Camu-Camu Fruit Extract Inhibits Oxidative Stress and Inflammatory Responses by Regulating NFAT and Nrf2 Signaling Pathways in High Glucose-Induced Human Keratinocytes

Round 1

Reviewer 1 Report

This manuscript reveals the mechanisms underlying oxidative stress reduction and anti-inflammatory effects of camu-camu fruit on high glucose-induced skin damage, showing that the camu-camu fruit modulated NF-κB/AP-1, MAPK, and NFAT signaling pathways related to inflammation by down regulating the expression of proinflammatory cytokines and chemokines. The topic is interesting and the experimental design was reasonable. I recommend its publication in Molecules after minor revision.

1. A list of abbreviations accompanying their explanations should be provided.
2. The references should be listed as the requirements of the Journal.

Author Response

Response to Reviewer 1 Comments

Point 1: A list of abbreviations accompanying their explanations should be provided.

Response 1: Thank you for your recommendation. A list of abbreviations accompanying their explanations was added in the revised manuscript. Please check Page 1,2 line 35-53.

Abbreviations:

AP-1: activator protein-1

ARE: antioxidant responsive element

DCFH-DA: 2’,7’-dichlorofluorescin diacetate

DPPH: 1,1-diphenyl-2-picrylhydrazyl

ERK: extracellular signal-regulated kinase

HaCaT: human skin keratinocyte cell line

HO-1: heme oxygenase 1

IL-8: interleukin 8

JNK: c-Jun N-terminal kinase

MAPK: mitogen-activated protein kinase

MDC: human macrophage-derived chemokine

NFAT: nuclear factor of activated T cells

NF-κB: nuclear factor-light-chain-enhancer of activated B cells

NQO1: NAD(P)H: quinone oxidoreductase1

Nrf2: nuclear factor E2-related factor 2

RANTES: regulated upon activation, normal T cell expressed, and secreted

ROS: reactive oxygen species

TARC: thymus- and activation-regulated chemokine

Point 2: The references should be listed as the requirements of the Journal.

Response 2: Thank you for your indication. The references were checked and corrected as the requirements of the Journal. Please check in the revised manuscript Page 13-16 line 425-542.

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Reviewer 2 Report

The authors in their article present research related to the anti-oxidative and anti-inflammatory effects of an extract derived from the fruit of Myrciaria dubia (camu-camu). These effects were examined toward oxidative stress-induced, high glucose treated HaCaT cells.  The extract was found to exert its activity through modulation of the NF-κB/AP-1, MAPK, and NFAT signaling pathways downregulating proinflammatory cytokines and chemokines. Additionally, the upregulation of Nrf2 and NQO1 were induced by camu-camu.

Overall, the authors present interesting findings adding to the current knowledge regarding the bioactivity of Myrciaria dubia. The manuscript is well written and research clearly presented. I have the following questions for the authors regarding their study:

The increase in fluorescence intensity between the control and glucose-treated cells in the histograms does not seem very significant. Could the authors explain the performed analysis of the fluorescence intensity. Did the authors consider different treatment periods with glucose to achieve a more pronounced increase in ROS generation.

In Figure 2B and 2C please add information regarding the probe used to determine ROS generation, either in the figure description or to the axis of the graph or histograms.

In Figure 5 in the determination of p-IKBα levels with Western blot, the changes in the levels of p-IKBα between samples are undistinguishable. Do the authors have a better quality repetition of this experiment that they could include.

The authors propose the use of the camu-camu extract as an alternative to treatment with cyclosporin A. Since the constituent of camu-camu, ellagic acid, has been demonstrated to exert anti-inflammatory and anti-oxidant activity, could the authors comment on the applicability of the cam-camu extract in comparison to ellagic acid in skin inflammation treatment.

I would suggest adding a graphical scheme of the obtained results at the end of the manuscript.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The authors have thoroughly addressed my questions and I therefore find the manuscript appropriate for publication.