lines 42-43).
Question 3: Line 38. “Low levels of ROS” or physiological levels of ROS?
Response 3: Thank you for your suggestion. We revised “Low levels of ROS” to “physiological normal levels of ROS” as you suggested (line 44).
Question 4: Line 70. “AST is the most potent antioxidant active substance currently available in nature”. This statement needs to be supported by bibliography.
Response 4: Thank you for your suggestion. We rewritten this section in the manuscript (lines 77-78).
Question 5: Introduction sections should present astaxanthin. What is astaxanthin dietary source? Astaxanthin dietary concentrations? What was astaxanthin concentrations and astaxanthin doses chosen?
Response 5: Thank you for your suggestion. We have added the analysis and appropriate references in the manuscript (lines74-75, 78-82 and 92-93).
Question 6: Line 88. “Interestingly, ROS levels and SOD activity recovered in a dose-dependent manner after AST treatment”. In my opinion, concentration-dependent is more suitable than dose-dependent in this in vitro context. Please checking along the manuscript.
Response 6: Thank you for your suggestion. We revised dose-dependent to concentration-dependent as you suggested in the manuscript (lines 101 and 113).
Question 7: Line 122. “The colon length was lower in the model group than in the normal group…” Model group, normal group……what means??? In figures I see control group and AOM/DSS group. Please checking along the manuscript.
Response 7: Thank you for your suggestion. We revised it in the manuscript (lines 114, 135-138, 156 and 167).
Question 8: I can not understand a lot of sentences along the manuscript. Per example “Similarly, the expression of p-p65 was increased and suppressed by AST treatment (Figure 5 163 C-D) (line 165)” What means??? English should be reviewed.
Response 8: Thank you for your suggestion. We apologized for our careless mistakes. Thanks for your reminder. We revised it in the manuscript (lines 181-184).
Question 9: The title of the figure legends should be improved. “Figure 5. Effects of AST on major proteins and inflammatory factors” This title is too much ambiguous.
Response 9: Thank you for your suggestion. We modified the title of the figure legends according to your request (lines 187-188).
Question 10: “The contents of proinflammatory cytokines IL-6, IL-1β and TNF-α. CTR was the control group, - was the AOM/DSS model group, and 50–200 mg/kg was the AST dose group. The presentation of AOM/DSS groups without/with treatment should be improved in the figures.
Response 10: Thank you for your suggestion. We revised it in the manuscript (line 192).
Question 11: Discussion section should be improved. Authors should mention the novelty of these findings considering the important bibliography on astaxanthin-inflammation. In this way, several references could be considered (doi: 10.1080/10408398.2021.1983766; doi: 10.1186/s12876-014-0212-z).
Response 11: Thank you for your suggestion. We have improved the discussion section as you suggested in the manuscript (lines 207-216).
Question 12: Materials section should describe astaxanthin source.
Response 12: Thank you for your suggestion. We have added astaxanthin source in the manuscript (line 233).
Question 13: Line 278. “All the data are expressed as the mean ± standard deviation (mean ± sem)” What happen with the number of experiments/determinations? This important aspects is not described in the manuscript.
Response 13: Thank you for your suggestion. We revised this section in the manuscript (lines 315-318).
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe outcome of research is very interesting, well written with well-presented data; however, find below my comments, which can improve the manuscript.
1. In the Title, addition of the hyphen between “Colitis” and “Associated”.
2. All abbreviations/acronym, including P-, should be defined in the Abstract and in the text upon first use.
3. In the Results section, the related panels of Figure 1 (A-F) should be clearly mentioned after ROS levels and SOD activity as follows, for example: ROS levels (P < 0.001, Fig 1A-E) and so on for SOD activity. For clear and obvious decrease in ROS generation based on Dichlorofluorescein (DCF) intensity, the histograms shown in Fig A-D should be presented into the same plot. In the bar graphs of Fig 1E-F, the addition of TNF-a should be clearly indicated. The authors should explain why ERK phosphorylation, which is mainly involved in cell growth, was monitored while along with JNK, the other MAPK family member well-known to be activated in response to stress stimuli is p38. Therefore, it will be more convincing to monitor p38 phosphorylation in TNF-a-treated SW480 cells and in AOM/DSS-induced mice than ERK phosphorylation. In the Figures 2A, 2D, 5A, and 5C, the molecular weight (kDa) of each targeted protein must be indicated along each Western blot. In each Figure (Western blot image, bar graph), the treatment with TNF-a must be clearly indicated. In the legend of Figure 2, remove graphs from “ (A) The representative Western blotting graphs”. Also in the legend, the authors omitted to mention the detection of the total forms of JNK and ERK for relative densities analysis. The authors must clarify this part of the legend “- was the AOM/DSS model group, and 50-200 mg/kg” while the cell lysates were used after cell exposure to TNF-a. For a better flow of data presentation/description and related Figures, the authors should move the panels E-I of the Figure 3 to the corresponding text of the sub-section 2.2.2. In the sub-section 2.2.3., the related Figure of the text “The expression of Ki67…. (P < 0.001)” (l148-149) is missing. In the Figure 5, the authors must improve the legend “Western blotting of representative proteins of..” as the term representative should be related to Western blot and not to the targeted proteins. Same remark for “representative proteins of p-p65 and b-actin”. Of note, to confirm the antioxidant effect of AST, the detection of nuclear factor erythroid 2-related factor 2 (Nrf2) is relevant for this study. The authors are strongly recommended to monitor Nrf2 expression in both in vitro and in vivo models.
4. The Discussion is too poor and too short. For instance, the authors should further discuss their results, the choice of targeted signaling proteins in the process of inflammation induced by TNF-a in cancer cells and AOM/DSS model, with those reported in the literature. Other reported molecular mechanisms underlying the antioxidant and anticancer properties of AST must be discussed as well with their findings.
5. In the Materials and Methods, primary antibodies (catalog number, dilution) used should be listed.
6. In the Conclusion, the authors should avoid using “could” because the current strong and relevant findings must be listed and not probabilities or hypotheses. This section must be revised and improved.
Comments on the Quality of English Language
Moderate editing of English language is required.
Author Response
Question 1: In the Title, addition of the hyphen between “Colitis” and “Associated”.
Response 1: Thank you for your suggestion. We added the hyphen between “Colitis” and “Associated” in the title (line 4).
Question 2: All abbreviations/acronym, including P-, should be defined in the Abstract and in the text upon first use.
Response 2: Thank you for your suggestion. We revised it in the manuscript (lines 21-27).
Question 3: In the Results section, the related panels of Figure 1 (A-F) should be clearly mentioned after ROS levels and SOD activity as follows, for example: ROS levels (P < 0.001, Fig 1A-E) and so on for SOD activity. For clear and obvious decrease in ROS generation based on Dichlorofluorescein (DCF) intensity, the histograms shown in Fig A-D should be presented into the same plot. In the bar graphs of Fig 1E-F, the addition of TNF-a should be clearly indicated. The authors should explain why ERK phosphorylation, which is mainly involved in cell growth, was monitored while along with JNK, the other MAPK family member well-known to be activated in response to stress stimuli is p38. Therefore, it will be more convincing to monitor p38 phosphorylation in TNF-a-treated SW480 cells and in AOM/DSS-induced mice than ERK phosphorylation. In the Figures 2A, 2D, 5A, and 5C, the molecular weight (kDa) of each targeted protein must be indicated along each Western blot. In each Figure (Western blot image, bar graph), the treatment with TNF-a must be clearly indicated. In the legend of Figure 2, remove graphs from “(A) The representative Western blotting graphs”. Also in the legend, the authors omitted to mention the detection of the total forms of JNK and ERK for relative densities analysis. The authors must clarify this part of the legend “- was the AOM/DSS model group, and 50-200 mg/kg” while the cell lysates were used after cell exposure to TNF-a. For a better flow of data presentation/description and related Figures, the authors should move the panels E-I of the Figure 3 to the corresponding text of the sub-section 2.2.2. In the sub-section 2.2.3., the related Figure of the text “The expression of Ki67…. (P < 0.001)” (l148-149) is missing. In the Figure 5, the authors must improve the legend “Western blotting of representative proteins of..” as the term representative should be related to Western blot and not to the targeted proteins. Same remark for “representative proteins of p-p65 and b-actin”. Of note, to confirm the antioxidant effect of AST, the detection of nuclear factor erythroid 2-related factor 2 (Nrf2) is relevant for this study. The authors are strongly recommended to monitor Nrf2 expression in both in vitro and in vivo models.
Response 3: Thank you for your suggestion. We revised the sentence in the manuscript (line 100). We sincerely thank the reviewer for careful reading. For clear and obvious decrease in ROS generation based on Dichlorofluorescein (DCF) intensity, the histograms may be presented into the same plot. However, due to our measurement method, it can only be presented as a separate graph. We added the TNF-a in the bar graphs of Fig 1E-F (line 104). Regarding Monitoring Phosphorylated ERK, we agree with your comments. Your suggestion provides a direction for our next research. Unfortunately, due to the limited time and funding, we did not supplement the experimental validation. We added the molecular weight (kDa) of each targeted protein along each Western blot in the Figures 2A, 2D, 6A, and 6C. We revised it in each Figure (Western blot image, bar graph) as you suggested (lines 104 and 118). We revised it as you suggested in Figure 2 (lines 118-120). We apologize for not explaining this clearly in the manuscript. We referred to Maresin2 negatively regulates DC's maturation via the MAPK/NF-κB pathway in DCs for relative densities analysis. We revised this part of the legend in the manuscript (lines 122-124). We moved the panels E-I of the Figure 3 to the corresponding text of the sub-section 2.2.2 as you suggested (lines 160-165). We apologize for not explaining this clearly in the manuscript. Figure 5 is a related graph of Ki67 expression (lines 171-176). We modified the legend according to your request (lines 118-120 and 187-188). Thank you very much for pointing out this important issue. We agree with your comments. Your suggestion provides a direction for our next research. Unfortunately, due to the limited time and funding, we did not supplement the experimental validation. However, in follow-up experiments, we will conduct relevant research in this direction.
Question 4: The Discussion is too poor and too short. For instance, the authors should further discuss their results, the choice of targeted signaling proteins in the process of inflammation induced by TNF-a in cancer cells and AOM/DSS model, with those reported in the literature. Other reported molecular mechanisms underlying the antioxidant and anticancer properties of AST must be discussed as well with their findings.
Response 4: Thank you for your suggestion. We revised this section in the manuscript (lines 206-216).
Question 5: In the Materials and Methods, primary antibodies (catalog number, dilution) used should be listed.
Response 5: Thank you for your suggestion. We added the information on antibodies in the manuscript (lines 241-246).
Question 6: In the Conclusion, the authors should avoid using “could” because the current strong and relevant findings must be listed and not probabilities or hypotheses. This section must be revised and improved.
Response 6: Thank you for your suggestion. We have rewritten this paragraph based on your suggestion (lines 321-332).
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for Authors
The last version of the manuscript considers my comments and suggestions but a few additional corrections should be considered.
Line 41. “….and the 41 main enzymes involved in the production of ROS are superoxide dismutase (SOD), xan-42 thine oxidase (XO) and NADPH oxidase (NOX)” SOD is not directly involved in ROS production such as XO and NOX.
Line 44. “Physiological normal levels of ROS” Physiological levels of ROS is enough.
Comments on the Quality of English Language
Without comments
Author Response
Question 1: Line 41. “….and the 41 main enzymes involved in the production of ROS are superoxide dismutase (SOD), xan-42 thine oxidase (XO) and NADPH oxidase (NOX)” SOD is not directly involved in ROS production such as XO and NOX.
Response 1: Thank you for your suggestion. We have revised this sentence to “In normal cells, catalase (CAT), glutathione peroxidase (GSH-PX), and superoxide dismutase (SOD) partially help to clear ROS and maintain cellular redox homeostasis” (lines 41-43).
Question 2: Line 44. “Physiological normal levels of ROS” Physiological levels of ROS is enough.
Response 2: Thank you for your suggestion. We revised “physiological levels of ROS” as you suggested (line 44).
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authors
I would like to thank the authors for improving the manuscript Protective effects of Astaxanthin against oxidative stress: Attenuation of TNF-a-induced oxidative damage in SW480 cells and AOM/DSS-induced colitis-associated cancer in C57BL/6 mice. However, some parts still need to be improved and corrected. Once again, there are too many grammatical errors. Therefore, the manuscript cannot be considered for publication as it stands.
Kindly, find below my major and minor comments:
1. In most Figures, it is still not clearly indicated the continuous cell exposure to TNF-a with or without the addition of different concentrations of AST. In addition, in the 4.2.1. Cell culture and Treatment, there is only description of the maintenance of the cell line in culture but there is no description of cell treatment with TNF-a and AST. This method must be described.
2. In the legend of Figure 1, although the cells were intact to be analysed by flow cytometry, it is mentioned that “While the cell lysates were used after cell exposure to TNF-a”. This sentence must be corrected and when it is correctly mentioned in the legend Figure 2, this sentence is incomplete. Therefore, both legends of Figures 1 & 2 must be improved. In the legend of Figure 6, the sentence “(E-G) The contents of proinflammatory cytokines IL-6, IL-1b and TNF-a.” is incomplete. Of note, these Fig. 6E-G should move to the “2.2.5. AST reduces inflammatory cytokines in AOM/DSS-induced mice”.
3. The Discussion is still too poor and nothing regarding the results related to inflammatory cytokines have been mentioned and discussed.
4. At this stage of this review process, two authors have been added. As there was no additional experiments performed or additional results described, I would like to know their contribution to this revised paper.
Comments on the Quality of English Language
Moderate editing of English is required.
Author Response
Question 1: In most Figures, it is still not clearly indicated the continuous cell exposure to TNF-a with or without the addition of different concentrations of AST. In addition, in the 4.2.1. Cell culture and Treatment, there is only description of the maintenance of the cell line in culture but there is no description of cell treatment with TNF-a and AST. This method must be described.
Response 1: Thank you for your suggestion. We have indicated in the legend based on your suggestion that cells are continuously exposed to TNF-a with or without the addition of different concentrations of AST (lines 104-105 and118). In addition, in the 4.2.1, methods for treating cells with TNF-a and AST have also been added as you suggested (lines 258-265).
Question 2: In the legend of Figure 1, although the cells were intact to be analysed by flow cytometry, it is mentioned that “While the cell lysates were used after cell exposure to TNF-a”. This sentence must be corrected and when it is correctly mentioned in the legend Figure 2, this sentence is incomplete. Therefore, both legends of Figures 1 & 2 must be improved. In the legend of Figure 6, the sentence “(E-G) The contents of proinflammatory cytokines IL-6, IL-1b and TNF-a.” is incomplete. Of note, these Fig. 6E-G should move to the “2.2.5. AST reduces inflammatory cytokines in AOM/DSS-induced mice”.
Response 2: Thank you for your suggestion. We have changed “While the cell lysates were used after cell exposure to TNF-a” to “After the cell modeling intervention was completed, cell proteins were extracted from cell lysate for follow-up experiments” (lines 107-108 and 123-125). In the legend of Figure 7, We have revised the sentence “The contents of proinflammatory cytokines IL-6, IL-1b and TNF-a” to “The contents of proinflammatory cytokines IL-6, IL-1β and TNF-α in mice” according to your suggestion (lines 200-201). And these figures 6 E-G have been moved to “2.2.5. AST reduces inflammatory cytokines in AOM/DSS-induced mice” as you suggested (lines198-203).
Question 3: The Discussion is still too poor and nothing regarding the results related to inflammatory cytokines have been mentioned and discussed.
Response 3: Thank you for your suggestion. We revised it in the manuscript as you suggested (lines 208-211).
Question 4: At this stage of this review process, two authors have been added. As there were no additional experiments performed or additional results described, I would like to know their contribution to this revised paper.
Response 4: Thank you for your suggestion. The two authors made significant contributions to this revision and joined the team later on. In this revision, the two authors provided many solutions, so their names were added.
Author Response File: Author Response.pdf
Round 3
Reviewer 2 Report
Comments and Suggestions for Authors
I would like to thank the authors for taking into account most of my comments, which improved the quality of the manuscript. However, the Discussion is still too poor to be published as it stands. The addition of the two authors during the review process can be only justified by tremendous positive changes, which are unfortunately not noticeable.
Comments on the Quality of English Language
Moderate grammatical and spelling errors
Author Response
Question 1: I would like to thank the authors for taking into account most of my comments, which improved the quality of the manuscript. However, the Discussion is still too poor to be published as it stands. The addition of the two authors during the review process can be only justified by tremendous positive changes, which are unfortunately not noticeable.
Response 1: Thank you for your suggestion. We sincerely thank the reviewer for careful reading. We have carefully corrected the grammatical mistakes in the whole manuscript according to your comments. We apologize for our careless mistakes. Thanks for your reminder. We did our best to polish the language in the revised manuscript with the help of American Journal Expert (AJE), a language polishing agency. We hope the revised manuscript will be acceptable to you. We revised the Discussion as you suggested (lines 207-259).
Round 4
Reviewer 2 Report
Comments and Suggestions for Authors
I would like to thank the authors for considering my comments and for brilliantly improving the Discussion.
Comments on the Quality of English Language
Moderate editing of English language is still required.
