Loquat Leaf Extract Inhibits Oxidative Stress-Induced DNA Damage and Apoptosis via AMPK and Nrf2/HO-1 Signaling Pathways in C2C12 Cells

Round 1

Reviewer 1 Report

In this study, Kwon et al. investigated the potential of loquat leaf (LE) extract in protecting C2C12 cells from H₂O₂-induced oxidative stress. LE co-treatment reversed ROS increase, DNA fragmentation and apoptosis in cells treated with certain concentration of H₂O₂. Authors owed this to prearranged upregulation of AMPK and Nrf2 signalings by LE.

Format and language are good. Some issues must be addressed before manuscript acceptance.

Major concerns:

1. Authors used 0.8 mM (0.00273%) H₂O₂ for mechanistic study. It induced oxidative stress, DNA damage and apoptosis. However, neither phosphorylation levels of AMPK and ACC (Fig 5b and e) nor (p)-Nrf2/Keap1/HO-1 axis (Fig 7b and c) was increased by 0.8 mM H₂O₂. Therefore, cytotoxicity of 0.8 mM H₂O₂ was not dependent on AMPK or Nrf2 signaling even LE relied on higher AMPK activity to restore cell viability (Fig 5c and d). LE pretreatment itself activated AMPK signaling and antioxidant signaling beforehand and exerted general protective effect on C2C112 cells to defend external stress. Hence, results in Fig 5, 7 and 9 were over-interpreted and accordingly the whole manuscript even the title should be revised intensively.

2. Authors focused on AMPK in H₂O₂-treated C2C12 because “previous studies have suggested that the Sirt1/AMPK pathway is activated 296 by H2O2-induced oxidative stress in C2C12 murine myoblasts [13]” (row 296-297). However, in this referred paper (Immun. Inflamm. Dis. 2021, doi:10.1002/iid3.490.) the only used cell line was 16HBE, a human bronchial epithelial cell line. C2C12 was mentioned once in the Discussion section and at there the referred paper involving H₂O₂, AMPK and C2C12 was Molecules. 2019 Oct 29;24(21):3903. doi: 10.3390/molecules24213903. Authors did not refer the correct paper to support their hypothesis. This let me suspect if all other papers are appropriately cited.

3. As described in row 134, cell were lysed in 1% Triton X-100 before DCF loading. DCF is cell permeable and does not require permeabilization. I double check this in the Certificate of Analysis provided by R&D Systems where DCF-DA was purchased from. I doubt if cells were still intact and alive after incubating with such high concentration of Triton and if result in Fig 2a really reflected ROS levels in different groups of cells.

4. FACS analysis of JC-1 fluorescence in Fig 4a and b and Fig 9a and b had issue. Both aggregates and monomers of JC-1 were high in control cells. Theoretically, healthy cells with high mitochondrial membrane potential (MMP) have more JC-1 aggregates and scatter points should locate at the upper left quadrant. JC-1 aggregates turn to be monomers in apoptotic cells with low MMP and at this moment part of scatter points move to the lower right quadrant. Authors should either optimize this experiment and perform it again or just show JC-1 aggregates in peak diagrams and quantify proportion of JC-1 aggregates.

5. What’s the relationship between Fig 10 and main topic? Was myotube differentiation after three-day LE treatment related to protective effect happened within one day?

Minor concerns:

1. Please specify the subunit (α1, 2; β1, 2; γ1, 2, 3) of AMPK measured in Fig 5a, b and e. Although p-AMPKα (T172) was mentioned in Materials and Methods section.

2. Please specify phosphorylated residues in AMPK, ACC and Nrf2 that were detected by corresponding antibodies in both Figures and Materials and Methods sections (p-ACC and p-Nrf2).

3. Please provide catalog numbers of antibodies in Materials and Methods section.

4. ACC is the kinase substrate of AMPK so p-ACC (S79) is a better indicator of AMPK agonist like compound c. In Fig 5a, p-ACC increase was more obvious than p-AMPK. Total ACC and p-ACC should be included in Fig 5b and e.

Author Response

Response to Reviewer 1 Comments

Dear editor Ms. Marija Pavic and reviewers：

Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled “Loquat Leaf Extract Inhibits Oxidative Stress-Induced DNA Damage and Apoptosis via AMPK and Nrf2/HO-1 Signaling Pathways in C2C12 Cells” (ID: applsci-2056674). These 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 corrections which we hope meet with approval. Revised portions are marked in red on the paper. The main corrections in the paper and the response to the reviewer’s comments are as flowing:

• Point 1: Authors used 0.8 mM (0.00273%) H₂O₂for mechanistic study. It induced oxidative stress, DNA damage and apoptosis. However, neither phosphorylation levels of AMPK and ACC (Fig 5b and e) nor (p)-Nrf2/Keap1/HO-1 axis (Fig 7b and c) was increased by 0.8 mM H₂O₂. Therefore, cytotoxicity of 0.8 mM H₂O₂ was not dependent on AMPK or Nrf2 signaling even LE relied on higher AMPK activity to restore cell viability (Fig 5c and d). LE pretreatment itself activated AMPK signaling and antioxidant signaling beforehand and exerted general protective effect on C2C112 cells to defend external stress. Hence, results in Fig 5, 7 and 9 were over-interpreted and accordingly the whole manuscript even the title should be revised intensively.

Response 1: Thanks for the advice. In a similar papers published by our labs, when C2C12 cells were treated with H₂O₂, there was no increase in p-AMPK [1]. We used compound C, an AMPK inhibitor, and H₂O₂+LE+compound C significantly decreased p-AMPK, p-Nrf2, and HO-1, and increased Keap1 compared to H₂O₂+LE (Fig. 5e and Fig. 7c). Therefore, we thought that loquat leaf extract inhibits oxidative stress-induced DNA damage and apoptosis through AMPK and Nrf2/HO-1 signaling pathways in C2C12 cells.

 

• Point 2: Authors focused on AMPK in H₂O₂-treated C2C12 because “previous studies have suggested that the Sirt1/AMPK pathway is activated 296 by H₂O₂-induced oxidative stress in C2C12 murine myoblasts [13]” (row 296-297). However, in this referred paper (Immun. Inflamm. Dis. 2021, doi:10.1002/iid3.490.) the only used cell line was 16HBE, a human bronchial epithelial cell line. C2C12 was mentioned once in the Discussion section and at there the referred paper involving H₂O₂, AMPK and C2C12 was Molecules. 2019 Oct 29;24(21):3903. doi: 10.3390/molecules24213903. Authors did not refer the correct paper to support their hypothesis. This let me suspect if all other papers are appropriately cited.

Response 2: We are very sorry for the mistake. We changed reference 13 in the manuscript to a reference on myoblasts. In addition, all other references have been checked, and except for number 13, they have been correctly cited.

 

• Point 3: As described in row 134, cells were lysed in 1% Triton X-100 before DCF loading. DCF is cell permeable and does not require permeabilization. I double check this in the Certificate of Analysis provided by R&D Systems where DCF-DA was purchased from. I doubt if cells were still intact and alive after incubating with such high concentration of Triton and if result in Fig 2a really reflected ROS levels in different groups of cells.

Response 3: Thanks for the advice. We checked the protocol and corrected the manuscript.

 

• Point 4: FACS analysis of JC-1 fluorescence in Fig 4a and b and Fig 9a and b had issue. Both aggregates and monomers of JC-1 were high in control cells. Theoretically, healthy cells with high mitochondrial membrane potential (MMP) have more JC-1 aggregates and scatter points should locate at the upper left quadrant. JC-1 aggregates turn to be monomers in apoptotic cells with low MMP and at this moment part of scatter points move to the lower right quadrant. Authors should either optimize this experiment and perform it again or just show JC-1 aggregates in peak diagrams and quantify proportion of JC-1 aggregates.

Response 4: Thanks for your comment. According to the results of JC-1 studies in other papers, the control is located in the upper right quadrant. And when apoptosis occurs, it moves to the lower right [1-5].

Figure 3 of Reference 2

 

• Point 5: What’s the relationship between Fig. 10 and main topic? Was myotube differentiation after three-day LE treatment related to protective effect happened within one day?

Response 5: Thanks for your comment. In addition to studying the mechanism, which is the main subject of apoptosis, we studied the effect of LE on cell differentiation as shown in Fig. 10. This is because the decrease in differentiation of muscle cells in the human body and the increase in sarcopenia are related to the increase in the concentration of oxidants such as ROS, inflammation, and apoptosis. And this is the result of a study conducted to confirm the research results of our laboratory and other scientists that it is possible to prevent sarcopenia by increasing the induction of muscle cell differentiation through antioxidant and anti-inflammatory actions. As a result, LE of C2C12 cells significantly increased the degree of differentiation in a concentration-dependent manner by day 3.

 

• Minor concerns
• Point 1: Please specify the subunit (α1, 2; β1, 2; γ1, 2, 3) of AMPK measured in Fig 5a, b and e. Although p-AMPKα (T172) was mentioned in Materials and Methods section.

Response 1: Thanks for the advice. We modified it by adding the subunit of AMPK measured in Fig. 5a, b, and e of the manuscript.

 

• Point 2: Please specify phosphorylated residues in AMPK, ACC and Nrf2 that were detected by corresponding antibodies in both Figures and Materials and Methods sections (p-ACC and p-Nrf2).

Response 2: Thanks for the advice. We added the phosphorylated residues of AMPK, ACC, and Nrf2 to the Figures and Materials and Methods sections of the manuscript.

 

• Point 3: Please provide catalog numbers of antibodies in Materials and Methods section.

Response 3: Thanks for the advice. We have added antibody catalog numbers to the manuscript's materials.

 

• Point 4: ACC is the kinase substrate of AMPK so p-ACC (S79) is a better indicator of AMPK agonist like compound C. In Fig 5a, p-ACC increase was more obvious than p-AMPK. Total ACC and p-ACC should be included in Fig 5b and e.

Response 4: Thanks for the suggestion. Thank you very much for your very good comments and suggestions. However, regarding this part, this manuscript is one of my last research papers because the first authour who led this research has already graduated from the graduate school, and I am closing my lab very soon due to mandatory retirement. Therefore, additional experiments are not possible, and we ask for your understanding.

 

We tried our best to improve the manuscript and made some changes in the manuscript. These changes will not influence the content and framework of the paper. And here we did not list the changes but marked them in red in the revised paper. We appreciate for Editors/Reviewers’ warm work earnestly and hope that the correction will meet with approval.

 

Once again, thank you very much for your comments and suggestions.

 

[References for the responses]

1. Park, C.; Ji, S.Y.; Lee, H.; Choi, S.H.; Kwon, C.Y.; Kim, S.Y.; Lee, E.T.; Choo, S.T.; Kim, G.Y.; Choi, Y.H.; et al. Mori ramulus suppresses hydrogen peroxide-induced oxidative damage in murine myoblast C2C12 cells through activation of AMPK. Int. J. Mol. Sci. 2021, 22, doi:10.3390/ijms222111729.
2. Lin, H.Y.; Han, H.W.; Wang, Y.S.; He, D.L.; Sun, W.X.; Feng, L.; Wen, Z.L.; Yang, M.K.; Lu, G.H.; Wang, X.M.; et al. Shikonin and 4-hydroxytamoxifen synergistically inhibit the proliferation of breast cancer cells through activating apoptosis signaling pathway in vitro and in vivo. Chin. Med. 2020, 15, 23, doi:10.1186/s13020-020-00305-1.
3. Jiang, S.; Zhu, R.; He, X.; Wang, J.; Wang, M.; Qian, Y.; Wang, S. Enhanced photocytotoxicity of curcumin delivered by solid lipid nanoparticles. Int. J. Nanomedicine 2017, 12, 167-178, doi:10.2147/ijn.S123107.
4. Liu, Z.; Xiong, L.; Ouyang, G.; Ma, L.; Sahi, S.; Wang, K.; Lin, L.; Huang, H.; Miao, X.; Chen, W.; et al. Investigation of copper cysteamine nanoparticles as a new type of radiosensitiers for colorectal carcinoma treatment. Sci. Rep. 2017, 7, 9290, doi:10.1038/s41598-017-09375-y.
5. Lin, W.; Zhao, J.; Cao, Z.; Zhuang, Q.; Zheng, L.; Cai, Q.; Chen, D.; Wang, L.; Hong, Z.; Peng, J. Livistona chinensis seed suppresses hepatocellular carcinoma growth through promotion of mitochondrial-dependent apoptosis. Oncol. Rep. 2013, 29, 1859-1866, doi:10.3892/or.2013.2319.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript by Kwon et al. describes the protective effect of Loquat leaf ethanol extract (LE) against H₂O₂ induced ROS generation, DNA damage, mitochondria dysfunction and apoptosis. Authors showed the effect of LE in C2C12 myoblast and in 3 days differentiated cells. In general, this kind of studies are critical for the identification of natural compounds that may contribute to the development of a treatment of oxidative stress induced muscle damage. Study is well designed, and the data are convincing but there are few comments.

 

Comments:

Figure 2C: Why is there no effect of 5ug/ml LE+H₂O₂ treatment on y-H2AX level, maybe authors can include graphs for quantification? Interestingly, in Figure 2a and b, there is some effect on DNA damage at 5ug/ml LE+H₂O₂ treatment but not on y-H2AX level. 

 

Figure 3b: According to images shown, chromatin condensation is not clear to me, it would be helpful for readers if authors can provide better quality images. 

 

Line 281-283, authors claim that ‘LE restores mitochondrial dysfunction’, based on Figure 4 data, it is only partial correction not full restoration. It will be easier to interpret if authors can include graphs for cytochrome quantification.

 

Figure 5, Section 3.5: citation ‘number 13’ doesn’t support what authors mentioned, study by Xu et al (13) was done in 16HBE cells not myoblasts. 

 

Figure 5E: why authors did not use compound C with LE treatment to demonstrate the effect of the inhibitor itself. If H₂0₂ treatment suppresses p-AMPK expression, why is there remaining p-AMPK after H2O2+LE+Compound C treatment, this needs clarification. 

 

Author Response

Response to Reviewer 2 Comments

Dear editor Ms. Marija Pavic and reviewers：

Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled “Loquat Leaf Extract Inhibits Oxidative Stress-Induced DNA Damage and Apoptosis via AMPK and Nrf2/HO-1 Signaling Pathways in C2C12 Cells” (ID: applsci-2056674). These 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 corrections which we hope meet with approval. Revised portions are marked in red on the paper. The main corrections in the paper and the response to the reviewer’s comments are as flowing:

• Point 1: Figure 2C: Why is there no effect of 5 μg/ml LE+H₂O₂ treatment on γ-H2AX level, maybe authors can include graphs for quantification? Interestingly, in Figure 2a and b, there is some effect on DNA damage at 5 μg/ml LE+H₂O₂ treatment but not on y-H2AX level.

Response 1: Thanks for your comment. In Fig. 2c, western blot analysis at 5 μg/ml LE+H₂O₂ did not significantly reduce the expression of g-H2AX in response to DNA double-strand breaks. However, in the comet analysis, another analysis for DNA damage, it was confirmed that the smear pattern of nuclear DNA was slightly reduced at 5 μg/ml LE+H₂O₂. And it seems that we need to be more careful about graphing the expression change of g-H2AX in western blot analysis. This is because if this result is graphed, the western blot data of other figures should be displayed in the same way, so it would be better not to include graphs for quantification. We ask for your deep understanding.

• Point 2: Figure 3b: According to images shown, chromatin condensation is not clear to me, it would be helpful for readers if authors can provide better quality images. 

Response 2: Thanks for your suggestion. We have changed the Fig. 3b in the manuscript to a higher quality image.

• Point 3: Line 281-283, authors claim that ‘LE restores mitochondrial dysfunction’, based on Figure 4 data, it is only partial correction not full restoration. It will be easier to interpret if authors can include graphs for cytochrome quantification.

Response 3: Thanks for the advice. We corrected the manuscript to "suggest that LE partially restores mitochondrial dysfunction". In addition, we will not present a graph because if we provide a graph for cytochrome c quantification here, the same graph should be provided for other western data. As mentioned in Point 1, it would be better to avoid additional graphing in this case as well.

• Point 4: Figure 5, Section 3.5: citation ‘number 13’ doesn’t support what authors mentioned, study by Xu et al (13) was done in 16HBE cells not myoblasts. 

Response 4: We are very sorry for the mistake. We changed reference 13 in the manuscript to a reference on myoblasts.

• Point 5: Figure 5E: why authors did not use compound C with LE treatment to demonstrate the effect of the inhibitor itself. If H₂O₂ treatment suppresses p-AMPK expression, why is there remaining p-AMPK after H₂O₂+LE+Compound C treatment, this needs clarification. 

Response 5: Thanks for your advice. In Fig. 5c, we confirmed that cell viability was not affected when LE alone and compound C were treated alone, so we did not conduct LE+compound C experiments. We confirmed that H₂O₂ treatment did not inhibit p-AMPK expression, and confirmed that p-AMPK significantly decreased after H₂O₂+LE+compound C treatment compared to H₂O₂+LE.

We tried our best to improve the manuscript and made some changes in the manuscript. These changes will not influence the content and framework of the paper. And here we did not list the changes but marked them in red in the revised paper. We appreciate for Editors/Reviewers’ warm work earnestly and hope that the correction will meet with approval.

Once again, thank you very much for your comments and suggestions.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

My concerns are largely addressed. While recalling the graduated student for additional experiments using leisure time is logical when I was in the graduate school, I am fine with no more data to appropriately address some of my concerns given author’s lab will be closed soon. I hope authors can resolves three more issues:

1. Response 5 to my major comment 5: If literatures support the explanation that “LE induces muscle cell differentiation through antioxidant and anti-inflammatory actions”, please place it in the discussion and provide reference(s). Otherwise, readers may raise the same question.

2. Concern 1 of the other reviewer let me raise two more questions: (1) Cytosolic, nuclear and mitochondrial proteins were analyzed by WB in this study. However, corresponding lysis buffer information was not provided. It was written in row 150 of the manuscript version 2 that “and lysed using lysis buffer”. Specific kit or solution and method are required to separate cytosolic and mitochondrial for measuring cytochrome c leakage. High concentration of SDS as detergent is important for extraction of histone proteins. Authors should provide protocols in 2.7. Western Blot Analysis; (2). authors need to include quantification of tail moment (% tail DNA*tail length) of migrated DNA for Fig 2b to clear show rescue of DNA damage by LE is dose dependent. Draw bar graph of average tail moment or use the criteria: no damage (<5%); low damage (5-20%); moderate (20-40%); high damage (40- 80%) or extreme damage (> 80%) for assessment. Variation of DNA tail in comet assay is not small. Authors just picked up representative pictures with a couple of cells. Usually I quantify more than 50 cells in each treatment group.

Author Response

Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled “Loquat Leaf Extract Inhibits Oxidative Stress-Induced DNA Damage and Apoptosis via AMPK and Nrf2/HO-1 Signaling Pathways in C2C12 Cells” (ID: applsci-2056674). These 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 corrections which we hope meet with approval. Revised portions are marked in red on the paper. The main corrections in the paper and the response to the reviewer’s comments are as flowing:

Point 1: Response 5 to my major comment 5: If literatures support the explanation that “LE induces muscle cell differentiation through antioxidant and anti-inflammatory actions”, please place it in the discussion and provide reference(s). Otherwise, readers may raise the same question.

Response 1: Thank you for your comments. We have added several references to the manuscript's discussion of the antioxidant and anti-inflammatory effects of LE. And we also added a reference on the induction of muscle cell differentiation through antioxidant activity.

Point 2: Concern 1 of the other reviewer let me raise two more questions: (1) Cytosolic, nuclear and mitochondrial proteins were analyzed by WB in this study. However, corresponding lysis buffer information was not provided. It was written in row 150 of the manuscript version 2 that “and lysed using lysis buffer”. Specific kit or solution and method are required to separate cytosolic and mitochondrial for measuring cytochrome c leakage. High concentration of SDS as detergent is important for extraction of histone proteins. Authors should provide protocols in 2.7. Western Blot Analysis; (2). authors need to include quantification of tail moment (% tail DNA*tail length) of migrated DNA for Fig 2b to clear show rescue of DNA damage by LE is dose dependent. Draw bar graph of average tail moment or use the criteria: no damage (<5%); low damage (5-20%); moderate (20-40%); high damage (40- 80%) or extreme damage (> 80%) for assessment. Variation of DNA tail in comet assay is not small. Authors just picked up representative pictures with a couple of cells. Usually I quantify more than 50 cells in each treatment group.

Response 2: Thank you for your valuable comments. (1) We re-write the protocols in 2.7. Western Blot Analysis. (2) The images were analyzed using OpenComet software v1.3.1 (Available at http://cometbio.org. Accessed: 12.27.2022.). The degree of DNA damage was graded on a scale of 0―4 counting at least 30 cells in each image using the following criteria: no damage (<5%) scored 0; low damage (5-20%) scored 1; moderate damage (20-40%) scored 2; high damage (40-80%) scored 3; extreme damage (> 80%) scored 4. And the results of Comet assay analysis were graphically displayed in Figure 2c and Figure 6c.

We tried our best to improve the manuscript and made some changes in the manuscript. These changes will not influence the content and framework of the paper. And here we did not list the changes but marked them in red in the revised paper. We appreciate for Editors/Reviewers’ warm work earnestly and hope that the correction will meet with approval.

Once again, thank you very much for your comments and suggestions.

 

 

 

Author Response File: Author Response.pdf