Galloyl–RGD, Derived from a Fusion of Phytochemicals and RGD Peptides, Regulates Photoaging via the MAPK/AP-1 Mechanism in Human Dermal Fibroblasts

Abstract

Galloyl–RGD is a novel compound that combines gallic acid with RGD peptides (arginine, glycine, and asparaginic acid) to overcome the problems associated with gallic acid, such as instability at high temperatures and low solubility. In this study, we investigated the effects and molecular mechanisms of action of galloyl–RGD on UVB-induced skin photoaging in human dermal fibroblasts-neonatal (HDF-n). Galloyl–RGD increased collagen synthesis by inhibiting UVB-induced MMP-1 via inhibiting extracellular signal-regulated kinase and Jun N-terminal kinase and their downstream mitogen-activated protein kinase signaling, which are known to be representative photoaging mechanisms. The results of this study will be helpful for understanding the anti-photoaging effect and mechanism of galloyl–RGD and its future applications in the cosmetic and pharmaceutical industries.

1. Introduction

Skin aging can be broadly divided into intrinsic and extrinsic types. Intrinsic aging occurs when the body naturally deteriorates with age and the production of reactive oxygen species (ROS) increases. Extrinsic aging factors include smoking, air pollution, environmental hormones, and ultraviolet rays, which are known to be the most direct causes of skin photoaging that damage cellular DNA. Exposure of the skin to ultraviolet B (UVB) radiation can cause wrinkling, dryness, and pigmentation, which are the main characteristics of photoaging, by inducing oxidative cell damage [1].

UVB irradiation induces oxidative stress in dermal fibroblasts characterized by ROS overproduction. ROS accelerate the phosphorylation of mitogen-activated protein kinase (MAPK) subunits, such as extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK), and p-38 [2]. Downstream targets of this signaling pathway, such as ERK, JNK, and p38, are phosphorylated, and transcription factors such as activator protein-1 (AP-1) are activated, which together promote the expression of matrix metalloproteinases (MMPs), thereby increasing collagen degradation. [3].

It was also reported that UV radiation downregulated the expression of the transforming growth factor beta (TGF-β), which is involved in the synthesis of procollagen type I, by the production of ROS [4]. In addition, the nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) is a key molecular target of inflammatory photoaging, which activates NF-κB in skin cells upon UVB exposure. Activated NF-κB promotes the expression of matrix metallo proteinase-1 (MMP-1) and causes dermal tissue transformation by degrading type I collagen in the dermal layer [5,6].

Many studies have reported skin damage caused by UVB irradiation. At the same time, research is continuously being conducted to identify safer and more effective ingredients for raw cosmetic materials.

Gallic acid (3, 4, 5-trihydroxybenzoic acid) is a phytochemical derived from various herbs. It is an endogenous plant polyphenol found abundantly in tea, grapes, berries, and other fruits, as well as in wine [7,8]. It has various biological properties, including antioxidant, anticancer, antiviral, and anti-inflammatory effects [9,10,11]. In addition, it regulates photoaging by reducing ROS and MMP-1 in UVB-exposed fibroblasts, increasing TGF-β1 and inhibiting MMP-1 in hairless mice [12]. However, despite having a good effect, it is difficult to apply it as a cosmetic ingredient because it is unstable under heat and has low solubility [13].

In a previous study, a novel compound called galloyl–RGD was synthesized by combining an RGD peptide with gallic acid to overcome the problems of gallic acid. According to our previous research results, it was found that galloyl–RGD exhibits excellent antioxidant effects and ROS scavenging activity and inhibits α-MSH-induced melanogenesis in melanocytes by regulating ERK signaling [14]. Pigmentation is one of the symptoms observed in aging skin. Galloyl–RGD can suppress pigmentation by inhibiting melanin production; however, the direct effect or molecular mechanism of skin aging is unknown. Therefore, in this study, we investigated the effect and molecular mechanism of galloyl–RGD on UVB-induced skin photoaging in human dermal fibroblast-neonates (HDF-n).

2. Materials and Methods

2.1. Chemicals and Reagents

Galloyl–RGD was prepared by combining gallic acid with arginine, glycine, and asparaginic acid (RGD peptide) and was obtained from Bio-FD&C Co., Ltd. (Hwasun, Republic of Korea). 1,2-diphenyl-2-picrylhydrazyl (DPPH), (3-4,5-dimethylthiazol-2-yl)-2,5,-diphenyltetrazolium bromide (MTT), and PD98059 were purchased from Sigma-Aldrich (St. Louis, MO, USA). SP600125 was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). Antibodies against MMP-1 (sc-58377), JNK (sc-571), p38 (sc-7149), and NF-κB (sc-8008) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against c-Fos (#2250), p-c-Fos (#5348), c-Jun (#9165), p-c-Jun (#3270), ERK (#9102), p-ERK (#9101), p-JNK (#4668), and p-p38 (#4511) were purchased from Cell Signaling Technology (Beverly, MA, USA). TGF-β1 (ab92486) was purchased from Abcam (Cambridge, UK).

2.2. DPPH Radical Scavenging Activity

The DPPH radical scavenging assay is a simple, convenient, and widely used antioxidant screening method. The assay was performed as previously described, with some modifications [15]. Each well of 96-well plates contained 100 µL of various samples and 100 µL of the solution of 0.2 mM DPPH. After incubating the plate at room temperature for 30 min, the absorbance was measured at 515 nm using a UV/Vis microplate reader (Thermo Fisher Scientific, Multiskan Sky, Seoul, Republic of Korea).

2.3. ABTS Radical Scavenging Activity

The ABTS radical scavenging activity is the same as that of the DPPH radical; however, DPPH is a free radical, whereas ABTS is a cation [16]. The assay was performed as previously described [17], with some modifications. A total of 7.4 mM ABTS and 4.9 mM potassium persulfate were dissolved in distilled water and allowed to react in the dark for 16 h at room temperature to form ABTS cation radicals. Prior to use in the assay, the ABTS radical cations were diluted with distilled water for an initial absorbance of approximately 0.7 ± 0.02 at 734 nm. Each well contained 100 µL of various samples and 100 µL of the solution of ABTS cation radical. After incubating the plate for 15 min in the dark at room temperature, the absorbance was measured at 734 nm using a UV/Vis microplate reader (Thermo Fisher Scientific, Multiskan Sky, Seoul, Republic of Korea).

2.4. Cell Cultures

Human dermal fibroblast-neonatal (HDF-n, cat. no. 2310) was purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). The cells were cultured in Fibroblast Medium (cat. no. 2301), which contained 10% fetal bovine serum (FBS) and fibroblast growth supplement, under 5% CO₂ at 37 °C.

2.5. Cell Viability

HDF-n cells were seeded at a density of 4 × 10³ cells/well in 96-well plates and incubated at 37 °C in 5% CO₂ for 24 h. The cells were incubated with galloyl–RGD for 24 h. After incubation, 100 µL of MTT solution (5 mg/mL in phosphate-buffered saline [PBS]) was added to each well and incubated for 3 h. The medium was discarded and 200 µL of DMSO was added. The absorbance was measured at 570 nm using a microplate reader (EZ read 400, Biochrom, Cambridge, UK).

2.6. UVB Irradiation

The HDF-n cells were cultured in 24-well plates for 24 h until they reached approximately 80% confluence. The cells were then washed with PBS and exposed to UV-B light (10 mJ/cm²) using a Biolink BLX-312 UV crosslinker (Vilber Lourmat, Marne-la-Vallee, France). The UVB irradiation dose used was non-cytotoxic.

2.7. Collagenase Inhibition Assay

Calcium chloride (4 mM) dissolved in 0.1 M Tris-HCl buffer (pH 7.5) was the final buffer assessed. The substrate 4-phenylazo-benzyloxy-carbonyl-Pro-Leu-Gly-Pro-D-Arg (0.3 mg/mL) was dissolved in the buffer and then 250 µL of this solution was injected into the test tube with 100 µL of adequate concentration of the sample. Collagenase was dissolved in the final buffer to achieve a concentration of 0.2 mg/mL, and 150 µL of this solution was added. After incubation at 37 °C for 30 min, the reaction was stopped by adding 6% citric acid. The reaction mixture was then separated using ethyl acetate. The absorbance of the supernatant was measured at 320 nm. Epigallocatechin gallate (EGCG) was used as a positive control.

2.8. Type I Procollagen Synthesis

The HDF-n cells were seeded at a density of 3 × 10⁴ cells/well in 24-well plates and incubated for 24 h. After incubation, the cells were washed with PBS and exposed to 10 mJ/cm2 of UVB light. The cell culture medium was replaced with a fresh medium, and the cells were treated with galloyl–RGD for 72 h. The supernatant was collected from each well and centrifuged at 12,000 rpm for 10 min prior to use. The amount of procollagen type I was determined using a procollagen type I C-peptide assay kit (Takara Bio, Tokyo, Japan).

2.9. MMP-1 Activity

The HDF-n cells were seeded at a density of 3 × 10⁴ cells/well in 24-well plates and incubated for 24 h. After incubation, the cells were washed with PBS and exposed to 10 mJ/cm² of UVB light. The sample used to measure MMP-1 activity was obtained in the same manner as for type I procollagen. MMP-1 activity was determined using the Human Pro MMP-1 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA).

2.10. Western Blot Analysis

HDF-n cells were seeded at a density of 1.5 × 10⁵ cells/well in a 6-well plate and incubated for 24 h. The cells were treated with galloyl–RGD for 1–72 h. The cell culture medium was acetone-downregulated and used to measure MMP-1 protein expression. Cell lysates were centrifuged at 13,000 rpm for 5 min at 4 °C. The supernatant was collected and the protein concentration was determined using the Bradford assay. After that, the protein (5–20 µg) was separated by electrophoresis on a 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel and transferred to nitrocellulose (NC) membranes. Membranes were blocked for 1 h at room temperature (15–25 °C) with 5% skim milk in Tris-buffered saline and 0.1% Tween 20 (TBST) solution. Then, the membranes were incubated with the following specific primary antibodies in 5% skim milk in TBST for 1 h at room temperature: MMP-1 (1:500), JNK (1:500), p38 (1:200), TGF-β1 (1:500), NF-κB (1:200), c-Fos (1:1000), p-c-Fos (1:1000), c-Jun (1:1000), p-c-Jun (1:1000), ERK (1:1000), p-ERK (1:1000), p-JNK (1:1000), p-p38 (1:1000), and GAPDH (1:10,000) antibodies. The blots were washed four times with TBST for 10 min each time. The membranes were then incubated with a secondary antibody (1:5000–1:10,000) for 1 h. Protein bands were visualized using an enhanced chemiluminescence (ECL) kit (Advansta, San Jose, CA, USA).

2.11. Statistical Analysis

Statistical analysis of the experimental data points was performed using ANOVA and Duncan’s multiple range test using SPSS software (version 22.0, IBM, Armonk, Chicago, IL, USA) and Student’s t-test. Statistical significance was set at p < 0.05.

3. Results

3.1. Antioxidant Effects of Galloyl–RGD

Galloyl–RGD was synthesized using solid-phase peptide synthesis, which can quickly and efficiently synthesize substances by binding amino acids to solid-phase supports (Figure 1a). The chemical structure of galloyl–RGD is shown in Figure 1b. It has a molecular weight of 498.44. DPPH is a water-soluble free radical that forms a stable purple color; when it reacts with an antioxidant, the color dissipates. This principle is used to search for antioxidants [18]. Galloyl–RGD increased the DPPH radical scavenging activity in a concentration-dependent manner (Figure 1c). In particular, at a concentration of 100 µM, it exhibited about 62.2% scavenging activity. In addition, galloyl–RGD exhibited an EC50 of 82.5 µM and had superior DPPH radical scavenging activity than L-ascorbic acid (positive control). The ABTS radical scavenging ability is based on the principle that the absorbance of cationic radicals of ABTS is reduced by antioxidant-active substances [19]. After the formation of radicals by the oxidation of potassium persulfate and ABTS, the antioxidant ability of each sample was determined by measuring its radical scavenging activity. Galloyl–RGD increased the ABTS radical scavenging activity in a concentration-dependent manner (Figure 1d). In particular, at a concentration of 100 µM, it exhibited about 88.0% scavenging activity. In addition, galloyl–RGD exhibited an EC50 of 50.6 µM and had superior ABTS radical scavenging activity than L-ascorbic acid (positive control).

3.2. Anti-Photoaging Effects of Galloyl–RGD

Galloyl–RGD was not cytotoxic at concentrations of 10–100 µM for 72 h and there was no significant difference in viability compared to the control (Figure 2a). Type 1 collagen is the most abundant type of collagen in human skin and controls skin aging in the dermis [20]. Collagenase is an enzyme that breaks down collagen, and its inhibition may represent a potential strategy for the prevention or treatment of UV radiation-induced photoaging [21]. To determine the effect of galloyl–RGD on collagen synthesis, HDF-n cells were irradiated with UVB and then treated with galloyl–RGD at concentrations of 25–100 µM for 72 h. Galloyl–RGD exhibited a collagenase inhibitory ability of about 41.1% at a concentration of 100 µM (Figure 2b). The IC50 value for collagenase was 130.0 µM. To measure the effect of galloyl–RGD on collagen synthesis, Type I collagen synthesis was evaluated after UVB irradiation and the treatment of HDF-n cells with galloyl–RGD. The UVB-irradiated control group showed reduced collagen synthesis by approximately 23.3% compared to the normal control group. Galloyl–RGD increased the procollagen synthesis decreased by UVB irradiation in a concentration-dependent manner (Figure 2c). In particular, at a concentration of 100 µM, the collagen synthesis was increased by about 13.7% compared to the control group.

Several studies have shown that matrix metalloproteinases (MMPs) play important roles in skin aging. MMP-1 is an enzyme produced in dermal fibroblasts that breaks down collagen and is involved in wrinkle formation [22,23,24]. To measure the effect of galloyl–RGD on MMP-1 activity, HDF-n cells were irradiated with UVB and then treated with galloyl–RGD at a concentration of 25–100 µM for 72 h. The UVB-irradiated control group showed a 90.4% increase in MMP-1 activity compared to that in the normal group. The galloyl–RGD-treated group reduced the MMP-1 activity increased by UVB irradiation in a concentration-dependent manner (Figure 2d). In particular, at a concentration of 100 µM, MMP-1 activity was reduced by about 55.2% compared to the control group. In addition, the galloyl–RGD treatment group reduced the protein expression that MMP-1 increased by UVB irradiation in a concentration-dependent manner (Figure 2e,f). These results suggest that the inhibition of MMP-1 by galloyl–RGD can increase collagen production.

3.3. Signaling Pathway of Galloyl–RGD on Photoaging Effects

The MAPK signaling pathway is an important transmitter of extracellular signals to the cell nucleus and is known to be one of the important signal transduction pathways activated by UVB treatment [2]. The three major components of MAPKs are ERK, JNK, and p38, and the phosphorylation of these proteins activates MAPK signaling cascades [25] (Johnson et al. 2002). To confirm the effect of galloyl–RGD on MAPK expression, ERK, JNK, and p-38 expression was measured at 1 h after UV irradiation and galloyl–RGD treatment. UVB increases collagen degradation by phosphorylating signaling pathways, such as those of ERK, p38, and JNK, and activating transcription factors, such as AP-1 [26]. The c-Fos and c-Jun proteins form heterodimers and act as a transcription factor called AP-1 in the nucleus.

As shown in Figure 3, the UVB-irradiated control group showed increased protein expression of phosphorylated ERK, JNK, and p38 compared with the normal control group. The protein expression of phosphorylated ERK and JNK was increased by UVB irradiation and was significantly inhibited by galloyl–RGD treatment. However, the phosphorylation of p38 was not inhibited. To confirm the effect of galloyl–RGD on AP-1 expression, c-Fos/c-Jun expression was measured at 4 h after UV irradiation and galloyl–RGD treatment. The UVB-irradiated control group showed increased protein expression of p-c-Fos and p-c-Jun compared with the normal control group. The increased expression of p-c-Fos and p-c-Jun upon UVB irradiation was significantly inhibited by galloyl–RGD treatment. These results indicated that galloyl–RGD inhibited AP-1 through the regulation of ERK and JNK in the MAPK pathway.

TGF-β is a multifunctional cytokine involved in cellular functions such as cell growth and differentiation and the biosynthesis of extracellular connective tissue and is a procollagen expression regulator in HDF-n cells [27,28,29]. In addition, UVB irradiation leads to the activation of transcription factors such as AP-1 and MAPK as well as to the increased expression of protein kinases such as NF-κB. When the effect of galloyl–RGD on the expression of TGF-β1 and NF-κB was evaluated after 1.5 h of treatment, galloyl–RGD did not affect the protein expression of TGF-β1 and NF-κB (Figure 3e,f).

3.4. Effect of MAPK (ERK/JNK) Inhibitor on MMP-1

We used PD98059 (an ERK inhibitor) and SP600125 (a JNK inhibitor) to confirm whether galloyl–RGD inhibited MMP-1 activity via the MAPK pathway. The ERK and JNK inhibitors decreased the MMP-1 activity increased by UVB irradiation by 98.3% and 32.4%, respectively (Figure 4a,b). In addition, the ERK/JNK inhibitor and galloyl–RGD co-treated groups were inhibited by 19.9% and 37.3%, respectively, compared to the inhibitor-only group. These results suggested that galloyl–RGD exerts anti-photoaging effects by inhibiting collagen degradation via the MAPK pathway. The protein expression of MMP-1 was also inhibited by ERK and JNK (Figure 4c–e). Taken together, these results indicate that galloyl–RGD exerts an anti-photoaging effect by inhibiting collagen degradation via decreasing AP-1 and MMP-1 through the MAPK pathway.

4. Discussion

Photoaging of the skin is a complex biological process induced by oxidative stress. Continuous UVB irradiation accelerates photoaging mainly due to excessive ROS generation. In the photoaging process, UVB-induced ROS production upregulates the expression of MMPs, which degrade collagen and other ECM proteins. UVB-induced MMP-1 overexpression initiates collagen breakdown by cleaving type I and plays an important role in the physiological mechanisms of skin photoaging [30]. Therefore, the development of antioxidants that can inhibit free radicals and MMP-1 inhibitors that prevent collagen breakdown is an area of interest in anti-aging research.

In the present study, galloyl–RGD significantly increased DPPH and ABTS radical scavenging activities, indicating that galloyl–RGD may act as a potential antioxidant. Several studies have found that UVB-generated ROS activate the MAPK signaling pathway through the phosphorylation of ERK, JNK, and p38 kinase and then stimulate MMP gene transcription by activating the AP-1 transcription factor [26,31]. Thus, the activation of MAPK/AP-1 signaling is thought to play a dominant role in inducing MMP-1. To understand the inhibitory mechanism of MMP-1 secretion, we investigated the effects of galloyl–RGD on MAPK/AP-1 signaling and phosphorylation. The results showed that galloyl–RGD dose-dependently reduced ERK and JNK phosphorylation, but not p38. p38 MAPK is involved in stress responses such as cell growth and survival, differentiation, and apoptosis depending on cell type and stimulus. The MAPK pathway is very complex, with multiple regulatory factors that regulate each other’s expression or activity. We speculate that galloyl–RGD may alter the interaction between them to increase p-p38 expression. Galloyl–RGD also attenuated the expression of p-c-Fos and p-c-Jun. The ameliorating effect of galloyl–RGD on MMP-1 overexpression is likely regulated by the inhibition of MAPK/AP-1 signaling and was validated using ERK and JNK inhibitors. As expected, UVB-induced MMP-1 activity and protein expression levels were significantly inhibited by treatment with the ERK- and JNK-specific inhibitors PD98059 and SP600125, respectively.

Oxidative stress and inflammation are involved in several processes that play important roles in skin photoaging. Excessive ROS generation induced by UVB activates MAPK to induce an NF-κB-dependent inflammatory response [32]. In addition, because collagen provides elasticity and strength to the skin, promoting collagen synthesis is essential for suppressing skin photoaging. TGF-β1 is an important multifunctional cytokine involved in the regulation of ECM-related genes that regulate the synthesis of collagen [33] (Wells and Discher, 2008). However, in the mechanism of photoaging, gallolyl-RGD did not affect TGF-β1 and NF-κb expression. Similarly, gallic acid increased UVB irradiation-reduced TGF-β1 expression in hairless mice but had no significant effect on human dermal fibroblasts [12]. In addition, gallic acid is known to exhibit anti-inflammatory effects by inhibiting NF-κB in Raw264.7 cells [34]. However, the effect of gallic acid on NF-kB expression in UV-B-treated human dermal fibroblasts has not yet been reported. Additionally, our study results showed that TGF-β1 expression was not affected by UV radiation. There may be various reasons for this result. In general, cytokines do not act independently but rather interact with other cytokines and factors, which may cause this result. Another factor is that protein expression may change over time. Therefore, a study on this is necessary, and a study on the mechanistic differences between gallic acid and galloyl–RGD in UVB-induced inflammatory photoaging would be interesting in the future.

In conclusion, our study showed that galloyl–RGD can prevent UVB-induced photoaging and is involved in collagen synthesis via the MAPK pathway and downstream signaling regulation (Figure 5). Taken together, these findings suggest that galloyl–RGD is a potential candidate for application as an antiphotoaging agent in the cosmetic and pharmaceutical industries. However, in order to understand the mechanism of galloyl–RGD more comprehensively, research on the level of the tissue inhibitor of metalloproteinase (TIMP), a direct inhibitor of MMPs, and its various mechanisms should be conducted. Also, additional in vivo and clinical trials are needed for its application in products such as functional cosmetics and food.