Ann Dermatol.  2016 Jun;28(3):352-359. 10.5021/ad.2016.28.3.352.

Ampelopsis japonica Makino Extract Inhibits the Inflammatory Reaction Induced by Pathogen-Associated Molecular Patterns in Epidermal Keratinocytes

• Choi MR
• Choi DK
• Kim KD
• Kim SJ
• Kim DI
• Im M
• Lee Y
• Seo YJ
• Kim CD
• Lee JH

• KMID: 2164643
• DOI: http://doi.org/10.5021/ad.2016.28.3.352

Abstract

BACKGROUND
Keratinocytes are the major cells in epidermis, providing barrier components such as cornified cells through the sophisticated differentiation process. In addition, keratinocytes exerts their role as the defense cells via activation of innate immunity. It has been known that pathogen-associated molecular patterns (PAMPs) including double-strand RNA and nucleotides can provoke inflammatory reaction in keratinocytes.
OBJECTIVE
The aim of this study is to evaluate the effect of Ampelopsis japonica Makino extract (AE) on PAMPs-induced inflammatory reaction of keratinocytes.
METHODS
The effects of AE were determined using poly (I:C)-induced inflammation and imiquimod-induced psoriasiform dermatitis models.
RESULTS
In cultured keratinocytes, AE significantly inhibited poly(I:C)-induced expression of inflammatory cytokines, such as interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor-α. AE significantly inhibited poly(I:C)-induced release of caspase-1 active form (p20), and down-regulated nuclear factor-κB signaling pathway. In imiquimod-induced psoriasiform dermatitis model, topical application of AE resulted in significant reduction of epidermal hyperplasia.
CONCLUSION
These results suggest that AE may be a potential candidate for the treatment of skin inflammation.

Keyword

Ampelopsis; Inflammation; Keratinocytes; Pathogen-associated molecular pattern molecules

MeSH Terms

Ampelopsis*
Cytokines
Dermatitis
Epidermis
Hyperplasia
Immunity, Innate
Inflammation
Interleukin-6
Interleukin-8
Interleukins
Keratinocytes*
Necrosis
Nucleotides
Pathogen-Associated Molecular Pattern Molecules*
RNA
Skin
Cytokines
Interleukin-6
Interleukin-8
Interleukins
Nucleotides
Pathogen-Associated Molecular Pattern Molecules
RNA

Figure

• 

  Fig. 1 Cytotoxicity of Ampelopsis japonica Makino extract (AE). (A) SV40Tag-transformed human epidermal keratinocytes (were treated with AE at the indicated concentrations for 24 hours. Cell viability was measured by MTT assay. (B) Lactate dehydrogenase (LDH) assay. After treatment with AE, culture medium was collected and LDH activity was determined. The mean values±standard deviations are averages of triplicate measurements. *p<0.01.

• 

  Fig. 2 Effect of Ampelopsis japonica Makino extract (AE) on poly(I:C)-induced inflammatory reaction in keratinocytes. SV40Tag-transformed human epidermal keratinocytes were pretreated with AE at the indicated concentrations for 1 hour, and then stimulated with 1 µg/ml poly(I:C) for 2 hours. The messenger RNA (mRNA) level was determined by quantitative real-time polymerase chain reaction. Data are expressed as fold induction. The mean values±standard deviations are averages of triplicate measurements. IL: interleukin, TNF-α: tumor necrosis factor-α. *p<0.01.

• 

  Fig. 3 Effect of Ampelopsis japonica Makino extract (AE) on poly(I:C)-induced cytokine release from keratinocytes. SV40Tag-transformed human epidermal keratinocytes were pretreated with AE at the indicated concentrations for 1 hour, and then stimulated with 1 µg/ml poly(I:C) for 24 hours. Released cytokines were measured by enzyme-linked immunosorbent assay. The mean values±standard deviations are averages of triplicate measurements. IL: interleukin, TNF-α: tumor necrosis factor-α. *p<0.01.

• 

  Fig. 4 (A) Effect of Ampelopsis japonica Makino extract (AE) on poly(I:C)-induced inflammasome activation in keratinocytes. SV40Tag-transformed human epidermal keratinocytes (SV-HKEs) were pretreated with AE at the indicated concentrations for 1 hour, and then stimulated with 1 µg/ml poly(I:C) for 24 hours. Culture medium was collected and concentrated, then subjected to Western blot. The protein level for active form of caspase-1 (p20) was decreased by AE treatment. Ponceau S staining was used for loading control. (B) Effect of AE on poly(I:C)-induced nuclear factor (NF)-κB activation in keratinocytes. SV-HKEs were pretreated with AE at the indicated concentrations for 1 hour, and then stimulated with 1 µg/ml poly(I:C) for 45 minutes. Activation of NF-κB signaling was determined by Western blot. The protein level for phoshorylated-p65 (p-p65) NF-κB subunit was decreased by AE treatment. Actin was used for internal control.

• 

  Fig. 5 Inhibition of imiquimod-induced psoriasiform dermatitis by Ampelopsis japonica Makino extract (AE). (A) BALB/c mice were topically applied with 5% imiquimod cream (Aldara; 3M Health Care Ltd., Leicestershire, United Kingdom) for 7 days. AE (1 mg/ml dissolved in polyethylene glycol) was pretreated 1 hour before imiquimod application. Skin specimens were investigated by hematoxylin and eosin staining (upper panels). Immunohistochemistry analysis was performed to determine the interleukin (IL)-1β level (middle panels), and to determine the cell proliferation in epidermis using anti-proliferating cell nuclear antigen (PCNA) antibody (lower panels). Bar=50 µm. (B) Epidermal thickness was measured. Data are the mean values±standard deviations (n=5). *p<0.01.

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