Topically Applied Molecular Hydrogen Normalizes Skin Parameters Associated with Oxidative Stress: A Pilot Study

Round 1

Reviewer 1 Report

1. The topic has good potential, particularly regarding the role of antioxidants in retinal degeneration. However, the manuscript reads more like a summary of known facts rather than offering new insights or critical analysis. I’d expect a stronger narrative or hypothesis-driven discussion.
2. The abstract feels generic and doesn’t clearly convey the core findings or take-home messages. It would benefit from a more focused summary of the review’s main conclusions and a clearer statement on the gaps this paper addresses.
3. Several sections repeat similar content (e.g., the mechanisms of oxidative stress are rephrased multiple times). This redundancy weakens the impact. Consider streamlining the text to avoid repeating ideas unnecessarily.
4. Figures 1 and 2 are informative but appear to be adapted from previously published concepts without much novelty. A schematic specifically created for this paper that integrates the review’s key points would strengthen the visual impact.
5. It’s not clear how the authors selected the articles included in the review. 
6. The table summarizes antioxidant therapies but lacks context—such as the stage of disease or specific models used. Including a column for “Evidence strength” or “Preclinical vs. clinical” would make it more useful.
7. Although oxidative stress is repeatedly mentioned, there is minimal discussion on specific molecular pathways (e.g., Nrf2, MAPK) beyond surface-level descriptions. 
8. The paper mostly summarizes supportive findings. Including contradictory or inconclusive results would add credibility and demonstrate the authors’ critical engagement with the literature.
9. The conclusion only briefly touches on gaps in the field. 
10. While the English is generally understandable, some awkward phrasing and grammar slips remain (e.g., inconsistent verb tenses, overly long sentences). 

1. The topic has good potential, particularly regarding the role of antioxidants in retinal degeneration. However, the manuscript reads more like a summary of known facts rather than offering new insights or critical analysis. I’d expect a stronger narrative or hypothesis-driven discussion.
2. The abstract feels generic and doesn’t clearly convey the core findings or take-home messages. It would benefit from a more focused summary of the review’s main conclusions and a clearer statement on the gaps this paper addresses.
3. Several sections repeat similar content (e.g., the mechanisms of oxidative stress are rephrased multiple times). This redundancy weakens the impact. Consider streamlining the text to avoid repeating ideas unnecessarily.
4. Figures 1 and 2 are informative but appear to be adapted from previously published concepts without much novelty. A schematic specifically created for this paper that integrates the review’s key points would strengthen the visual impact.
5. It’s not clear how the authors selected the articles included in the review. 
6. The table summarizes antioxidant therapies but lacks context—such as the stage of disease or specific models used. Including a column for “Evidence strength” or “Preclinical vs. clinical” would make it more useful.
7. Although oxidative stress is repeatedly mentioned, there is minimal discussion on specific molecular pathways (e.g., Nrf2, MAPK) beyond surface-level descriptions. 
8. The paper mostly summarizes supportive findings. Including contradictory or inconclusive results would add credibility and demonstrate the authors’ critical engagement with the literature.
9. The conclusion only briefly touches on gaps in the field. 
10. While the English is generally understandable, some awkward phrasing and grammar slips remain (e.g., inconsistent verb tenses, overly long sentences). 

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

1. The topic has good potential, particularly regarding the role of antioxidants in retinal degeneration. However, the manuscript reads more like a summary of known facts rather than offering new insights or critical analysis. I’d expect a stronger narrative or hypothesis-driven discussion.

Response:

Thank you for this valuable comment. We acknowledge that the previous version of the manuscript had a predominantly descriptive tone. In response, we have implemented several key revisions aimed at strengthening the manuscript’s critical and analytical framework:

A new subsection entitled 3.8. Limitations and Context-Dependent Effects of Molecular Hydrogen (lines 769 – 791, page 22) has been added to the Discussion. This subsection integrates clinical and meta-analytical findings from non-dermatological disciplines, including respiratory medicine, aging biology, and exercise physiology, to illustrate that the therapeutic effects of molecular hydrogen are highly context-dependent and influenced by factors such as tissue type, delivery method, and study design. This allows for a more nuanced and interdisciplinary interpretation of our findings.

The same subsection also explicitly discusses the limitations of our own study, such as the absence of a control group, small sample size, short observation period, and lack of biochemical oxidative stress markers. We also emphasize the need for future research to incorporate expanded diagnostic protocols (e.g., Courage + Khazaka® instrumentation) and more heterogeneous populations. This transparent self-assessment supports a more critical evaluation of our results.

The Introduction have been revised to broaden the biological scope of the manuscript. In particular, we included emerging evidence on the effects of molecular hydrogen not only in the retina but also in periodontal tissues, cancer therapy, and the central nervous system. These additions highlight the systemic and multifaceted nature of H₂ action, reinforcing the relevance of studying its topical application in the skin from a whole-body perspective (lines 56-102, pages 2-3).

The central hypothesis—that molecular hydrogen can modulate key skin parameters via oxidative and inflammatory pathways—is now clearly articulated in both the Abstract and the Introduction. Its validity is critically examined in light of the wider body of evidence from both dermatological and non-dermatological fields.

2. The abstract feels generic and doesn’t clearly convey the core findings or take-home messages. It would benefit from a more focused summary of the review’s main conclusions and a clearer statement on the gaps this paper addresses.

Response 2:

Thank you for this valuable feedback. We have revised the Abstract to provide a clearer and more focused summary of the study’s key findings. The revised abstract emphasizes the following points: a clear hypothesis is stated, the number of participants and the nature of the treatment are specified. Quantitative results are summarized, including improvements in porphyrins, pigmentation irregularities, pore size, wrinkle severity, and reduction in biological skin age. The study’s limitations are acknowledged, including the absence of a control group and a short follow-up period. The revised abstract highlights the gap in the literature regarding standardized clinical assessments of topical hydrogen treatments and positions this study as addressing that gap. These revisions make the abstract more concise, informative, and focused, aligning it with the main conclusions of the study and highlighting its novelty and significance.

3. Several sections repeat similar content (e.g., the mechanisms of oxidative stress are rephrased multiple times). This redundancy weakens the impact. Consider streamlining the text to avoid repeating ideas unnecessarily.

Response 3:

Thank you for the comment. We carefully revised the manuscript to eliminate redundancies. Repetitive content was removed or rephrased, and each section now contributes information, improving clarity and focus.

4. Figures 1 and 2 are informative but appear to be adapted from previously published concepts without much novelty. A schematic specifically created for this paper that integrates the review’s key points would strengthen the visual impact.

Response 4:

Thank you for the valuable suggestion. In response, we have created an original scheme (now included as Figure 6, page 17) specifically designed for this manuscript. The figure integrates the mechanistic pathways and clinical effects of molecular hydrogen on the skin.

This scheme visually summarizes the hypothesized sequence of events:

External stressors such as UV radiation, pollutants, diet, and psychological stress lead to the formation of reactive oxygen and nitrogen species (ROS/RNS); Molecular hydrogen selectively neutralizes harmful species (e.g., •OH, ONOO⁻). Hydrogen modulates key signalling pathways involved in oxidative stress and inflammation, including the Nrf2, MAPK (ERK, JNK, p38), and NF-κB pathways. This molecular modulation contributes to observed clinical outcomes such as reduced porphyrins, pore size, wrinkle severity, and skin discoloration (brown and red spots).

The scheme serves to visually connect the molecular basis of hydrogen's action with the measurable improvements observed in our clinical analysis, thereby strengthening the mechanistic and translational relevance of the study.

5. It’s not clear how the authors selected the articles included in the review.

Response 5:

We acknowledge the importance of clearly describing our literature selection process. In response, we have added a description of the methodology used for selecting articles to the Materials and Methods section 2.8. Literature Search and Selection Criteria (page 6).

6. The table summarizes antioxidant therapies but lacks context—such as the stage of disease or specific models used. Including a column for “Evidence strength” or “Preclinical vs. clinical” would make it more useful.

Response 6:

We have addressed this comment by creating a new table (Table 5, page 18) that summarizes key studies on molecular hydrogen’s effects on skin health and oxidative stress. This table includes an additional column for “Evidence strength” (preclinical vs. clinical) and “Model/Disease stage” (e.g., in vitro, animal, human studies). The table has been placed in the Discussion section, immediately after the detailed description of molecular pathways and Figure 6, which integrates clinical findings and mechanistic insights. This positioning ensures logical flow from clinical results to molecular mechanisms and literature summary, enhancing the manuscript's clarity and context.

7. Although oxidative stress is repeatedly mentioned, there is minimal discussion on specific molecular pathways (e.g., Nrf2, MAPK) beyond surface-level descriptions.

Response 7:

The revised Introduction provides a clearer and more concise mention of key molecular pathways, including the activation of Nrf2 and suppression of NF-κB by molecular hydrogen (lines 114-120). However, we recognize that a more detailed discussion of these pathways is essential.

We have expanded the Discussion section to provide a more detailed analysis of the molecular pathways by which hydrogen exerts its effects on the skin. Specifically, we included descriptions of: Nrf2 pathway activation and upregulation of antioxidant enzymes (HO-1, NQO1, GST), NF-κB pathway suppression, resulting in reduced pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), emerging evidence of MAPK modulation, including ERK, JNK, and p38 pathways relevant to skin aging and oxidative stress responses. These additions strengthen the mechanistic foundation of our findings and provide essential context for interpreting the clinical improvements observed (lines 628 – 652 and Figure 6, page 16-17).

Furthermore, we created Table 5, a comprehensive summary of key studies on molecular hydrogen’s effects in skin health and oxidative stress. This table includes a new column for “Evidence strength” (preclinical/clinical/Review) and “Model/Disease stage” to clearly indicate the type of study and its context. This addition not only addresses the need for deeper discussion of specific pathways (Comment 7) but also incorporates and fully addresses Comment 6, ensuring that both the mechanistic insights and the literature summary are logically integrated and enhance the clarity and scientific rigor of the manuscript.

8. The paper mostly summarizes supportive findings. Including contradictory or inconclusive results would add credibility and demonstrate the authors’ critical engagement with the literature.

Response 8:

In the revised discussion, we have incorporated findings from clinical and mechanistic studies that report inconsistent or non-significant effects of hydrogen-based interventions. This addition provides a more balanced and critical assessment of the current evidence, highlighting the variability of outcomes depending on study design, population, delivery method, and disease context. We believe this enhances the credibility and scientific depth of the manuscript (lines 769-791, page 22)

9. The conclusion only briefly touches on gaps in the field.

Response 9:

We expanded the Conclusion (page 22) to more clearly highlight current gaps in the field, including the lack of standardized clinical protocols for topical hydrogen application, the absence of long-term safety data, and the need for biomarker-based evaluations. These additions strengthen the contextual relevance of our findings and outline specific directions for future research. The revised conclusion now more clearly addresses the broader implications and limitations of current knowledge.

10. While the English is generally understandable, some awkward phrasing and grammar slips remain (e.g., inconsistent verb tenses, overly long sentences).

Response 10:

We appreciate the reviewer’s note regarding language clarity. The manuscript has been carefully revised for grammar, style, and clarity by a Prof. Jerzy Pałka with scientific writing experience.

 

General Revisions (Figures):

During the revision process, we conducted a detailed review of all figures and their captions. We discovered that in a few isolated instances, identical images had been inadvertently used in both “before” and “after” panels. These errors were unintentional and have now been fully corrected. We replaced the duplicate images with the correct original photographs corresponding to each condition (e.g., post-treatment), ensuring that all visual data accurately reflect the experimental outcomes.

In addition, all figure legends were revised for clarity, consistency, and completeness.

We thank the Reviewer for helping us to improve the scientific accuracy of the manuscript.

Reviewer 2 Report

In the present study the authors have tested the antioxidant effects of molecular hydrogen (H2) in skin. H2 was tested in three different populations, young, middle and old. H2 acts by binding to hydroxyl radicals but does not neutralize the beneficial radicals. H2, due to its low MW can easily penetrate SC and cellular membranes thus reaching the mitochondria and the nucleus. Further, H2 can upregulate the Nrf2 pathway and downregulate the NF-kB pathway.

Previously, it was found that hydrogen-rich baths and facial cleansing routines increased significantly in aesthetic practices.

Here, the authors found that H2 generated through water electrolysis in a specific device improved the skin surface in multiple parameters exerting potential antioxidant but also antiaging effects.

The study is interesting but there are some points that need to be addressed.

Specifically,

1. In Line 292 the authors report on the desquamation and attribution to dehydration etc. This is not correct desquamation is due to pH regulated epidermal proteolytic cascade (for an updated version please see Sotiropoulou et al. 2022 Reconstructing the epidermal proteolytic cascades in health and disease. J Pathol 257: 545-560)
2. The authors reported that H2 treatment improved the inflammation in an AD patient (Figure 2). AD patients should be described in detail in Materials and Methods. The way the Materials and Methods are written indicates that all participants are healthy.
3. What are the quantification parameters in Figure 3? How do they correlate with Table 3? The values in Figure 3 are not in accordance with Table 3, e.g. porphyrins in Table 3 are <30 and in Table 3 >75. The details of quantification should be added in text and in Materials and Methods.
4. The same for Table 4 and Figure 5. On the contrary, comparison of Table 5 with Figure 6 appears to be correct. Figure 6 depicts the data from Table 5.

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.

1. In Line 292 the authors report on the desquamation and attribution to dehydration etc. This is not correct desquamation is due to pH regulated epidermal proteolytic cascade (for an updated version please see Sotiropoulou et al. 2022 Reconstructing the epidermal proteolytic cascades in health and disease. J Pathol 257: 545-560)

Response:

Thank you for pointing out the inaccurate description of the desquamation process. We have revised the original text to accurately reflect this mechanism, adding a citation to the work by Sotiropoulou et al. (2022). (Lines 354-359, page 8, References [21], page 25)

2. The authors reported that H2 treatment improved the inflammation in an AD patient (Figure 2). AD patients should be described in detail in Materials and Methods. The way the Materials and Methods are written indicates that all participants are healthy.

Response 2:

We would like to clarify that the participant in question, despite having a documented diagnosis of AD, was in clinical remission at the time of the study and exhibited only symptoms of impaired barrier function. The presence of AD did not pose a contraindication for participation in molecular hydrogen treatment. The inclusion criteria were carefully considered to exclude active skin diseases or conditions that could affect the outcomes or pose safety concerns. Since the participant met the inclusion criteria and had no active contraindications, they were included in the study. To address this point and ensure clarity in the manuscript, we have revised the Materials and Methods section as follows (lines 154-158, page 4).

3. What are the quantification parameters in Figure 3? How do they correlate with Table 3? The values in Figure 3 are not in accordance with Table 3, e.g. porphyrins in Table 3 are <30 and in Table 3 >75. The details of quantification should be added in text and in Materials and Methods.

Response 3:

Regarding the quantification parameters in Figure 3 and their alignment with Table 3 we have carefully revised both the table (page 12)  and the figure (page 12) to ensure consistency and clarity in the presentation of the data (lines 505-539, page 13-14). Details of the quantification process have been added to the text (lines 517-521, page 13) and to the Materials and Methods 2.6 Skin Analysis and Quantification Parameters section (lines 265-273, page 6).

4. The same for Table 4 and Figure 5. On the contrary, comparison of Table 5 with Figure 6 appears to be correct. Figure 6 depicts the data from Table 5.

Response 4:

We have carefully revised both the table 4 (page 15) and the figure 5 (page 16) to ensure consistency and clarity in the presentation of the data. Details of the quantification process have been added to the Materials and Methods 2.6 Skin Analysis and Quantification Parameters section (lines 265-273, page 6).

General Revisions (Figures): 

During the revision process, we conducted a detailed review of all figures and their captions. We discovered that in a few isolated instances, identical images had been inadvertently used in both “before” and “after” panels. These errors were unintentional and have now been fully corrected. We replaced the duplicate images with the correct original photographs corresponding to each condition (e.g., post-treatment), ensuring that all visual data accurately reflect the experimental outcomes. 

We apologize for mistake.

In addition, all figure legends were revised for clarity, consistency, and completeness. 

We thank the Reviewer for helping us to improve the scientific accuracy of the manuscript. 

Round 2

Reviewer 1 Report

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