Glutathione Trisulphide Improves Skin Brightness with Anti-Melanogenesis Effects and Maintains Intracellular Persulphide Levels

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

All the required comments are in the manuscript

Comments for author File: Comments.pdf

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

Q1. Methodology does not appear here in the abstract.

A1. We wish to thank the reviewer for your suggestion. We have added the methodology in abstract as followed.  

 

In this study, B16-F0 cells were used to evaluate the melanin and intracellular per-sulphide levels by alpha-melanocyte stimulating hormone (α-MSH). Moreover, the effects of GSSSG on melanin production.

 

Q2 There were some type and grammar errors in the text.

We sincerely appreciate your careful review and thoughtful corrections to both the comments and the manuscript. Your attention to detail was incredibly helpful, and thanks to your support, the content has improved significantly. I have rewritten all the type and grammar errors. Thank you very much for your kind assistance.

 

Reviewer 2 Report

Comments and Suggestions for Authors

In my opinion, the paper can be accepted in its form. All scientific aspect is okay for me.

Author Response

We sincerely appreciate your careful review and thoughtful corrections to both the comments and the manuscript. Your attention to detail was incredibly helpful, and thanks to your support, the content has improved significantly. I have rewritten all the type and grammar errors. Thank you very much for your kind assistance.

Reviewer 3 Report

Comments and Suggestions for Authors

The study aimed to elucidate the effect of melanogenesis on intracellular persulphide abundance and synthesis, and the efficacy of glutathione trisulphide in inhibiting melanogenesis in melanoma cells. The results indicated that α-MSH-induced melanogenesis significantly increased melanin production and decreased intracellular persulphide levels. The expression of persulphide synthesis genes, including Cars2 and Cbs, was significantly downregulated by α-MSH. Glutathione trisulphide significantly suppressed α- MSH-induced melanin production and restored the intracellular persulphide levels reduced by α-MSH and upregulated Cars2 and Cbs expression.

1. Several publish papers are worked on the finding of glutathione or glutathione disulphide decreasing melanogenesis or melanin content in the skin. This study studying the effects of glutathione trisulphide on skin brightness with anti-melanogenesis effects. The advance of this study must state clearly especially for the similar study design.
2. Review this work and related papers, it seems that increasing the sulphide levels in melanocytes can reduce melanin concentration. This study should conduct more experiments to confirm that glutathione trisulphide can indeed reduce melanin in the skin.
3. The Figures of the manuscript should be clearly presented and explained for facilitate reading and understanding of the readers.
4. The statistical analysis methods should be described in the text.
5. The experimental results should present the results of statistical analysis.
6. The results were insufficient discussed. The discussion section should be written in more depth and compared with more relevant papers
7. There were some type and grammar errors in the text. Please check the manuscript carefully.

Comments on the Quality of English Language

1. There were some type and grammar errors in the text. Please check the manuscript carefully.

Author Response

The study aimed to elucidate the effect of melanogenesis on intracellular persulphide abundance and synthesis, and the efficacy of glutathione trisulphide in inhibiting melanogenesis in melanoma cells. The results indicated that α-MSH-induced melanogenesis significantly increased melanin production and decreased intracellular persulphide levels. The expression of persulphide synthesis genes, including Cars2 and Cbs, was significantly downregulated by α-MSH. Glutathione trisulphide significantly suppressed α-MSH-induced melanin production and restored the intracellular persulphide levels reduced by α-MSH and upregulated Cars2 and Cbs expression.

 

Q1. Several publish papers are worked on the finding of glutathione or glutathione disulphide decreasing melanogenesis or melanin content in the skin. This study studying the effects of glutathione trisulphide on skin brightness with anti-melanogenesis effects. The advance of this study must state clearly especially for the similar study design.

A1. We strongly appreciate the reviewer's comment on this. We have added the additional information about GSH in vitro study and discuss the advance in discussion part as followed. (p10. Line 269)

 

Several published studies have reported that GSH or GSSG can reduce melanogenesis or melanin content in the skin [5]. In our study, we focused on GSSSG and its potential anti-melanogenic effects. In previous study, the melanin-suppressing effects of GSH was observed at concentrations in the mM range, whereas in our study, GSSSG showed significant inhibitory effects even at several tens of μM levels [33]. This difference may be attributed to distinct mechanisms by which intracellular supersulphide levels are regulated, suggesting a unique role of GSSSG in modulating melanogenesis not found in GSH and GSSG.

 

Q2. Review this work and related papers, it seems that increasing the sulphide levels in melanocytes can reduce melanin concentration. This study should conduct more experiments to confirm that glutathione trisulphide can indeed reduce melanin in the skin.

A1. We wish to thank the reviewer for your insightful comment. I completely agree that further experiments are necessary to confirm the melanin-reducing effect of glutathione trisulphide in the skin. In this submission, we have categorized the manuscript as a communication to share our preliminary findings. We plan to investigate the skin-related effects in more detail in future studies.

 

Q3. The Figures of the manuscript should be clearly presented and explained for facilitate reading and understanding of the readers.

A3. Thank you very much for your comments. In response to the reviewer’s comment, I have added statistical bars to the figure to enhance clarity and facilitate understanding . I hope this revision meets your expectations.

Q4. The statistical analysis methods should be described in the text.

A1. We greatly appreciate the reviewer’s valuable comment on this point. In response, we have added the statistical analysis methods to the manuscript as follows:

Statistical analyses were performed using EZR version 4.1.2 (Saitama Medical Centre, Saitama, Japan). All data are presented as mean ± SD. Comparisons between the α-MSH group and other groups were conducted using one-way analysis of variance (ANOVA), followed by Dunnett’s test. Statistical significance was defined as *p < 0.05, **p < 0.01, and ***p < 0.001.

 

Q5. The experimental results should present the results of statistical analysis.

We wish to thank for your comment. We consider that the statistical analyses are adequately presented in the Figures, Figure legends, and the Materials and Methods section.

If the reviewer’s intention is to request explicit mention of p-values in the main text, we would like to maintain the current format, as including all statistical values may complicate the text due to the large number of experimental conditions.

 

Q6. The results were insufficient discussed. The discussion section should be written in more depth and compared with more relevant papers

A6. We sincerely thank the reviewer for pointing out the need for a more in-depth discussion. In response, we have added further considerations regarding the following points, including comparisons with previous studies and interpretations of our current findings:

 

・About albumin persulphide(p10. Line 243)

Previous reports on albumin persulphide have demonstrated its inhibitory effect on melanin production through the suppression of ROS and NO. In the present study, we further investigated the effects of GSSSG on key melanogenesis-related factors such as Tyr and Mitf. Although further studies are needed to clarify whether there are differences in the effects between albumin persulphide and GSSSG, our findings suggest that persulphide compounds may exert these biological effects.

 

・About the difference with GSH, GSSSG, K.A and GSSSG(p10. Line 269)

Several published studies have reported that GSH or GSSG can reduce melano-genesis or melanin content in the skin [5]. In our study, we focused on GSSSG and its potential anti-melanogenic effects. In previous study, the melanin-suppressing effects of GSH was observed at concentrations in the mM range, whereas in our study, GSSSG showed significant inhibitory effects even at several tens of μM levels [33]. This dif-ference may be attributed to distinct mechanisms by which intracellular supersulphide levels are regulated, suggesting a unique role of GSSSG in modulating melanogenesis not found in GSH and GSSG. Furthermore, our study revealed that GSSSG suppresses tyrosinase enzymatic activity and exhibits antioxidant effects (Figure 5). These findings suggest that GSSSG may exert higher anti-melanogenic activity than conventional whitening agents such as GSH or K.A, possibly due to its ability to comprehensively activate multiple mechanisms involved in melanogenesis suppression (Figure 3-a).

 

Q7. There were some type and grammar errors in the text. Please check the manuscript carefully.

A1. Thank you very much for your insightful comment. We have carefully re-reviewed the manuscript in response to your comment regarding typographical and grammatical errors. Corrections have been made, and the changes are tracked in the revision history. We hope the revised version meets your expectations.