Nutr Res Pract.  2016 Aug;10(4):371-376. 10.4162/nrp.2016.10.4.371.

Adenophora remotiflora protects human skin keratinocytes against UVB-induced photo-damage by regulating antioxidative activity and MMP-1 expression

• Kim HK

• KMID: 2390138
• DOI: http://doi.org/10.4162/nrp.2016.10.4.371

Abstract

BACKGROUND/OBJECTIVES
Chronic ultraviolet (UV) exposure-induced reactive oxygen species (ROS) are commonly involved in the pathogenesis of skin damage by activating the metalloproteinases (MMP) that break down type I collagen. Adenophora remotiflora (AR) is a perennial wild plant that inhabits Korea, China, and Japan. The present study investigated the protective effects of AR against UVB-induced photo-damage in keratinocytes.
MATERIALS/METHODS
An in vitro cell-free system was used to examine the scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical and nitric oxide (NO). The effect of AR on ROS formation, antioxidant enzymes, elastase, MMP-1 level, and mRNA expression of MMP-1 were determined in UVB-irradiated human keratinocyte HaCaT cells.
RESULTS
AR demonstrated strong DPPH free radical and NO scavenging activity in a cell-free system exhibiting IC50 values of 1.88 mg/mL and 6.77 mg/mL, respectively. AR pretreatment dose-dependently attenuated the production of UVB-induced intracellular ROS, and antioxidant enzymes (catalase and superoxide dismutase) were enhanced in HaCaT cells. Furthermore, pretreatment of AR prevented UVB-induced elastase and collagen degradation by inhibiting the MMP-1 protein level and mRNA expression. Accordingly, AR treatment elevated collagen content in UVB-irradiated HaCaT cells.
CONCLUSION
The present study provides the first evidence of AR inhibiting UVB-induced ROS production and induction of MMP-1 as a result of augmentation of antioxidative activity in HaCaT human keratinocytes. These results suggest that AR might act as an effective inhibitor of UVB-modulated signaling pathways and might serve as a photo-protective agent.

Keyword

Adenophora remotiflora; UVB-irradiation; HaCaT cell; antioxidative effect; MMP-1

MeSH Terms

Campanulaceae*
Cell-Free System
China
Collagen
Collagen Type I
Humans*
In Vitro Techniques
Inhibitory Concentration 50
Japan
Keratinocytes*
Korea
Metalloproteases
Nitric Oxide
Pancreatic Elastase
Plants
Reactive Oxygen Species
RNA, Messenger
Skin*
Superoxides
Collagen
Collagen Type I
Metalloproteases
Nitric Oxide
Pancreatic Elastase
RNA, Messenger
Reactive Oxygen Species
Superoxides

Figure

• 

  Fig. 1 Antioxidant effect of AR on DPPH radical and nitric oxide scavenging in a cell-free system. (A) DPPH radical scavenging activity of AR. (B) Nitric oxide scavenging activity of AR. The level of DPPH radical was measured spectrophotometrically at 515 nm. The NO scavenging capacity was assessed by Griess assay. The IC50 values for DPPH radical and NO scavenging activities were 1.88 mg/mL and 6.77 mg/mL, respectively. Each bar represents the mean ± SD (n = 6). The bars with a different letter are significantly different from each other at the level of P < 0.05.

• 

  Fig. 2 Effect of AR on UVB-induced cell cytotoxicity and ROS formation of human keratinocytes. (A) Cells were pretreated with AR prior to UVB irradiation (30 mJ/cm2) and harvested 24 h later. Cytotoxicity was determined by LDH leakage assay. (B) Intracellular ROS levels induced by UVB were determined by the DCF-DA method. Cells, treated with AR prior to UV irradiation, were incubated with 20 µM of DCF-DA for 30 min, and harvested after 24 h. ROS formation was analyzed with a fluorometer (excitation; 486 nm, emission; 530 nm). Each bar represents the mean ± SD (n = 3). The bars with a different letter are significantly different from each other at the level of P < 0.05.

• 

  Fig. 3 Effect of AR on antioxidative enzyme activity. Cells were pretreated with AR for 24 h prior to UVB irradiation (30 mJ/cm2) and harvested 24 h later. (A) Catalase activity was measured using a colorimetric assay kit. One unit of catalase was defined as the amount of enzyme required to decompose 1 µM of H2O2 per minute. (B) SOD activity was measured using a colorimetric assay kit. Enzyme activity is expressed as average enzyme activity unit per mg protein ± SD (n = 3). The bars with a different letter are significantly different from each other at the level of P < 0.05.

• 

  Fig. 4 Effect of AR on UVB-induced degradation of elastin and collagen, and collagen synthesis in human keratinocytes. Cells were pretreated with AR for 24 h prior to UVB irradiation (30 mJ/cm2) and harvested 24 h later. (A) Elastase activity was measured spectrophotometrically with N-Succ-(Ala)3-p-nitroanilide as a substrate, and the release of p-nitroaniline was monitored. (B) MMP-1 level was determined using an ELISA kit. (C) Collagen production was determined using a SirCol collagen assay kit. (D) Expression of MMP-1 mRNA was determined by quantitative real time RT-PCR. GAPDH was used as an internal control. Each bar represents the mean ± SD (n = 3). The bars with a different letter are significantly different from each other at the level of P < 0.05.

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