Berberine prevents UV-induced MMP-1 and reduction of type I procollagen expression in human dermal fibroblasts.

Abstract

Matrix metalloproteinases (MMPs) induction and type I procollagen reduction in photoaging of the skin due to exposure to ultraviolet (UV) irradiation. Therefore, regulation of these genes has been suggested to be a useful tool to abolish skin aging. In this study, antioxidative plant ingredients used in traditional Chinese medicine, berberine (BBR) was investigated for their capacity to regulate MMP-1 and type I procollagen expression in human dermal fibroblasts. Our results showed that both basal and UV-induced MMP-1 expression was decreased by BBR. On the other hand, type I procollagen expression was dose-dependently increased by it. In addition, UV-induced reduction of type I procollagen expression is recovered by it. Therefore, we suggest that BBR may be a possible candidate for anti-skin aging agent.

[C] 2007 Elsevier GmbH. All rights reserved.

Keywords: Berberine: MMP-1; Type I procollagen: Human dermal fibroblast; UV irradiation

Introduction

Berberine is an isoquinoline derivative alkaloid that has been isolated from Berberis aquifolium (Oregon grape), Berberis aristata (tree turmeric), Berberis vulgaris (barberry), and Hydrastis canadensis (goldenseal) (Ikram, 1975). Berberine has many pharmacological effects including cell cycle arrest, anti-oxidant and anticancer effect (Anis et al., 2001; Chi et al., 1994; Kuo et al., 1995). In particular, we previously reported that TPA-induced MMP-9 and IL-6 expression are inhibited by berberine in human keratinocytes (Kim et al., 2007).

Ultraviolet (UV) irradiation results in numerous and diverse clinical skin changes such as wrinkling, sunburn, immuno suppression, cancer, and premature skin aging (photoaging) (Gilchrest and Yaar, 1992). Photodamage to skin connective tissues is responsible for the characteristic aged appearance of photodamaged skin (Fisher et al., 1997).

UV irradiation triggered initial events and in subsequent signaling resulting in enhanced expression of two major members of the matrix metalloproteinase family, the interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3), in human dermal fibroblasts (Brenneisen et al., 2000).

In addition, UV irradiation disrupts the skin collagen matrix by two interdependent pathways: stimulating collagen degradation and inhibiting procollagen production (Fisher et al., 1996). UV irradiation activates MAP kinase signaling, which in turn induces transcription factor AP-1, which stimulates expression of matrix metalloproteinases (MMP) including MMP-1 and MMP-3 (Brenneisen et al., 2000; Di Girolamo et al., 2003).

In our study, we explored the anti-aging property of BBR. We observed that basal and UV-induced MMP-1 expression is dose-dependently decreased by BBR, whereas basal and UV-induced type I procollagen is increased by it in human dermal fibroblasts. Also, these effects are similar with the effects of EGCG derived from green tea. Therefore, like EGCG, we demonstrate that BBR may be a possible candidate for anti-skin aging agent in human dermal fibroblasts.

Material and methods

Cell culture

Primary human dermal fibroblasts were obtained from foreskin of healthy volunteers, age 20-30 y. The skin was minced, followed by incubation with collagenase (1 mg/ml in DMEM) for 1-2h at 37[degrees]C. Then collagenase was removed by washing with Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Rockville, MD). The isolated cells were allowed to attach on plastic plates and cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 2mM glutamine, l00 IU/ml penicillin, and 100[micro] g/ml streptomycin (Life Technologies, Rockville, MD). After six and eight passages the fibroblasts were used for experimentation.

UV irradiation and berberine treatments

Philips TL 20W/12 RS fluorescent sun lamps with an emission spectrum between 275 and 380 nm (peak, 310-315nm) were used as a UV source, and a Kodacel filter (TA401/407; Kodak, Rochester, NY) was used to block UVC, which has wavelengths of < 290 nm. UV strength was measured using a Waldmann UV meter (model 585100) (Seo et al., 2003). The cells were treated with 100mJ/[cm.sup.2] of UV for experiments.

For experiments, cells were maintained in culture medium without FBS for 24 h. Serum-starved cells was treated with berberine (stock solution; l0mM, Sigma, St. Louis, MO) at the indicated concentration for 72 h or pretreated with berberine for 1 h prior to UV treatment and then further incubated for 72 in serum-free media with berberine. Three independent normal human dermal fibroblasts cells were used in this study.

Western blotting

Western blotting was performed as described previously (Park et al., 2004). Cells were lysed with lysisbuffer [50 mM Tris-HCl, pH 7.4, 150mM NaCI, 1 mM EDTA, 1 mM EGTA, 10[micro]g/ml aprotinin, 10[micro]g/ml leupeptin, 5mM phenylmethylsulfonyl fluoride (PMSF), and 1mM DTT] containing 1% Triton X-100. Insoluble debris was removed by centrifugation at 12,000 rpm for 10min, and protein content was determined using Bradford reagent (Bio-Rad, Hercules, CA). Equal amounts of protein were resolved on gradient 8% SDS-PAGE gels and then electrophoretically transferred to PVDF membranes (Immobilon P; Amersham, Buckinghamshire, UK). Membranes were subsequently blocked with 5% skim milk in TBS/T (20 mM Tris--HC1, pH 7.6, 137mM NaCI, and 0.05% Tween 20) and incubated with anti-human MMP-1 (Oncogen, Boston, MA) and anti-human type I procollagen (SP1.D8, Developmental Studies Hybridoma Bank, Iowa City, IA). Blotting proteins were visualized by enhanced chemiluminescence (Amersham Bioscience, Buckinghamshire, UK).

Zymography

To assess the gelatinolytic activities of MMP-2 in culture media, Zymography was performed in 10% Zymogram gel (Invitrogen, Carlsbad, CA). Briefly, samples were suspended in loading buffer [10% SDS, 25% glycerol, 0.25 M Tris (pH 6.8), and 0.1% Bromo-phenol Blue] and, without prior denaturation, run on 10% Zymogram gel. After electrophoresis, gels were washed to remove SDS and incubated for 30min in renaturing buffer (50mM Tris, 5mM Ca[Cl.sub.2], 0.02% Na[N.sub.3], 1% Triton X-100). The gels were then incubated for 24 h at 37 [degrees]C in developing buffer [50 mM Tris (pH7.8), 5mM Ca[Cl.sub.2], 0.15M NaCI, and 1% Triton X-100J. Gels were subsequently stained with 0.5% Coomassie Brilliant Blue G-250 and destained in 30% methanol, 10% acetic acid to detect gelatinolytic activity (Kim et al., 2007).

Statistical analysis

Statistical significance was determined using the Student t-test. Results are presented as means [+ or -] SEM. All p values quoted are two-tailed and significance was accepted when p was < 0.05.

Results and discussion

Although many studies have reported the function of BBR, it has not been reported the effects of BBR on MMP-1 and type I procollagen expression in human dermal fibroblasts. Therefore, we investigated the effect of BBR on basal level of MMP-1 and type I procollagen expression. The structure of BBR is given in Fig. 1A.Cells were treated with BBR at the indicated concentration for 72h. BBR dose-dependently suppressed the basal level of MMP-1 expression (Fig. 1B). The basal level of MMP-1 expression was significantly decreased to 68.2 [+ or -]4.4%, 30.7 [+ or -] 1.6%, 18.4 [+ or -] 5.5%, and 13.3 [+ or -] 3.2% of control level by 1,5, 10 and 20 [micro]M BBR treatment, respectively.

[FIGURE 1 OMITTED]

On the other hand, the basal level of type I procollagen expression was dose-dependently increased by BBR (Fig. 1C). Type 1 procollagen expression was significantly increased by 244.9 [+ or -] 7.8%, 418.7 [+ or -] 55% and 510.8 [+ or -] 80.8% of control level by 5, 10 and 20 [micro] M BBR treatment, respectively. Cell viability was not affected by BBR treatment at the indicated concentrations (data not shown). Previously, BBR has antioxidant effect including nitric oxide scavenging, superoxide scavenging, and inhibition of iNOS expression (Shirwaikar et al., 2006; Pan et al., 2005). Based on the reports, we demonstrated that MMP-1 reduction and type I procollagen induction may be mediated by antioxidant activity of BBR in human dermal fibroblasts.

Next, we investigated the effects of BBR treatment on UV-induced MMP-1 and reduction of type I procollagen expression. MMP-1 expression was significantly increased by 100 mJ/[cm.sub.2] UV treatment (Fig. 2A). UV-induced MMP-1 expression was significantly increased by 219.6 [+ or -] 35.7% of control level. On the other hand, UV-induced MMP-l expression was prevented to 131.3[+ or -] 18.9% of control level by 20 [micro] M BBR treatment (Fig. 2A). In contrast, type I procollagen expression was decreased by 100mJ/[cm.sup.2] UV treatment (Fig. 2B). UV irradiation decreased type I procollagen expression to 52.6 [+ or -] 5.7% of control level. BBR prevented the UV-induced reduction of type I procollagen expression (Fig. 2B). Treatment with 20 [micro]M BBR suppressed UV-induced reduction of type 1 procollagen expression to 107.7 [+ or -] 22.2% of control level (Fig. 2B).

[FIGURE 2 OMITTED]

It has been well known that MMP-l induction and type I procollagen reduction were mediated by UV irradiation as well as these phenomena accelerated in aged and photoaged human skin. Increased expression of MMP-1 is involved with collagen degradation in UV-irradiated and aged/photoaged human skin (Fisher et al., 1997; Steinbrenner et al., 2003), leading to wrinkle formation and aged appearance. Based on these reports, the balance of MMP-1 and type I procollagen expression play an important role in aged and photoaged skin. Here, we suggest that BBR has a dual function on MMP-l and type I procollagen expression in human dermal fibroblasts.

In previous study, we reported that EGCG decreased the level of MMP-1 and MMP-3 expression (Lee et al., 2005) and also inhibited UVA-induced MMP-l expression through inhibition of Jun transcriptional activity in human skin (Song et al., 2004). Therefore, we compared with the effects of EGCG and BBR on MMP-1 and type I procollagen expression in human dermal fibroblasts. As shown in Fig. 2C, both EGCG and BBR dose-dependently decreased MMP-l expression. In contrast, type I procollagen expression was increased by both EGCG and BBR (Fig. 2D). Our results showed that BBR-induced type I procollagen expression was higher than one of EGCG at 5 [micro]M BBR and EGCG treatments in human dermal fibroblasts.

In conclusion, our results showed that basal and UV-induced MMP-1 expression is significantly decreased, while basal and UV-induced reduction of type I procollagen expression is increased by BBR. We demonstrate that BBR may be used as an effective ingredient for anti-skin aging products, which can prevent MMP-l expression and the degradation of type I procollagen.

Acknowledgements

This study was supported by grant of the Korea Health 21 R & D Project, Ministry of Health & Welfare, Republic of Korea (03-PJI-PGI-CH14-0001) and by a research agreement with the AmorePacific R & D Center.

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SangminKim(a), (b), (c), Jin HoChung(a), (b), (c), *

(a) Department of Dermatology, Seoul National University College of Medicine, Seoul, Republic of Korea

(b) Laboratory of Cutaneous Aging Research, Clinical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea

(c) Institute of Dermatological Science, Medical Research Center, Seoul National University, Seoul, Republic of Korea

* Corresponding author. Department of Dermatology, Seoul National University Hospital, 28 Yungon-dong, Chongno-Gu, Seoul 110-744, Republic of Korea. Tel.: [phone]; fax: [phone].

E-mail addresses: [e-mail] (S. Kim), [e-mail] (J.H. Chung).