An Aqueous Extract of Beta vulgaris subsp. Vulgaris Beetroot Group Reduces Lipid Accumulation in Human Keratinocyte Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript examines the biological activity of an aqueous extract obtained from Beta vulgaris leaves on lipid metabolism and stress responses in human keratinocytes, with a focus on lipotoxicity, oxidative stress, and endoplasmic reticulum homeostasis. Overall, the manuscript presents a coherent and comprehensive data set with generally sound experimental approaches. However, several methodological limitations, data interpretation issues, and insufficient mechanistic validation weaken the strength of the conclusions. Therefore, I recommend substantial revision, as significant clarification, additional testing, and revision of the statements are necessary to make the manuscript suitable for publication.

Abstract:
The abstract overstates its mechanistic conclusions, implying causal links between BvE treatment and signaling pathways such as AMPK and PPARα without direct functional confirmation. The authors should soften their statements to reflect associative rather than definitive mechanistic evidence.

Introduction:
The introduction is too long and contains sections that could be shortened without loss of clarity. Simplifying the discussion of thermoregulation and systemic metabolic diseases would allow for a better focus on the central research question.

The rationale for choosing beet leaf extract over root extracts, which are more frequently studied, should be more clearly explained, including potential differences in metabolite composition and their relevance for dermatological use.

The AMPK/PLIN2 axis hypothesis requires a clearer distinction between previous data obtained in other cell types and the novelty of testing this pathway specifically in keratinocytes.

Results:
Figure 2: The functional significance of the identified metabolites is not well integrated into subsequent analyses. The authors should explicitly link key metabolites, such as betaine or sugars, to the observed biological effects.

Figure 3: The Western blot analysis of UPR markers lacks a positive control for an ER stress inducer, limiting the interpretation of whether BvE actively reduces stress or simply reduces basal expression levels.

Section 2.3: The conclusion that BvE suppresses UPR signaling across all branches may be exaggerated, as only protein abundance is measured. Additional functional analyses, such as reporter activity or downstream transcriptional targets, are needed.

Figure 4: The DCFH-DA assay is suitable for measuring ROS, but lacks specificity for different types of ROS. The authors should acknowledge this limitation and avoid overinterpreting oxidative stress modulation.

Figure 4: The observed decrease in catalase and GPx-4 levels with BvE treatment contradicts the logic of an antioxidant effect. Explain this effect.

Figure 5: How was fluorescence intensity normalized to cell count or protein content?

Section 2.6: The interpretation of MAGL regulation is somewhat speculative. Additional measurements of lipolytic activity or free fatty acid release are needed to confirm claims regarding lipid metabolism.

Figure 5: TLC-based lipid analysis has limited resolution compared to lipidomic approaches. Authors should acknowledge this limitation and avoid overgeneralizing lipid class redistribution.

Figure 6: AMPK activation is inferred from the pAMPK/AMPK ratio, but upstream or downstream functional assays are not presented. Authors should consider including data on pharmacological inhibition or genetic modulation.

Section 2.8: The conclusion that BvE "supports" fatty acid oxidation is not directly proven, as no measurements of β-oxidation flux or oxygen consumption are presented.

Figure 6: Mitochondrial membrane potential data are suggestive and should be supplemented with additional functional assays, such as ATP production or the Seahorse assay, to confirm mitochondrial effects.

The overall quality of the figures should be substantially improved, as several images appear low in resolution and lack sufficient clarity for accurate interpretation. I strongly recommend increasing the resolution of all figures, enhancing contrast where necessary, and enlarging the font size of labels and legends.

Methods:
The reproducibility of the extract composition is poorly documented. Authors should provide information on batch-to-batch variability or quality control measures beyond betanin quantification.

The selection of a single BvE concentration (1 μg/mL) for most experiments, based solely on viability, may not reflect dose-dependent biological effects. At least one additional concentration should be included.

The statistical analysis lacks detailed information on the assays used, the assumptions tested, and whether corrections for multiple comparisons were applied. This information should be clearly stated.

The use of HCT15 cells as a secondary epithelial model is appropriate, but the rationale for their choice and their relevance to skin biology should be clarified.

Discussion:
The discussion is comprehensive and integrates the findings with existing literature, but often goes beyond the available data. Several mechanistic interpretations, particularly those related to PLIN2 phosphorylation and CMA-mediated degradation, were not experimentally tested in this study and should be presented as hypotheses.

The statement of "first evidence" should be tempered unless a systematic literature search confirms the absence of previous studies of beetroot extracts in keratinocytes.

The translational implications for dermatological applications are interesting but speculative. The authors should highlight the limitations of in vitro models and avoid overextending clinical relevance.

The potential contribution of individual extract components is discussed, but fractionation or comparative analysis was not performed. This limitation should be clearly acknowledged.

The manuscript lacks a distinct Conclusion section, which is essential for clearly summarizing the main findings and their broader implications. 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The paper effectively maps the downstream consequences of BvE treatment but without identifying the Molecular Initiating Event. We  know antioxidants reduce ROS. The claim that this works via the AMPK/PLIN2 axis is supported, but this pathway is already well-known for betaine. This work still has potential for IJMS, but authors need to show what makes this specific complex mixture superior to its individual components.

The following points must be addressed before further consideration:

Data shows BvE downregulates UPR markers like GRP78, PERK, and ATF6 even in the absence of an inducer. Lowering the cell’s basal surveillance machinery is not inherently protective and could be a sign of general transcriptional or translational inhibition; this requires explicit clarification.

The abstract is currently sound like a laundry list of "Extract did X and Y". It needs to be reframed to highlight the dual action of chemical ROS scavenging and metabolic TAG reprogramming. Furthermore, the transition from H₂O₂ stress to PA/OA lipotoxicity is too abrupt. The authors should discuss how saturated fatty acids act as a primary source of ER-driven ROS.

The authors must repeat the PLIN2 and lipid droplet assays in the presence of an autophagy inhibitor. If the BvE effect is lost, it would provide necessary proof that the extract works via Chaperone-Mediated Autophagy (CMA).

A significant loss of Complex I is reported following BvE treatment under lipotoxic conditions. Attributing this to remodeling is hypothetical. Without measuring mitophagic flux, using Bafilomycin A1 or LC3-II/Parkin co-localization, it is just as likely the extract is inducing selective mitochondrial degradation.

The authors should compare 1 μg/mL of the extract against a synthetic version containing only the measured amounts of Betanin and Betaine. This will determine if the mentioned compositional complexity and synergy are actually necessary for the observed phenotype.

The extract is added only during the final 24 hours of a 48 hour lipid challenge. While a reduction in TAG is shown, the lipid burden stays significantly higher than control levels. Labeling this therapeutic rebalancing is a stretch without a kinetic study showing the actual rate of lipid clearance.

The extract contains high levels of glucose, fructose, and glutamine. The authors need to prove that the effects are not simply due to keratinocytes using these metabolites as an alternative energy source, which would naturally alter the AMPK and lipid profiles.

Evidence must be provided to show the extract prevents palmitic acid-induced ER membrane rigidification. This is the missing link between the lipid redistribution data and the observed UPR suppression.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This study investigated the effects of an aqueous extract from Beta vulgaris subsp. vulgaris Beetroot Group (BvE) on lipid metabolism and stress responses in human keratinocytes. Interesting results have been obtained, and the manuscript is well written. However, there are some issues need to be addressed before it may be considered for publication.

1. More information about the Beta vulgaris should be introduced in the introduction section.
2. For the chemical characterization of BvE, (1) LC-MS analysis can be performed to tentatively identify the chemical structures of compounds in BvE; (2) the content of anthocyanidin, nitrate, phenolic acids and flavonoids, etc. can be determined; (3) the 13C NMR may be supplemented with 1H NMR analysis results.
3. Are there any positive controls for the tests? Is it possible to perform animal study?
4. The naturally growth Beta vulgaris may be included in the study for comparison.
5. The bioactive constituents, such as betaine, can be tested along with BvE.
6. In the “BvE reduces H₂O₂-induced oxidative stress”, the ascorbic acid (Vitamin C) may be tested for comparison.
7. If necessary, a separated conclusion section can be added, and the shortcomings of present work and future study can be emphasized.
8. The “Beta vulgaris” in the abstract should be in Italics font. The “(BvE)” can be removed from the subtitle. The “4.500 rpm” can be changed to “4,500 rpm”, and its conversion to centrifugal force “g” can be supplemented. The approximate temperature “room temperature” can be indicated in the text.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have adequately addressed the major concerns raised during the first round of review, and the manuscript has been substantially improved. Although minor issues related to language polishing and figure readability remain, these can be addressed during the final editorial stage. In its current form, I recommend acceptance of the manuscript for publication.

Author Response

Thank you for the revision

Reviewer 2 Report

Comments and Suggestions for Authors

Authors have attended all my comments. The manuscript is ready for publication.

Author Response

Thank you for the revision

Reviewer 3 Report

Comments and Suggestions for Authors

Most of the comments have been addressed, and the manuscript has been carefully revised. Although some of the suggested experiments can not be supplemented in current study, the authors may discuss them in the Discussion or Conclusion section.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf