ARTICLE
Auteur(s) : Mei-Fen Shih1,
Jong-Yuh Cherng2
1Department of Pharmacy, Chia-Nan University of
Pharmacy and Science, 60 Erh-Jen Road, Sec.1, Pao-An, Jen-Te
Hsiang, Tainan, 717, Taiwan ROC
2Department of Chemistry and biochemistry, National
Chung-Cheng University, Chia-Yi, Taiwan, ROC
accepté le 4 Février 2008
UV irradiation damages human skin and causes premature skin
ageing (photoageing) characterized by thickening, rough texture,
coarse wrinkles, and mottled pigmentation [1]. Damage to human skin
due to repeated exposure to ultraviolet-A (mainly 350-390 nm)
or B (290-320 nm) radiation is considered as a process
distinct from skin damage occurring during ageing [2]. Photoaged
skin is biochemically characterized by a predominance of abnormal
elastic fibers in the dermis and by a dramatic decrease in distinct
collagen types. Matrix metalloproteinases (MMPs) are a family of
structurally related zinc and calcium-dependent endopeptidases,
which are crucial factors involved in the connective tissue
remodeling accompanying UV irradiation-induced skin damage [3, 4].
UVB is known to induce the expressions of MMP1, MMP3, and MMP9 in
the normal human epidermis in vivo [5]. MMP1 and MMP3 are thought
to be the two major contributors to photoageing [5, 6]. The
up-regulation of UVA-induced MMP1 in dermal fibroblasts is also
demonstrated through a PKC-dependent pathway [7].
Chlorella, a type of fresh water grown unicellular green algae,
has been shown to possess many biological effects, such as
promoting the growth rate of animals [8], boosting immune function
[9-12], accelerating dioxin secretion [13], preventing
stress-induced ulcers [14], preventing high fat-diet induced
dyslipidemia [15-17], and streptozocin-induced diabetic
hyperglycemia [18, 19]. In addition, Chlorella inhibited MMP1
activity in vitro from human peripheral blood mononuclear cells
[20]. However, the effects of an aqueous extract fraction of
Chlorella on preventing UV-induced skin ageing have not been
studied before. In this study we used PMA, a PKC activator, and UVB
irradiation as skin ageing inducing devices to investigate effects
of Resilient Factor (the aqueous extract of Chlorella) on MMP1
production, protein expressions of MMP1 and elastin, and mRNA
expressions of MMP1, TIMP1, and procollagen in normal skin
fibroblast cells.
Materials and methods
PMA (phorbol 12-myristate 13-acetate, Sigma, St. Louis, USA),
Ilomastat (GM6001, Chemicon, USA), PKC inhibitor peptide
(RFARKGALRQKNV, Upstate, USA) and UVB lamp (302 nm, Ultra-Violet
products Model UVM-57, Cambridge, UK) were purchased from local
representatives. Chlorella dry powder was purchased from Gong-Bih
Enterprise Co., LTD. (Taipei, Taiwan). Vitamin C (A5960) and
Vitamin E (T3634) were purchased from Sigma (St. Louis, USA).
Resilient Factor preparation
Resilient Factor (RF) was obtained from boiling water extract of
Chlorella powder (10% w/v). After being extracted for 30 min,
the extracting material was centrifuged at 10,000 rpm for
20 min, the resulting supernatant was collected and freezing
dried for storage.
Cell culture
Normal skin fibroblast 966SK (BCRC 60153) cells were obtained from
Bioresource Collection and Research Center (Hsinchu, Taiwan) and
cultured in Eagle-MEM supplemented with 1 mM sodium pyruvate
and 10% FBS. The cells were maintained at 37 °C in a
humidified atmosphere of 5% CO2-95% air. When cells reached above
80% confluence, subculture was conducted at a split ratio of 1:5.
MMP1 production determination
Our preliminary data indicated that PMA-induced MMP1 expression
reached the maximal difference between treated and untreated groups
(referred to as basal MMP) after 5h treatment (data not shown),
this time point was thus chosen. Culture media of skin fibroblasts
were collected 5 hours after PMA (100 nM) incubation with or
without various doses of RF, GM6001 (0.4 nM), or vitamin C
(0.15 mM). PMA is the activator of PKC and is used for
investigating effects of Resilient Factor on MMP1 production. Cells
were also irradiated with a total dose of
247.5 mJ/cm2 and cell culture media were collected
5 h after UVB exposure. Production of MMP1 was measured by
using a fluorimetric assay kit (Human active MMP1 fluorimetric
assay, R&D system, Minneapolis, USA). In brief, aliquots of
media were transferred into MMP1 antibody pre-coated black
microplates and were incubated at room temperature for 3 h and
subsequently re-incubated with fluorogenic substrate for
17-20 h at 37 °C in a humidified environment. The
relative fluorescence units were determined by using a fluorescence
plate reader set with an excitation wavelength of 320 nm and
an emission wavelength of 405 nm.
Western blot analysis
966SK skin fibroblast cells were cultured at approximately 80%
confluence in 100 mm dishes and treated with PMA alone or
co-administration with 20 mg/mL RF, GM6001 (0.4 nM), a
nonselective MMP inhibitor, or PKC inhibitor (0.2 μM). Five
hours later, the cells were harvested and lysed with
radioimmunoprecipitation assay (RIPA, Sigma, USA) buffer
(50 mM Tris-HCl, pH7.5, 1% Nonidet P-40 [NP-40], 150 mM
NaCl, 0.1% sodium dodecyl sulfate [SDS], 0.5% deoxycholate and
1 mM phenylmethylsulfonyl fluoride [PMSF]). For UVB treatment,
cells were exposed to UVB light with a total dose of
247.5 mJ/cm2 and cells were harvested and lysed
5 h later as described above. Approximately 50 μg of cell
lysate was boiled at 95 °C for 5 min in the sample buffer. The
samples were then separated by 10% SDS-PAGE, followed by protein
blotting onto a polyvinylidene fluoride (PVDF) membrane (BioRad,
Hercules, CA, USA). The protein-blotted membranes were blocked with
5% (w/v) fat-free dry milk in PBS with 0.05% Tween 20 (PBS-T)
overnight at 4 °C. They were then incubated with anti-MMP1 (St.
Louis, Sigma, USA), elastin (St. Louis, Sigma, USA) or α-tubulin
antibody (St. Louis, Sigma, USA) at 1:1000 dilution in PBS-T
containing 1% bovine serum albumin overnight at 4 °C. After
washing three times for 5 min with PBS-T solution, blots were
further incubated for 1 h at room temperature with goat
anti-rabbit IgG antibody coupled to horseradish peroxidase
(Amersham Pharmacia Biotech, Sweden) at 1:2000 dilution in 5% skim
milk in PBS-T and washed three times in PBS-T before visualization.
The expressions of the proteins were detected by ECL detection
system (Amersham Pharmacia Biotech, Sweden).
RT-PCR
Skin fibroblast cells were treated as described in 2.4 Western blot
analysis. Total RNA was extracted by using a GENTRA RNA isolation
kit (R-5000A, Minneapolis, USA). The extract of total RNA was
reverse transcribed using a first strand cDNA synthesis kit for
reverse transcription-polymerase chain reaction (RT-PCR) (Access
RT-PCR system, Promega, Madison, USA). Semiquantitative PCR was
performed using primers for human MMP1 (forward, 5’-GAT TGC ACA GCT
TTC CTC CAC TGC-3’; reverse, 5’-GAT GTC TGC TTG ACC CTC AGA GAC
C-3’), TIMP1 (forward, 5’-TTC CGA CCT CGT CAT CAG GG-3’; reverse,
5’-ATT CAG GCT ATC TGG GAC CGC-3’), Pro-collagen (forward, 5’-CTC
CGG CTC CTG CTC CTC TTA-3’; reverse, 5’-GCA CAG CAC TCG CCC TCC
C-3’), and house keeping gene GAPDH (forward, 5’-CCA CCC ATG GCA
AAT TCC ATG GCA-3’; reverse, 5’-TCT AGA CGG CAG GTC AGG TCC
ACC-3’). The PCR conditions used were as follows: initial
denaturation (for 5 min at 94 °C), 28 cycles of
amplification (for 45 sec at 94 °C, for 45 sec at
60 °C, and for 2 min at 72 °C), and final extension
(for 10 min at 72 °C). Reaction products were
electrophoresed in 1.0% agarose gels and visualized with ethidium
bromide. Densitometric analysis was performed using the Alpha
Imager 2000 Documentation & Analysis System (Alpha Innotech
Corporation, San Leandro, USA).
Statistical analysis
Data from each group (n ≥ 6) on different experimental days were
analyzed for MMP1 assay. A two-tailed Student’s unpaired test was
used to compare the mean values of two populations of continuous
data. Electrophoresis and Western blotting gel data were performed
(n ≥ 3) and a representative one is shown in results.
Results
Inhibitory effects of Resilient Factor (RF) on PMA-induced MMP1
level
MMP1 level in fibroblast culture media was significantly increased
5 h after PMA treatment (figure 1, p < 0.05). The
induction of MMP1 was significantly reduced when PMA was
co-administered with 20 mg/mL (p < 0.05) and 30 mg/mL
(p < 0.005) of RF. Neither co-administration of PMA and vitamin
C nor co-administration of PMA and GM6001 produced the same results
as those obtained by co-administration of PMA and RF.
Protein expression of MMP1 was induced after PMA treatment for 5
hours, this was prevented by co-administration of RF, PKC inhibitor
and GM6001 (figure
2). Although PMA treatment did not significantly enhance
MMP1 mRNA expression at the same incubation time, RF reduced the
MMP1 mRNA expression level (p < 0.01, figure 3). Under the same
condition, PKC inhibitor (p < 0.01) and GM6001 (p < 0.01)
also showed some protective effects on PMA-induced MMP1 mRNA
expression.
The effects of RF on UVB irradiation-induced MMP1 production,
protein and mRNA levels
MMP1 levels were increased in cell culture media significantly
after UVB exposure (p < 0.05, figure 4). RF treatment
suppressed the UVB-increased MMP1 production (figure 4) and protein
expression in a dose-dependent manner (figure 5). However, the
prevention effect was not observed in vitamin C or E-treated groups
(figure 4).
Expression of MMP1 mRNA was significantly reduced in the presence
of 10 mg/mL and 20 mg/mL of RF during UVB exposure (p
< 0.01 and p < 0.001, respectively, figures 6A and B).
Effects of RF on elastin expression following UVB
irradiation
One of the major matrix proteins in the skin tissue is elastin. The
amount of this protein represents the youthful appearance of skin
[21]. In the presence of RF, there was higher elastin protein
expression in UVB-exposed skin fibroblast cells (p < 0.05, figure 5).
Effects of RF on TIMP1 and pro-collagen mRNA expression
TIMP1 is thought to counteract MMP1 action in the skin matrix. The
expression of TIMP1 mRNA was not significantly affected by UVB
irradiation (figures 6A
and C). However, the expression was higher in 10 mg/mL
and 20 mg/mL RF treated groups (p < 0.05). The expression
of pro-collagen mRNA was slightly but not significantly suppressed
after UVB irradiation (figures 6A and D). There
was a restoration when cells were incubated with 20 mg/mL RF during
UVB exposure (p < 0.05).
Discussion
Among the long-term detrimental effects of sunlight is skin
photoageing, which is a well-documented consequence of exposure to
UVA and UVB radiation. MMPs play a key role in ECM remodeling
accompanying UV radiation-induced skin damage. It has been shown
that UV irradiation significantly affects the coordinated
regulation of various MMPs and TIMPs [22]. Moreover, the enzyme
activities of MMPs are post-transcriptionally controlled by
activation of latent proenzymes as well as by interactions with
their specific inhibitors, referred to as TIMPs. It has been
reported that the biosynthesis of MMP1 is up-regulated by PMA,
cytokines, and growth factors such as IL-1, TNF-α, IL-6, epidermal
growth factor, and platelet-derived growth factor, in a variety of
cells, including fibroblasts [23]. This is consistent with our
findings on PMA-induced MMP1 production and protein level, but not
on mRNA expression level (figure 3). The
inconsistency between PMA-induced MMP1 protein and mRNA expression
could be due to the presence of PKCξ isoform in 966SK fibroblasts.
PKCξ, a PKC isoform not inducible by PMA, is a component of
collagen matrix stimulatory pathway for MMP1 mRNA expression [24].
Basal MMP1 mRNA was strongly influenced by the presence of collagen
and was not induced by the treatment of PMA in dermal fibroblasts.
The isoform of PKCζ has been shown to be expressed at higher levels
in adults than in infant rats [25]. Our preliminary data indicate
that higher basal MMP1 levels are present in 966SK cell line (from
78 yr female) than that in 1090SK cell line (ATCC: CRL-2160, from
46 yr female) (data not shown).
Oxidative stress caused by UV irradiation, ozone, hydrogen
peroxide and free radicals is known to increase PKC activity [7].
Skin damage by oxidants may lead to activation of PKC and AP-1,
thus increasing MMPs expression and collagen degradation. Total PKC
activity in human skin fibroblasts increases during in vivo ageing
as a function of the donor’s age. During in vitro ageing PCK
activity also increased. MMP1 gene transcription and protein
expression increased up to 8 fold during in vivo ageing,
concomitant with the increase in PKC [22, 26]. PMA is an activator
of PKC and used to induce MMP in skin fibroblasts to mimic
physiologic and UVB-induced skin ageing in this study. Resilient
Factor, an aqueous extracted fraction of Chlorella, is shown to
possess inhibitory effects against PMA- and UVB-induced MMP1
production in this study. The inhibition of MMP1 by Chlorella has
also been demonstrated [20]. However, this is the first time that
Chlorella Aqueous Extract (referred to as Resilient Factor in this
study) is shown to prevent PMA- and UVB-induced MMP production in
skin fibroblasts. The inhibition is related to the reduction in
MMP1 protein product as well as in mRNA expression.
Elastin is an ECM protein in mammals where it is one of the main
components of skin, blood vessels such as the aorta and tissues of
the lung [27]. Elastin has specific cross-linkages, which are
responsible for the stability, elasticity and flexibility of skin
[21]. Collagen is another major component of ECM, which is
responsible for the skin tone. Collagen deficiency in photodamaged
skin may result from repetitive degradation of collagen by
UV-induced MMPs. RF increases elastin and pro-collagen mRNA
expression levels during UVB exposure suggesting that RF may
possess a potential role in preventing photoageing.
Taken together, RF prevents PMA- and UVB-induced MMP1 production
via the inhibition of MMP1 protein and mRNA expression.
UVB-suppressed elastin protein levels and pro-collagen mRNA level
in skin fibroblasts are also prevented by RF treatment.
Acknowledgements
We are very grateful to National Science Council of Taiwan for
providing the research funding (NSC 91-2626-B-041-001). Conflict of
interest: none.
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