Review Reports

Round 1

Reviewer 1 Report

The authors have submitted a revised version of their manuscript. The revisions have strongly improved the manuscript, however, there are still concerns to be addressed. Also, I would like to note that unfortunately, no marked manuscript or responses to the comments of the reviewer were provided.

Abstract:The abstract has sufficiently improved. Please introduce the abbreviation “RPE” after the first mention of retinal pigment epithelium. It is not necessary to write ARPE-19 in bold (applies to the whole manuscript).

Introduction

While the introduction has strongly improved, the references provided are still not really appropriate (please check especially 2,3, 6-10). E.g., 2 is about the association of metformin with AMD, not an appropriate reference that AMD is the leading course of blindness in the elderly. 3 is about the effects of VEGF inhibitors on RPE cells, which is not a correct reference for the involvement of RPE dysfunction in AMD, etc. Please carefully revise and cite the appropriate studies.

The authors write “in 2018, the mechanisms of RPE …”. Only in 2018?

While I appreciate that the authors now include the limitation of ARPE-19 in the introduction, I would advice to also include points that show the potential ability of ARPE-19 cells to be used as a model, e.g. the gene expression pattern of ARPE-19 concerning apoptosis is quite similar to native RPE cells.

Methods

The method section has been strongly improved. However, I am still missing a statement about the confluency of the cells at the time of experimentation.

Please include the sample size in all figure legends.

Discussion

Reference 2 is still not an appropriate refrence.

As pointed out above, the authors should mention that the gene expression of ARPE-19 cells in regard to cell death renders them rather suitable for this experimentation.

 

Author Response

Review 1:

 

Comments:

“English language and style are fine/minor spell check required”.

 

Answer:

Manuscript was revised extensively once more. American English Editor refined and corrected our manuscript to make it more clear, readable, and grammatically correct. We used Scribendi (online web-based professional copy editing company). Editor code, EM2274.

 

Comments:

Does the introduction provide sufficient background and include all relevant references? Must be improved (x)

Are all the cited references relevant to the research? Must be improved (x)

Are the methods adequately described? Can be improved (x)

Are the results clearly presented? Can be improved (x)

 

Answer:

Thankfully to Reviewer’s suggestion the indicated sections were supplemented and improved.

 

Comments:

“The authors have submitted a revised version of their manuscript. The revisions have strongly improved the manuscript, however, there are still concerns to be addressed. Also, I would like to note that unfortunately, no marked manuscript or responses to the comments of the reviewer were provided”.

 

Answer:

We thank the Reviewer for appreciating our work to improve the quality of revised version of our manuscript. Our work has been modified again. We tried to take into account all the comments of the Reviewer.

We apologize for the inconvenience, but we do not understand why the Reviewer did not receive marked manuscript and responses to the comments of the Reviewer.

 

Comments:

“Abstract: The abstract has sufficiently improved. Please introduce the abbreviation “RPE” after the first mention of retinal pigment epithelium. It is not necessary to write ARPE-19 in bold (applies to the whole manuscript)”.

 

Answer:

The indicated abbreviation was introduced after the first mention of the retinal pigment epithelium.

In the manuscript, the authors did not use bold font in the spelling of ARPE-19. We think this may be due to the fact that different text software is used around the world.

 

Comments:

Introduction

“While the introduction has strongly improved, the references provided are still not really appropriate (please check especially 2,3, 6-10). E.g., 2 is about the association of metformin with AMD, not an appropriate reference that AMD is the leading course of blindness in the elderly. 3 is about the effects of VEGF inhibitors on RPE cells, which is not a correct reference for the involvement of RPE dysfunction in AMD, etc. Please carefully revise and cite the appropriate studies”.

Answer:

 

Thanks to the Reviewer for the comment. We have analyzed the cited literature once again and in our opinion it was correct. Each citation has an own reference. In the case of citation 2 and 3 the authors in the introduction of their paper address the sentence we quoted. However, at the suggestion of the Reviewer, we have decided to add a few literature items, as well as substitute items that we think would actually be a better choice. Citations 6-10 have also been rechecked and are believed to be correct by the authors. Once again, we thank you for the opportunity to improve our manuscript and to provide our opinion on contentious issues.

 

Comments:

Introduction

“The authors write “in 2018, the mechanisms of RPE …”. Only in 2018?”

Answer:

By mentioning 2018, the authors intended to refer to the research of Gao et al. [14].

The sentence has been corrected so as not to be confusing to the reader.

 

Comments:

Introduction

“While I appreciate that the authors now include the limitation of ARPE-19 in the introduction, I would advise to also include points that show the potential ability of ARPE-19 cells to be used as a model, e.g. the gene expression pattern of ARPE-19 concerning apoptosis is quite similar to native RPE cells.”

Answer:

In accordance with the Reviewer's comment, the introduction was supplemented with a section commenting on the suitability of the ARPE-19 line as a research model.

 

Comments:

Methods

“The method section has been strongly improved. However, I am still missing a statement about the confluency of the cells at the time of experimentation”.

Answer:

We agree with Reviewer that the cell confluency is very important because it may influence cell metabolism and gene expression profile. In accordance with Reviewer’s suggestion, we added the missing information in Material and Method section. We added the following sentence in 2.1. Cell culture conditions and ARPEs-19 viability assessment section:

“…Both, cell number and viability were monitored by cell counting in the Automated Cell Counter TC20 (BioRad, Hercules, Ca, USA), after staining them with 0.2% trypan blue (Sigma-Aldrich, St Louis, MO, USA). All experiments were performed using cells that were approximately 80% confluent.”

 

Comments:

“Please include the sample size in all figure legends”.

 

Answer:

Figure legend has been corrected. The sample size has been added.

 

Comments:

Discussion

“Reference 2 is still not an appropriate reference”.

Answer:

Thank the Reviewer for the comment. In the discussion section changed the citation to the correct one.

 

Comments:

“As pointed out above, the authors should mention that the gene expression of ARPE-19 cells in regard to cell death renders them rather suitable for this experimentation”.

Answer:

Thank you Reviewer for your attention, the relevant sentence has been added.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Thank you for incorporating the comments and suggestions. 

Author Response

Review 2:

 

Comments:

“English language and style are fine/minor spell check required”.

 

Answer:

Manuscript was revised extensively once more. American English Editor refined and corrected our manuscript to make it more clear, readable, and grammatically correct. We used Scribendi (online web-based professional copy editing company). Editor code, EM2274.

 

Comments:

Are all the cited references relevant to the research? Can be improved (x)

Is the research design appropriate? Can be improved (x)

Are the methods adequately described? Can be improved (x)

Are the results clearly presented? Can be improved (x)

 

Answer:

Our work has been modified again. We have taken into account all the reviewer's comments improving the quality of our work.

 

Comments:

“Thank you for incorporating the comments and suggestions”.

 

Answer:

We thank the Reviewer for appreciating our work on improving the quality of the submitted manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

In this study, Strzalka-Mrozik et al. investigated the differences in expression patterns of pyroptosis genes in senescent human retinal pigment epithelial cells exposed to lutein. This study concludes that lutein modulates the expression level of pyroptosis-related genes, which may serve as an anti-pyroptotic agent in near future. Although, this study is relevant but not well-performed.

 

Major concerns:

 

The authors performed the viability assessment and senescence assay of ARPE-19 cells treatment and also, they performed Oligonucleotide Microarray Analysis on the extracted RNA from these cells and validated the expression pattern of 7 selected pyroptosis-related gene transcripts that were significantly differentially expressed using qPCR. However, without measuring their protein levels the authors performed bioinformatic analysis on the 7 selected pyroptosis-related proteins using the STRING online database.

 

Authors are requested to assess at least some of these proteins and their interactions to validate their bioinformatic analysis.

 

The authors are requested to provide the experimental data regarding the standardization of concentrations of H2O2 and lutein.

 

The Authors are requested to improve their figures and the way of data representation.

 

Minor concern:

There are misspellings and grammatical errors peppered throughout the manuscript. Recommend proofreading to correct these errors.

Author Response

Review 3:

 

Comments:

“Moderate English changes required”.

 

Answer:

Manuscript was revised extensively once more. American English Editor refined and corrected our manuscript to make it more clear, readable, and grammatically correct. We used Scribendi (online web-based professional copy editing company). Editor code, EM2274.

 

Comments:

Is the research design appropriate? Must be improved (x)

Are the methods adequately described? Can be improved (x)

Are the results clearly presented? Must be improved (x)

Are the conclusions supported by the results? Can be improved (x)

 

Answer:

Our work has undergone significant modifications. We have taken into account all the reviewer's comments improving the quality of our work.

 

Comments:

“In this study, Strzalka-Mrozik et al. investigated the differences in expression patterns of pyroptosis genes in senescent human retinal pigment epithelial cells exposed to lutein. This study concludes that lutein modulates the expression level of pyroptosis-related genes, which may serve as an anti-pyroptotic agent in near future. Although, this study is relevant but not well-performed”.

 

Answer

We would like to thank the Reviewer for this comment and for notice of value of our research. We hope that our explanations and modifications improve clarity of our paper and in its current form is acceptable for publication.

Comments:

Major concerns:

 

“The authors performed the viability assessment and senescence assay of ARPE-19 cells treatment and also, they performed Oligonucleotide Microarray Analysis on the extracted RNA from these cells and validated the expression pattern of 7 selected pyroptosis-related gene transcripts that were significantly differentially expressed using qPCR. However, without measuring their protein levels the authors performed bioinformatic analysis on the 7 selected pyroptosis-related proteins using the STRING online database.

Authors are requested to assess at least some of these proteins and their interactions to validate their bioinformatic analysis”.

 

Answer:

We would like to thank the Reviewer for this accurate comment.

We would like to explain that STRING is a database dedicated to known and predicted protein-protein interactions. This tool can be applied to predict functional interactions of proteins based on differentially expressed genes (DEGs) and seek potential interactions between DEGs. Proteins are considered functionally associated when they jointly contribute to a specific cellular process. We would like also to notice that bioinformatics has an essential role in identifying the underlying genetic basis of human disease. We showed probable protein-protein interactions based on previously selected DEGs by means of this analysis.

We would also like to explain that in our study we focused mainly on the changes in senescent cells that had been treated with lutein mostly at the mRNA level of selected genes associated with pyroptosis in order to understand the molecular mechanisms of action.

We agree with Reviewer that additional studies at protein level could support our results but we have managed our experiments with the available limited resources. We sincerely accept Reviewer's recommendation to implement those techniques in future studies. We are sorry for not able to include now.

Unfortunately, we currently are unable to perform additional analyzes as suggested by Reviewer, that is measuring of selected protein levels, because we do not have culture supernatants. When planning the next stage of research, we will take into account the Reviewer's suggestion.

We also emphasize this problem at the end of the discussion section.

 

Comments:

“The authors are requested to provide the experimental data regarding the standardization of concentrations of H2O2 and lutein”.

 

Answer:

In accordance with Reviewer’s suggestion, the tables below present the raw experimental data that were used for the analyzes on the basis of which the choice of concentrations of the tested compounds was selected.

We would also like to clarify that these results were used for statistical analysis and the results marked as outliers were excluded from the analysis.

 

Lutein (24 h)

OD570
C	0.1 µM	0.5 µM	1 µM	5 µM	10 µM	C
0.426	0.475	0.449	0.443	0.393	0.357	0.467
0.555	0.490	0.380	0.485	0.449	0.434	0.422
0.431	0.385	0.480	0.458	0.466	0.356	0.377
0.492	0.457	0.419	0.481	0.427	0.392	0.487
0.478	0.505	0.478	0.487	0.432	0.347	0.497
0.448	0.537	0.476	0.506	0.440	0.389	0.418
0.398	0.456	0.506	0.482	0.445	0.355	0.398
0.389	0.450	0.501	0.455	0.389	0.363	0.425
0.431	0.477	0.429	0.424	0.366	0.334	0.416
0.468	0.428	0.411	0.400	0.373	0.363	0.440
0.453	0.400	0.408	0.411	0.373	0.343	0.407
0.438	0.422	0.367	0.409	0.388	0.364	0.486
0.450	0.403	0.414	0.430	0.377	0.345	0.464
0.445	0.438	0.446	0.449	0.393	0.351	0.438
0.378	0.442	0.472	0.445	0.418	0.358	0.392
0.381	0.453	0.456	0.440	0.357	0.362	0.438
0.421	0.466	0.413	0.407	0.378	0.332	0.411
0.463	0.430	0.391	0.387	0.391	0.341	0.423
0.447	0.402	0.407	0.393	0.363	0.345	0.407
0.441	0.419	0.369	0.408	0.371	0.367	0.475
0.459	0.409	0.396	0.425	0.369	0.334	0.459
0.461	0.426	0.437	0.429	0.392	0.342	0.425
0.389	0.439	0.470	0.451	0.400	0.352	0.404
0.387	0.441	0.456	0.452	0.364	0.368	0.444

 

	C	0.1 µM	0.5 µM	1 µM	5 µM	10 µM
mean OD570	0.436	0.444	0.435	0.440	0.396	0.358
SD	0.036	0.036	0.040	0.033	0.031	0.022
% viability	100.00	101.70	99.59	100.79	90.84*	82.02*
% SD	8.33	8.19	9.26	7.45	7.16	5.11

*p<0.05 vs. C, Dunnett’s test following a significant one-way ANOVA result

 

H₂O₂ (30 min)

 

		OD570
C	100 µM	200 µM	300 µM	400 µM	500 µM	1000 µM	2000 µM	C
0.404	0.449	0.460	0.445	0.376	0.431	0.143	0.300	0.416
0.480	0.50	0.481	0.467	0.448	0.401	0.246	0.288	0.405
0.470	0.68	0.481	0.444	0.34	0.429	0.214	0.259	0.419
0.367	0.511	0.477	0.467	0.421	0.403	0.161	0.233	0.357
0.478	0.490	0.485	0.454	0.417	0.401	0.173	0.243	0.379
0.405	0.451	0.463	0.449	0.376	0.429	0.143	0.300	0.417
0.481	0.501	0.483	0.466	0.449	0.400	0.245	0.287	0.404
0.476	0.625	0.481	0.444	0.34	0.428	0.214	0.259	0.417
0.366	0.511	0.478	0.468	0.422	0.405	0.161	0.234	0.357
0.478	0.490	0.483	0.455	0.417	0.399	0.174	0.242	0.381
0.405	0.451	0.464	0.45	0.377	0.431	0.143	0.303	0.416
0.480	0.501	0.483	0.467	0.448	0.399	0.245	0.288	0.406
0.480	0.552	0.482	0.445	0.34	0.428	0.215	0.259	0.417
0.366	0.510	0.478	0.469	0.424	0.405	0.160	0.232	0.354
0.479	0.490	0.484	0.456	0.418	0.399	0.173	0.243	0.381

 

	C	100 µM	200 µM	300 µM	400 µM	500 µM	1000 µM	2000 µM
mean OD570	0.454	0.470	0.475	0.456	0.401	0.401	0.230	0.238
SD	0.031	0.022	0.009	0.010	0.040	0.002	0.017	0.005
% viability	100.00	103.65	104.61	100.62	88.37*	88.48*	50.67*	52.43*
% SD	6.84	4.79	1.90	2.20	8.72	0.54	3.74	1.18

*p<0.05 vs. C, Dunnett’s test following a significant one-way ANOVA result

 

H₂O₂ (60 min)

 

			OD570
C	100 µM		200 µM		300 µM		400 µM		500 µM		1000 µM		2000 µM		C
0.459	0.442		0.470		0.346		0.431		0.373		0.126		0.119		0.435
0.436	0.459		0.373		0.441		0.406		0.298		0.126		0.100		0.469
0.416	0.411		0.374		0.373		0.312		0.304		0.117		0.097		0.459
0.373	0.403		0.361		0.335		0.342		0.300		0.118		0.119		0.349
0.387	0.393		0.362		0.363		0.309		0.296		0.090		0.059		0.334
0.461	0.443		0.471		0.347		0.435		0.373		0.128		0.119		0.436
0.438	0.455		0.375		0.440		0.405		0.298		0.126		0.100		0.47
0.416	0.409		0.375		0.373		0.312		0.303		0.119		0.097		0.458
0.374	0.405		0.363		0.335		0.345		0.300		0.118		0.118		0.348
0.384	0.392		0.361		0.364		0.309		0.296		0.090		0.059		0.334
0.463	0.443		0.472		0.347		0.438		0.375		0.127		0.119		0.436
0.437	0.451		0.371		0.442		0.405		0.298		0.126		0.100		0.47
0.415	0.412		0.373		0.371		0.311		0.303		0.121		0.097		0.458
0.375	0.405		0.363		0.335		0.348		0.301		0.121		0.118		0.349
0.383	0.393		0.360		0.362		0.309		0.297		0.090		0.059		0.335
		C		100 µM		200 µM		300 µM		400 µM		500 µM		1000 µM		2000 µM
mean OD570		0.412		0.421		0.388		0.372		0.361		0.314		0.116		0.099
SD		0.047		0.025		0.043		0.038		0.052		0.031		0.014		0.023
% viability		100.00		102.23		94.26		90.22*		87.68*		76.31*		28.21*		23.95*
% SD		11.40		5.97		10.48		9.30		12.72		7.48		3.41		5.48

*p<0.05 vs. C, Dunnett’s test following a significant one-way ANOVA result

 

H₂O₂ (120 min)

 

		OD570
C	100 µM	200 µM	300 µM	400 µM	500 µM	1000 µM	2000 µM	C
0.474	0.458	0.47	0.441	0.413	0.43	0.133	0.175	0.433
0.484	0.473	0.455	0.451	0.435	0.432	0.133	0.172	0.402
0.495	0.468	0.444	0.364	0.438	0.405	0.127	0.157	0.479
0.495	0.476	0.409	0.391	0.44	0.38	0.127	0.138	0.433
0.421	0.452	0.368	0.362	0.429	0.418	0.154	0.14	0.463
0.476	0.459	0.472	0.442	0.414	0.43	0.133	0.176	0.433
0.483	0.475	0.456	0.451	0.437	0.433	0.132	0.179	0.401
0.483	0.438	0.435	0.363	0.441	0.389	0.127	0.157	0.479
0.493	0.479	0.408	0.395	0.44	0.379	0.126	0.138	0.439
0.421	0.454	0.366	0.363	0.431	0.421	0.148	0.138	0.463
0.477	0.459	0.472	0.442	0.413	0.431	0.133	0.176	0.433
0.486	0.477	0.457	0.451	0.437	0.433	0.133	0.172	0.404
0.48	0.439	0.440	0.361	0.441	0.387	0.128	0.157	0.479
0.494	0.481	0.410	0.399	0.441	0.369	0.126	0.139	0.441
0.422	0.456	0.366	0.364	0.432	0.42	0.151	0.138	0.463

 

	C	100 µM	200 µM	300 µM	400 µM	500 µM	1000 µM	500 µM
mean OD570	0.458	0.463	0.429	0.429	0.428	0.410	0.134	0.145
SD	0.031	0.014	0.039	0.026	0.010	0.023	0.009	0.009
% viability	100.00	101.16	93.64*	93.79*	93.50*	89.69*	29.30*	31.61*
% SD	6.70	3.05	8.42	5.69	2.28	5.09	2.03	2.03

*p<0.05 vs. C, Dunnett’s test following a significant one-way ANOVA result

 

Description and graphs of lutein and H₂O₂ cytotoxicity of the compounds are provided in the 3.1. ARPE-19 viability assessment section. Based on these results for further analyzes H₂O₂ at the concentration of 400 µM and the treatment time of 60 min, and also lutein at the concentration of 1.0 µM and the treatment time of 24 h were selected.

 

Comments:

“The Authors are requested to improve their figures and the way of data representation”.

 

Answer:

In accordance with Reviewer’s suggestion, we tried to improve the presentation of our results. We also improved the quality of figures. We hope that now our findings are clearly showed.

 

Comments:

Minor concern:

“There are misspellings and grammatical errors peppered throughout the manuscript. Recommend proofreading to correct these errors”.

Answer

We would like to inform that our manuscript was revised to make it more clear, readable and grammatically correct. Additional linguistic proofreading is also one of the steps in the procedure for publishing papers in MDPI, and therefore, in addition to our correction, there will be a linguistic evaluation at the publisher itself before the publication is made publicly available.

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors have addressed all reviewer's comments to the reviewer's satisfaction. 

Author Response

Review 1:

 

Comments:

“English language and style are fine/minor spell check required”.

 

Answer:

Manuscript was revised once more. Additional linguistic proofreading is also one of the steps in the procedure for publishing papers in MDPI, and therefore, in addition to our correction, there will be a linguistic evaluation at the publisher itself before the publication is made publicly available.

 

Comments:

“The authors have addressed all reviewer's comments to the reviewer's satisfaction”.

 

Answer:

We would like to thank the Reviewer for this comment and for notice of value of our research. We are glad that our responses to the reviewer's comments have been fully accepted.

Author Response File: Author Response.docx

Reviewer 3 Report

The authors are requested to improve the representation of the graphs and are also requested to remove the lines.

Author Response

Review 3:

 

Comments:

“English language and style are fine/minor spell check required”.

 

Answer:

Manuscript was revised once more. Additional linguistic proofreading is also one of the steps in the procedure for publishing papers in MDPI, and therefore, in addition to our correction, there will be a linguistic evaluation at the publisher itself before the publication is made publicly available.

 

Comments:

“The authors are requested to improve the representation of the graphs and are also requested to remove the lines”.

 

Answer:

We would like to explain that we analyzed our results using the STATISTICA 13.3 software (TIBCO Software Inc., Palo Alto, CA, USA). Also our graphs for data visualization (column and box plots) were generated in this program during the statistical analysis of the results. In accordance with Reviewer’s suggestion, we tried to improve the representation of the graphs. We also removed the lines. However, the graphical form cannot be changed significantly by the operator. And we do not have any other graphics program, e.g. GraphPad Prism.

We hope that our modifications improve clarity of our figures and manuscript in its current form is acceptable for publication.

Author Response File: Author Response.docx