Acer tataricum subsp. ginnala Inhibits Skin Photoaging via Regulating MAPK/AP-1, NF-κB, and TGFβ/Smad Signaling in UVB-Irradiated Human Dermal Fibroblasts

Abstract

Skin, the organ protecting the human body from external factors, maintains structural and tensile strength by containing many collagen fibrils, particularly type I procollagen. However, oxidative stress by ultraviolet (UV) exposure causes skin photoaging by activating collagen degradation and inhibiting collagen synthesis. Acer tataricum subsp. ginnala extract (AGE) is a herbal medicine with anti-inflammatory and anti-oxidative effects, but there is no report on the protective effect against skin photoaging. Therefore, we conducted research concentrating on the anti-photoaging effect of Acer tataricum subsp. ginnala (AG) in UVB (20 mJ/cm²)-irradiated human dermal fibroblasts (HDF). Then, various concentrations (7.5, 15, 30 µg/mL) of AGE were treated in HDF for 24 h following UVB irradiation. After we performed AGE treatment, the matrix metalloproteinase1 (MMP1) expression was downregulated, and the type I procollagen level was recovered. Then, we investigated the mitogen-activated protein kinases/activator protein 1 (MAPK/AP-1) and nuclear factor-κB (NF-κB) pathway, which induce collagen breakdown by promoting the MMP1 level and pro-inflammatory cytokines. The results indicated that AGE downregulates the expression of the MAPK/AP-1 pathway, leading to MMP1 reduction. AGE inhibits nuclear translocation of NF-κB and inhibitor of nuclear factor-κB (IκB) degradation. Therefore, it downregulates the expression of MMP1 and pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 increased by UVB. Besides, the TGFβ/Smad pathway, which is mainly responsible for the collagen synthesis in the skin, was also analyzed. AGE decreases the expression of Smad7 and increases TGFβRII expression and Smad3 phosphorylation. This means that AGE stimulates the TGFβ/Smad pathway that plays a critical role in promoting collagen synthesis. Thus, this study suggests that AGE can be a functional material with anti-photoaging properties.

Keywords: Acer tataricum subsp. ginnala; MAPK/AP-1; NF-kB; TGFβ/Smad; matrix metalloproteinase (MMP); type I procollagen.

Figure 1

Ultra-performance liquid chromatography (UPLC) chromatogram of Acer tataricum subsp. ginnala extract (A) and reference standards solution ginnalin A (B). Among the 10 identified peaks, peak no. 10 was identified as ginnalin A. Detailed information of the other peaks are listed in Table 1.

Figure 2

Cell viability after AGE treatment in HDFs. HDFs were treated with different concentrations of AGE, and incubated for 24 h. After incubation, MTT assay was performed to measure the cell viability of AGE (A). All data were expressed as the mean ± SD of at least three independent experiments. ** p < 0.01 versus control group.

Figure 3

Effect of AGE on type I procollagen and MMP-1 level in UVB-irradiated HDFs. HDFs were irradiated with UVB (20 mJ/cm²), followed by treatment with 7.5, 15, and 30 µg/mL of AGE. After 24 h of incubation, detection of type I procollagen (A) and MMP-1 (B) production were performed by using ELISA kits. Gene expression of COL1A1 (C) and MMP-1 (D) were determined by quantitative real-time (qRT)-PCR. All data were expressed as the mean ± SD of at least three independent experiments. ### p < 0.001 versus untreated cells, * p < 0.05, ** p < 0.01, and *** p < 0.001 versus UVB-irradiated cells.

Figure 4

Effects of AGE on MAPK signaling pathway in UVB-irradiated HDFs. HDFs were irradiated with UVB (20 mJ/cm²), followed by treatment with 7.5, 15, and 30 µg/mL of AGE. After 24 h incubation, protein extraction was performed. Phosphorylation and total protein ratios of ERK (B), JNK (C), and p38 (D) were measured using Western blot (A). All data were expressed as the mean ± SD of at least three independent experiments. ### p < 0.001 versus untreated cells, ** p < 0.01 versus UVB-irradiated cells.

Figure 5

Effects of AGE on the expression of AP-1 in UVB-irradiated HDFs. HDFs were irradiated with UVB (20 mJ/cm²), followed by treatment with 7.5, 15, and 30 µg/mL of AGE. After 24 h of incubation, protein extraction was performed. Protein expressions of c-fos (B), c-jun, and p-c-jun (C) were determined by Western blot (A). All data were expressed as the mean ± SD of at least three independent experiments. ## p < 0.01 and ### p < 0.001 versus untreated cells, * p < 0.05 versus UVB-irradiated cells.

Figure 6

Effects of AGE on the expression of NF-κB signaling pathway in UVB-irradiated HDFs. HDFs were irradiated with UVB (20 mJ/cm²), followed by treatment with 7.5, 15, and 30 µg/mL of AGE. After 24 h of incubation, protein extraction was performed. Protein expressions of IκB (B), nucleus, and cytosolic NF-κB (C) were determined by Western blot (A). All data were expressed as the mean ± SD of at least three independent experiments. ## p < 0.01 and ### p < 0.001 versus untreated cells, * p < 0.05, ** p < 0.01, and *** p < 0.001 versus UVB-irradiated cells.

Figure 7

Effects of AGE on the expression of inflammatory cytokines in UVB-irradiated HDFs. HDFs were irradiated with UVB (20 mJ/cm²), followed by treatment with 7.5, 15, and 30 µg/mL of AGE. After 24 h of incubation, gene expressions of TNFα (A), IL1β (B), and IL6 (C) were measured by qRT-PCR. All data were expressed as the mean ± SD of at least three independent experiments. ### p < 0.001 versus untreated cells, * p < 0.05, ** p < 0.01, and *** p < 0.001 versus UVB-irradiated cells.

Figure 8

Effects of AGE on the expression of TGFβ/Smad signaling pathway in UVB-irradiated HDFs. HDFs were irradiated with UVB (20 mJ/cm²), followed by treatment with 7.5, 15, and 30 µg/mL of AGE. After 24 h of incubation, protein extraction was performed. Protein expressions of TGFβ receptor II (B), p-Smad-3, Smad-3 (C), and Smad-7 (D) were determined by Western blot (A). All data were expressed as the mean ± SD of at least three independent experiments. # p < 0.05, ## p < 0.01, and ### p < 0.001 versus untreated cells, * p < 0.05 and ** p < 0.01 versus UVB-irradiated cells.