Papain Suppresses Atopic Skin Inflammation through Anti-Inflammatory Activities Using In Vitro and In Vivo Models
<p>Effects of papain (PN) on the clinical features of the skin of Dfb-induced atopic dermatitis (AD) mice. (<b>A</b>) Dermatitis scores were measured once a week for six weeks. The dermatitis score was defined as the sum of scores graded for each symptom. (<b>B</b>) Transepidermal water loss (TEWL) was measured by the end of six weeks. (<b>C</b>) Serum IgE level was measured using an ELISA kit. (<b>D</b>,<b>E</b>) Protein levels of TNF-α and IL-6 in dorsal skin tissue were determined using ELISA kits. (<b>F</b>,<b>G</b>) Total RNA was prepared from the dorsal skin tissue, and mRNA expression levels of TNF-α and IL-6 were determined via RT-qPCR. # <span class="html-italic">p</span> < 0.05, ## <span class="html-italic">p</span> < 0.01, ### <span class="html-italic">p</span> < 0.001 vs. control group; * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, and *** <span class="html-italic">p</span> < 0.001 vs. Dfb-induced AD group.</p> "> Figure 2
<p>Effects of PN on histological alterations in the skin of Dfb-induced AD mice. (<b>A</b>) Hematoxylin and eosin (H&E) staining of skin lesions in AD mice (scale bar = 200 μm). (<b>B</b>) Toluidine blue staining of skin lesions in AD mice (scale bar = 200 μm). (<b>C</b>) Determination of epidermal thickness. Through the H&E-stained sections, epidermal thickness was measured under a microscope. (<b>D</b>) Through the toluidine blue-stained sections, mast cell infiltration was shown as the average count in five fields. This can be confirmed through the brown arrow. Data represent mean ± standard deviation (SD) from three independent experiments. ### <span class="html-italic">p</span> < 0.001 vs. control group; *** <span class="html-italic">p</span> < 0.001 vs. Dfb-induced AD group.</p> "> Figure 3
<p>Effects of PN on AD-related cytokines and antioxidant makers in the skin of Dfb-induced AD mice. Total RNA was prepared from the dorsal skin tissue, and the mRNA expression levels of (<b>A</b>) IL-4, (<b>B</b>) IL-13, (<b>C</b>) TSLP, (<b>D</b>) IL-17A, (<b>E</b>) IL-17E, (<b>F</b>) IL-17F, (<b>G</b>) NRF-2, and (<b>H</b>) NQO1 were determined via RT-qPCR. Data are presented as the mean ± standard deviation from triplicate experiments. # <span class="html-italic">p</span> < 0.05, ## <span class="html-italic">p</span> < 0.01, ### <span class="html-italic">p</span> < 0.001 vs. control group; * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, and *** <span class="html-italic">p</span> < 0.001 vs. Dfb-induced AD group.</p> "> Figure 4
<p>Effects of PN on the activation of MAPKs and STAT1 in the skin of Dfb-induced AD mice. Total proteins were prepared from the dorsal skin, and Western blotting was performed for the determination of (<b>A</b>) p-IκBα, p-STAT1, STAT1, (<b>B</b>) p-ERK, ERK, p-JNK, JNK, p-p38, and p38 using specific antibodies. β-actin was used as an internal control. Densitometric analysis was determined via Bio-Rad Quantity One<sup>®</sup> 5.x Software. Data are presented as the mean ± standard deviation from triplicate experiments. ### <span class="html-italic">p</span> < 0.001 vs. control group; *** <span class="html-italic">p</span> < 0.001 vs. Dfb-induced AD group.</p> "> Figure 5
<p>Effects of PN on NF-κB activation in TNF-α/IFN-γ-stimulated HaCaT keratinocytes. (<b>A</b>) Viability of HaCaT keratinocytes was measured using MTT assay. *** <span class="html-italic">p</span> < 0.001 vs. non-treated group. Total proteins were prepared, and Western blotting was performed for the determination of (<b>B</b>) HO-1, (<b>C</b>) p-IκBα, and NF-κB p65. β-actin, α-tubulin, and PARP were used as internal control. ## <span class="html-italic">p</span> < 0.01, ### <span class="html-italic">p</span> < 0.001 vs. control group; * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, and *** <span class="html-italic">p</span> < 0.001 vs. TNF-α/IFN-γ-stimulated group.</p> "> Figure 6
<p>Effects of PN on the activation of MAPKs and STATs in TNF-α/IFN-γ-stimulated HaCaT keratinocytes. Total proteins were prepared, and Western blotting was performed for the determination of (<b>A</b>) p-STAT1, STAT1, p-STAT6, STAT6, (<b>B</b>) p-ERK, ERK, p-JNK, and JNK. β-actin was used as an internal control. Densitometric analysis was determined via Bio-Rad Quantity One<sup>®</sup> 5.x Software. Data are presented as the mean ± standard deviation from triplicate experiments. ### <span class="html-italic">p</span> < 0.001 vs. control group; * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, and *** <span class="html-italic">p</span> < 0.001 vs. TNF-α/IFN-γ-stimulated group.</p> "> Figure 7
<p>Effects of PN on oxidative damage in H<sub>2</sub>O<sub>2</sub>-induced HaCaT keratinocytes and Dfb-induced AD mice. (<b>A</b>) Viability of HaCaT keratinocytes after treatment with PN for 1 h and H<sub>2</sub>O<sub>2</sub> for 4 h, measured using Cell Counting Kit-8 assay. (<b>B</b>) HaCaT cells were labeled with a DCF-DA probe for fluorescent detection, and a representative ROS image was selected (10×). (<b>C</b>) Fluorescence microscopic image of ROS induced using H<sub>2</sub>O<sub>2</sub> after treatment with PN. Total proteins were prepared, and Western blotting was performed for the determination of (<b>D</b>) SOD1, SOD2, GPx-4, (<b>E</b>) NQO1, HO-1, and (<b>G</b>) Nrf2. β-actin was used as internal control. (<b>F</b>) Histological sections of dorsal skin tissue were immunohistochemically stained with HO-1 antibody, expressed as brown spots (Scale bar = 200 µm). Data are presented as the mean ± standard deviation from triplicate experiments. ### <span class="html-italic">p</span> < 0.001 vs. control group; ** <span class="html-italic">p</span> < 0.01, and *** <span class="html-italic">p</span> < 0.001 vs. TNF-α/IFN-γ-stimulated group or Dfb-induced AD group.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals and Reagents
2.2. Dfb-Induced AD Model
2.3. Dermatitis Severity Score
2.4. Measurement of Transepidermal Water Loss
2.5. IgE Measurement
2.6. Quantification of Cytokine Levels
2.7. Histological Analysis and Immunohistochemical Analysis
2.8. RT-qPCR Analysis
2.9. Cell Viability
2.10. Cell Culture and Sample Treatment
2.11. Western Blot Analysis
2.12. Intracellular Reactive Oxygen Species Assay
2.13. Statistical Analysis
3. Results
3.1. PN Alleviated the Severity of AD-like Skin Lesions and Inflammatory Response in Dfb-Induced AD Mice
3.2. PN Improved Histological Alterations in the Skin of Dfb-Induced AD Mice
3.3. PN Downregulated AD-Related Cytokines and Upregulated Anti-Oxidative Markers in Dfb-Induced AD Mice
3.4. PN Inhibited the Activation of STAT1 and MAPKs in Dfb-Induced AD Mice
3.5. PN Inhibited the NF-κB Signaling Pathway in TNF-α/IFN-γ-Induced HaCaT Keratinocytes
3.6. PN Inhibited the Activation of STATs and MAPKs in TNF-α/IFN-γ-Stimulated HaCaT Keratinocytes
3.7. PN Reduced Oxidative Damage in HaCaT Keratinocytes and Dfb-Induced AD Mice
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
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Kim, H.-M.; Kang, Y.-M.; Lee, M.; An, H.-J. Papain Suppresses Atopic Skin Inflammation through Anti-Inflammatory Activities Using In Vitro and In Vivo Models. Antioxidants 2024, 13, 928. https://doi.org/10.3390/antiox13080928
Kim H-M, Kang Y-M, Lee M, An H-J. Papain Suppresses Atopic Skin Inflammation through Anti-Inflammatory Activities Using In Vitro and In Vivo Models. Antioxidants. 2024; 13(8):928. https://doi.org/10.3390/antiox13080928
Chicago/Turabian StyleKim, Hye-Min, Yun-Mi Kang, Minho Lee, and Hyo-Jin An. 2024. "Papain Suppresses Atopic Skin Inflammation through Anti-Inflammatory Activities Using In Vitro and In Vivo Models" Antioxidants 13, no. 8: 928. https://doi.org/10.3390/antiox13080928