Placenta is a special organ that contains many nutrients such as growth factors, minerals, and bioactive peptides. Dipeptides of glycine and leucine are major components of porcine placenta extracts (PPE) that has been used as an alternative of human placenta extracts. In this study, we investigated whether major peptides of PPE, Glycyl-L-Leucine (Gly-Leu), L-Leucyl-Glycine (Leu-Gly), and L-Leucyl-L-Leucine (Leu-Leu), affect skin hydration and elasticity
Human skin is the largest organ of the body and extends to approximately 2 m2 in area (Tobin, 2006; Nichols and Katiyar, 2010). Skin protects the body against excessive water loss and dangerous external factors including pollutants, UV irradiation, and chemicals (Makrantonaki and Zouboulis, 2007; Bonte, 2011). Accumulated UV exposure leads to skin aging, which causes wrinkle formation, acute erythema, tanning, and loss of hydration and elasticity (Scharffetter-Kochanek
There are several factors that control skin moisturization and elasticity. First, hyaluronan (HA) regulates moisture, elasticity, and architecture of tissue, repairing tissue, promoting cell motility, and scavenging free radicals (Hsu and Chiang, 2009; Wen
Placenta is a specialized organ of pregnancy and is very important for growth and development of the fetus (Gude
In this study, we attempted to elucidate the effects of PPE, Glycyl-L-Leucine (Gly-Leu), L-Leucyl-Glycine (Leu-Gly), and L-Leucine-L-Leucine (Leu-Leu) dipeptides on skin moisturization and elasticity in normal human keratinocytes (NHEKs) and UVB-induced hairless mice.
PPE was purchased from Biofac A/S (Kastrup, Denmark). Gly-Leu, Leu-Gly, and Leu-Leu were purchased from Bachem AG (Bubendorf, Switzerland).
NHEKs from neonatal origin were purchased from Invitrogen (Carlsbad, CA, USA). NHEKs were cultured in EpiLife® medium (Life Techonologies, NY, USA) with 60 μM CaCl2, human keratinocyte growth supplement (Invitrogen), and 1% penicillin/streptomycin (Welgene, Gyeongsan, Korea). Cells were maintained at 37°C in a 5% CO2 incubator.
NHEKs were seeded into 96-well culture plates at 1×104 cells/well. After 24 h at 37°C, the media was replaced with EpiLife® media containing PPE, Gly-Leu, Leu-Gly, and Leu-Leu diluted to the appropriate concentrations for 24 h. Then cells were washed with DPBS, EZ-Cytox reagents (Daeil Lab Service, Seoul, Korea) were added, and the cells were incubated at 37°C for 1 h. The absorbance was measured using a microplate reader (Tecan, Mannedorf, Switzerland) at a wavelength of 450 nm.
Cells were lysed in extraction buffer (0.1 M Tris-HCl, pH 7.2, 1% TritonX-100, 200 mM NaCl, protease inhibitor cocktail) at 4°C for 30 min. The lysates were subjected to centrifugation at 13,000 rpm for 20 min, and the supernatant was obtained. Blots were incubated with antibodies against anti-TGase1 (Santa Cruz Biotechnology, CA, USA) and β-actin (Santa Cruz Biotechnology). After incubation, membranes were rinsed three times with TBS-T and were incubated with donkey antirabbit IgG antibody (Bethyl Laboratories, Montgomery, TX, USA) and goat anti-mouse IgG antibody (Bio-Rad, CA, USA) for 1 h at room temperature. Binding antibodies were detected using a WEST-ZOL® Plus Western Blot Detection System (INtRON Biotechnology, Sungnam, Korea) and visualized with ChemiDoc XRS (Bio-Rad, Hercules, CA, USA).
HA content was measured from culture media of the NHEK cultures with a Hyaluronan DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA). NHEKs were seeded into 96-well culture plates at 1×104 cells/well. After 24 h, the cells were washed with DPBS, and serum-free media was added. After starvation for 24 h, NHEK cells were cultured with various concentrations of PPE, Gly-Leu, and Leu-Gly. After 24 h, the HA concentration in the culture supernatant was measured.
The activity of porcine pancreatic elastase (Sigma, St. Louis, MO, USA) was examined using N-succinyl-(L-Ala)3-p-nitroanilide as the substrate. The reaction mixture contained 50 mM Tris-HCl buffer (pH 8.0), 1 U/mL elastase, and 0.5 mg/ml N-succinyl-(L-Ala)3-p-nitroanilide. The reaction mixture was pre-incubated for 30 min at 25°C before adding the substrate. The release of p-nitroaniline was measured at 410 nm using a 96-well reader. The percent inhibition of elastase was calculated as follows:
Total RNA was isolated from NHEK cells and mouse skin tissue with the Trizol reagent (Takara, Otsu, Japan). The quality and quantity of the RNA were determined by NanoDrop2000 (Thermo Scientific, Waltham, MA, USA). To synthesize cDNA, 1 μg quantities of total RNA were mixed with 100 pmol quantities of oligo (dT) (ELPIS, Daejeon, Korea), followed by denaturation at 65°C for 5 min and chilling on ice for 5 min. The annealed samples were then incubated with reverse transcriptase and 2 mM dNTPs (Fermentas, Waltham, MA, USA) for 1 h at 42°C. Reverse transcription was terminated by heating for 10 min at 70°C. For amplification, the cDNA was mixed with HiPi PCR Mix (ELPIS) and each of the following primer sets: HAS2: Forward: 5′-CAGAATCCAAACAGACAGTTC-3′, Reverse: 5′-TAAGGTGTTGTGTGTGACTG-3′; β-actin: Forward: 5′-GTGGGGCTGCCCCAGGCACCA-3′, Reverse: 5′-CTCCTTAATGTCACGCACGATTTC-3′. The resulting PCR products were visualized by electrophoretic separation on 3% agarose gels and staining with RedSafeTM Nucleic Acid Staining Solution (ELPIS). Specific primers for β-actin were added as a control.
Six-week-old female albino hairless mice (SKH-1) were purchased from Orient Bio (Seongnam, Korea). The hairless mice were acclimated for 1 week before starting the experiments and then divided into 6 groups of 10 mice each. The feeding environment was maintained under controlled temperature (24 ± 2°C) and humidity (55 ± 10%) and automatic lighting (12 h light and dark cycle). Feed was provided (Feed Lab Korea, Guri, Korea) to the hairless mice. Laboratory animal breeding management was based on the “Guide for the Care and Use of Laboratory Animals,” and all experiments were approved by the Institutional Animal Care and Use Committees of Gyeonggi Institute of Science & Technology (Suwon, Korea).
The UVB source was six fluorescent lamps (TL 20W/12RS SLV, wave length 290 to 390 nm, peak emission 315 nm; (Philips, Amsterdam, Netherlands), and the UVB irradiation intensity was measured with a UV meter (VARIOCONTROL, Waldmann ver.2.03, Villingen-Schwenningen, Germany). The mice were exposed to UVB irradiation three times per week. The starting dose of UVB irradiation was 75 mJ/cm2 during the first week and then increased weekly by 1 minimal erythema dose (MED) until reaching 3.3 MED, which was maintained until 8 weeks.
Skin hydration content and elasticity were measured on the dorsal skin of the mice using Corneometer (CK Electronics GmbH, Cologne, Germany) and Cutometer (CK Electronics).
After the end of the experiment, the dorsal skins of all animals were biopsied and placed in 10% formalin. The dorsal skin tissues were stained with haematoxylin and eosin (H&E) and Alcian blue. The stained tissues were photographed using a Nikon ECLIPSE Ti-E inverted fluorescent microscope (Nikon, Tokyo, Japan) and analyzed using NIS-Element BR 3.0 software (Nikon, Tokyo, Japan).
All of the data are expressed as mean ± SD. Statistical significance was determined by independent
To determine the cytotoxic effects of PPE, Gly-Leu, Leu-Leu, and Leu-Gly, we applied these compounds at different concentrations to NHEKs. Treatment with PPE, Gly-Leu, Leu-Leu, and Leu-Gly showed no cytotoxicity at concentrations up to 100 μg/ml and 10 μg/ml (Fig. 1).
To identify the effects of PPE, Gly-Leu, Leu-Leu, and Leu-Gly on keratinocyte differentiation, we measured TGase 1 protein expression level in NHEKs treated with PPE, Gly-Leu, Leu-Leu, and Leu-Gly. TGase 1 protein level was significantly increased with PPE in a dose-dependent manner (Fig. 2A). Gly-Leu and Leu-Gly treatment also increased TGase 1 protein level (Fig. 2B, 2D), but Leu-Leu treatment did not change TGase 1 protein level (Fig. 2C).
To investigate the effects of PPE, Gly-Leu, and Leu-Gly on synthesis of HA, HA was measured in NHEKs. Treatment of PPE increased HA level in a dose-dependent manner compared to the control (Fig. 3). Treatment with Gly-Leu and Leu-Gly also increased HA level at 10 μg/ml concentration. It was reported that increased mRNA level of HAS2 induced HA production in human keratinocytes (Kim
To determine the effects of PPE, Gly-Leu, and Leu-Gly on elastase activity, we measured elastase activity as described in the Methods section. PPE, Gly-Leu, and Leu-Gly treatment significantly reduced elastase activity in a dose-dependent manner (Fig. 5).
To analyze the effects of PPE, Gly-Leu, and Leu-Gly on skin hydration and elasticity
Epidermal thickness was increased and HA was decreased in UVB-induced hairless mouse skin. Oral supplement with PPE, Gly-Leu, and Leu-Gly significantly decreased epidermal thickness and increased the level of HA compared to those in the UVB-induced group (Fig. 7). We examined HAS2 mRNA level in dorsal skin using RT-PCR. HAS2 mRNA level was increased in the PPE intake group in a dose-dependent manner compared to the UVB-induced group. In addition, HAS2 mRNA level was increased in the Gly-Leu and Leu-Gly 10 mg/kg intake groups (Fig. 8).
Gly-Leu, Leu-Gly, and Leu-Leu dipeptides were included in PPE. When we analyzed PPE, large quantities of Gly-Leu, Leu-Gly, and Leu-Leu were contained, in amounts of 1,200∼2,400 mg/kg, 140∼600 mg/kg, and 250∼450 mg/kg, respectively. Glycine has been reported to increase collagen synthesis in rats (Chyun and Griminger, 1984). Based on this information, we tested whether PPE and glycine-containing peptides in PPE were effective for skin moisturization and elasticity.
We showed that treatment with PPE, Gly-Leu, and Leu-Gly increased keratinocyte differentiation (Fig. 2). In addition, synthesis of HA was increased by treatment with PPE, Gly-Leu, and Leu-Gly (Fig. 3). It was previously reported that synthesis of HA was inhibited by down-regulation of HAS2 expression (Rock
It has been reported that inhibition of HA synthesis decreased skin hydration and viscoelasticity by down-regulation of HAS2 in hairless mice (Rock
Gly-Leu and Leu-Gly dipeptides of fermented porcine placenta extract have been reported to have a reducing effect on fatigue (Nam
In summary, oral supplementation with PPE, Gly-Leu, and Leu-Gly could protect skin from UV-damage by restoring the synthesis of HA and reducing the inhibition of elastase. Also, Gly-Leu and Leu-gly peptides were shown to be functional ingredients of PPE. Therefore, we suggest that PPE and its major peptides, Gly-Leu and Leu-Gly, could be potential candidate materials for skin moisturization and elasticity as dietary supplements.
This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant NO: HN15C0102).