Porcine Placenta Extract Reduced Wrinkle Formation by Potentiating Epidermal Hydration

Background: Placenta extract is used as an alternative medicine especially in Asian countries as it is a rich reservoir of diverse bioactive molecules. The effects of placenta extract on skin conditions have been previously reported, however, the mechanism underlying for reduced wrinkle formation remains unclear. Objective: The primary objective of this study was to determine whether the continuous application of porcine placenta extract (PPE) alleviates wrinkle formation in humans and explore the underlying mechanism. Methods: Wrinkle formation, skin hydration, and skin elasticity were measured in 15 volunteers at weeks 0 and 6 after continuous application of a gel containing PPE. The production of type I collagen and hyaluronic acid from fibroblasts and keratinocytes, respectively, were measured using ELISA. Expression levels of ceramide synthase 3 (CERS3), filaggrin (FLG), transglutaminase 1 (TGM1), and kallikrein-7 (KLK7) were evaluated by quantitative real-time PCR. Results: The wrinkle index was significantly reduced to 72.1% after a 6-week of ap-plying the PPE gel, along with a significant increase in skin hydration to 126.5%. Type I collagen production from fibroblasts was enhanced slightly but significantly following treatment with PPE. PPE accelerated the expression of CERS3 (1.85-fold), FLG (1.35-fold), TGM1 (1.76-fold), and KLK7 (1.62-fold). Conclu-sion: Treatment with PPE alleviates wrinkle formation and simultaneously enhances skin hydration, which is induced via the accelerated expression of moisturizing-related proteins. These findings suggest that PPE is effective for combating dryness-induced wrinkle formation.


Introduction
As the skin is the outermost organ age-related changes such as wrinkles and pigmentation profoundly impact the quality of life, especially in women. Both extrinsic and intrinsic skin aging is known to occur with perpetual exposure to the external environment. Extrinsic skin aging primarily arises from ultraviolet (UV)-light exposure, directly enhancing reactive oxygen species, which induces the expression of matrix metalloproteinase (MMP)-1, MMP-3, and MMP-9. In contrast, intrinsically aged skin in UV-light-protected areas can be induced by the following factors: cell senescence with telomere shortening, mitochondrial DNA (mtDNA) mutations, oxidative stress, genetic mutations, and decreased levels of several hormones [1] [2] [3]. Wrinkle formation, which is a typical trait among age-related changes, can be observed in both UV-exposed and UV-protected sites. Based on previous studies, some underlying causes of wrinkle formation include reduced skin functions, skin dryness, UV exposure, and dermatitis [4] [5]. However, the treatment of wrinkles needs to be comprehensively addressed.
Clinical applications of human and/or porcine placenta extract (PPE) are widely accepted in oriental medicine as the placenta is a rich reservoir of diverse bioactive molecules. A previous study has shown that the injection of human placenta extract into wound boundaries accelerated decrease in wound size [6]. Moreover, oral administration of PPE reportedly reduces UVB-induced wrinkle formation in mice, along with the downregulation of MMP-2 mRNA expression [7]. In addition, studies using skin cells have revealed that human placenta extract accelerates keratinocyte and fibroblast proliferation [8] [9]. Accumulating evidence has highlighted the effects of placenta extract in the field of dermatology, and treatments with placenta are accepted as folk remedies to alleviate and prevent skin aging, especially in Asian countries where anti-aging is a major topic in dermato-cosmetology. However, it is necessary to elucidate the link between clinical improvement and molecular events induced by placenta extract. This study was designed to explore the effects of PPE on wrinkle formation, as well as to determine its underlying molecular mechanism.

Preparation of Porcine Placenta Extract (PPE)
After washing, the fresh porcine placenta underwent two freeze (−20˚C)-thaw (under 10˚C) cycles. The obtained freeze-thaw drip was heated at 65˚C for 6 h and subjected to vacuum-freeze drying, followed by removal of debris and filtration. Powdered PPE was reconstituted in sterilized water to a concentration of 50 mg/mL for in vitro testing. The gel formula containing 1.1 mg/mL PPE was kindly provided by Kasyu Industries Co., Ltd. (Fukuoka, Japan).

Evaluation of Elasticity, Water-Holding Property and Wrinkle Width
The protocol was reviewed and approved by the Ethical Committee for Research Cologne, Germany), respectively, at weeks 0 and 6. The laboratory conditions were maintained in the temperature range of 20˚C -22˚C, with a relative humidity of 50% ± 5%, during measurements. The data are expressed as the ratio (%) to week 0 in the control and treated sites, respectively.

Cytotoxicity
In brief, HaCaT cells were inoculated into 96-well plates at a cell density of 5 ×

Cell Proliferation
In brief, HaCaT cells were seeded into 96-well plates at a cell density of 2 × 10 3

Extracellular Matrix Production
To estimate the production of type I collagen from NHDFs and hyaluronic acid

RNA Isolation and Quantitative Real-Time PCR
HaCaT cells were seeded into 6-well plates at a cell density of 3 × 10 5 cells/well and maintained in a 5% CO 2 -humidified atmosphere at 37˚C for 24 h; then, the cells were further cultivated in the presence of KX-1 or its fractions for 24 h.
Cells were harvested and total RNA was prepared using an SV RNA isolation kit

Statistical Analysis
Data are expressed as the mean ± standard deviation (SD). Statistical analysis was performed using Student t-test. A value of p < 0.1 was considered statistically significant for in vivo tests. A value of p < 0.05, was assumed to be significant for in vitro tests.

Changes in Skin Properties
To evaluate the effects of PPE on skin conditions, various skin properties were assessed. The number of detectable wrinkles was reduced (Figure 1(a)) and the calculated wrinkle index was also significantly decreased to 72.1% of the control (Figure 1(b)). In contrast, conductance was significantly increased to 126.5 % at  (Figure 1(c)); no significant changes in gross elasticity (Figure 1(d)) and elastic recovery ( Figure   1(e)) were observed.

Cytotoxicity and Cell Proliferation
The neutral red assay was used to estimate the cytotoxicity of PPE. The results revealed that PPE did not significantly affect cell viability at concentrations ranging between 1 and 5% of PPE (Figure 2(a)). Likewise, no significant effects on cell proliferation were observed at 2% and 5% of PPE (Figure 2(b)). Thus, a concentration of 5% PPE was used for further experiments.

Extracellular Matrix Productions
To estimate the effects of PPE on extracellular matrix production, the production of type I collagen and hyaluronic acid was assessed in NHDF. Treatment with PPE induced a marginal but significant enhancement in type I collagen production (Figure 3(a)). In contrast, PPE did not impact hyaluronic acid production ( Figure 3(b)).

Expression of Proteins in HaCaT Cells
Next, we evaluated the effects of PPE on the mRNA expression of proteins in-

Discussion
In the skin, extrinsic and intrinsic skin aging occurs owing to perpetual exposure to the external environment. Wrinkles, which are typical among age-related changes, are caused by extrinsic and intrinsic factors, as reported previously [1] [2] [3] [4] [5]. However, the treatment of wrinkles needs to be further investigated.
Herein we revealed that PPE alleviated facial wrinkle formation (Figure 1(a) and Figure 1(b)). As previous studies have reported that wrinkle formation coincides with reduced hydration and elasticity levels [10] [11], we focused on epidermal water-holding properties and skin elasticity. In the present study, PPE increased the water content of the epidermis (Figure 1(c)). In contrast, skin elasticity indices were not affected following treatment with PPE (Figure 1(d) and Figure 1(e)), although PPE enhanced type I collagen production from fibrob-lasts (Figure 3(a)). These results suggest that the molecular mechanism underlying the reduction of wrinkle parameters is dependent on events in the epidermis, but not in the dermis. As various molecules such as hyaluronic acid, ceramides and natural moisturizing factors contribute to the water content in the epidermis, we evaluated the effect of PPE on the production of hyaluronic acid which is secreted from keratinocytes [12]. Hyaluronic acid secretion from Ha-CaT cells was not impacted by PPE (Figure 3(b)). Next, we examined the mRNA expression of CERS3, FLG, TGM1 and KLK7. As shown in Figure 4, PPE treatment accelerated all mRNA expression levels. CERS3 is predominantly expressed in keratinocytes and synthesizes C24-ceramides and ceramides with longer acyl chains [13]. Additionally, FLG is expressed in the granular layer of the epidermis and is ultimately degraded into free amino acids, which provide a natural moisturizing factor [14]. Therefore, PPE treatment may directly potentiate the skin barrier function and water-holding properties by accelerating the expressions of CERS3 and FLG mRNA. Conversely, it is postulated that well-balanced regulation of keratinocyte differentiation and corneocyte exfoliation is crucial for the construction of a healthy stratum corneum. TGM1 contributes to the assembly of the cornified envelope [15] and KLK7 plays a role in shedding corneocytes [16], indicating that these enzymes are pivotal participants in the formation of a healthy stratum corneum. As PPE accelerated the expression of TGM1 and KLK7, treatment with PPE is speculated to involve the construction of a healthy stratum corneum. Collectively, these results suggest that PPE is involved in restoring the skin barrier function and water-holding properties. In conclusion, treatment with PPE is effective for dryness-induced wrinkle formation via enhancement of skin hydration.