Nutr Res Pract.  2007 Dec;1(4):279-284.

Protective actions of Rubus coreanus ethanol extract on collagenous extracellular matrix in ultraviolet-B irradiation-induced human dermal fibroblasts

• Bae JY
• Lim SS
• Choi JS
• Kang YH

Abstract

Solar ultraviolet (UV) irradiation leads to distinct changes in the skin connective tissues by degradation of collagen, which is a major structural component in the extracellular matrix. UV irradiation induces the production of matrix metalloproteinases (MMP) capable of attacking native fibrillar collagen and responsible for inhibiting the construction of collagenous extracellular matrix. In this study, we attempted to investigate the protective actions of Rubus coreanus ethanol extract (RCE) on the MMP production and the consequent procollagen/collagen degradation in UV-B-irradiated human dermal fibroblasts. The analytical data showed that Rubus coreanus ethanol extract was mostly comprised of cyanidin 3-rutinoside. Pre-treatment of fibroblasts with this extract inhibited UV-B-induced production of MMP-1, MMP-8 and MMP-13 in dose-dependent manners. In addition, Western blot analysis and immunocytochemical staining assay revealed that RCE markedly augmented the cellular levels of procollagen/collagen declined in UV-B-exposed dermal fibroblasts. These results demonstrate that RCE blocks UV-B-induced increase of the collagen degradation by inhibiting MMP production. Thus, RCE may act as an agent inhibiting excessive dermal collagen degradation leading to the skin photoaging.

Keyword

Collagen; human dermal fibroblasts; MMP; Rubus coreanus; ultraviolet-B

MeSH Terms

Blotting, Western
Collagen*
Connective Tissue
Ethanol*
Extracellular Matrix*
Fibrillar Collagens
Fibroblasts*
Humans*
Matrix Metalloproteinases
Skin
Collagen
Ethanol
Fibrillar Collagens
Matrix Metalloproteinases

Figure

• 

  Fig. 1 LC-UV-ESI/MS/MS data recognizing cyanidin-3-rutinoside abundant in ripe fruits of Rubus coreanus. Total ion chromatography (A), mass spectra at 9.10min in TIC (B), tandem mass spectra of [M+H]+ (595) (C), HPLC chromatogram (PDA, photodiode-array detector) (D), ultra-violet spectra at 8.74 min (E) and structure of cyanidin 3-rutinoside (F).

• 

  Fig. 2 Cell viability in RCE-treated human dermal fibroblasts challenged with UV-B irradiation. Confluent fibroblasts were left untreated or stimulated with 100 mJ/cm2 prior to incubation for 48 h with RCE. Cell viability was measured using MTT assay and presented as means ± SEM from 3 independent experiments with multiple estimations. Values not sharing a letter are different at P<0.05.

• 

  Fig. 3 Inhibitory dose responses of RCE to induction of MMP-1, MMP-8 and MMP-13 in UV-B irradiated human dermal fibroblasts. After cells were pre-treated with 1-10 µg/mL RCE and exposed to 100 mJ/cm2 UV-B, conditioned culture media were collected and subjected to 10% SDS-PAGE, followed by Western blot analysis with respective primary antibody of MMP-1, MMP-8 and MMP-13. Bands are representative of 3 independent experiments.

• 

  Fig. 4 Inhibitory dose responses of RCE to reduction of type 1 procollagen levels in 100 mJ/cm2 UV-B-irradiated human dermal fibroblasts. Cell lysates were subjected to 6% SDS-PAGE and Western blot analysis with a primary antibody against type 1 procollagen (A). β-Actin protein was used as an internal control. Bands were representative of 3 independent experiments. In immunocytochemical experiments (B), cells were fixed and then incubated with goat anti-human type 1 procollagen. Antibody localization was detected with FITC conjugated donkey anti-goat IgG using a fluorescence microscopy. Representative fluorescent images were obtained from 3 separate experiments. Magnification: 100-fold.

• 

  Fig. 5 Augmentation of type 1 collagen levels in RCE-treated and 100 mJ/cm2 UV-B-irradiated human dermal fibroblasts. Cell lysates were electrophoresed on 6% SDS-PAGE, followed by Western blot analysis with a primary antibody against type 1 collagen (A). β-Actin protein was used as an internal control. Bands were representative of 3 independent experiments. For the immunocytochemical staining (B), fibroblasts were fixed and incubated with goat anti-human type 1 collagen. Antibody localization was detected with Cy-3 conjugated donkey anti-goat IgG using a fluorescence microscopy with rhodamine green filter. Representative fluorescent images were obtained from 3 separate experiments. Magnification: 100-fold.

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