Protective effects of Quercus acuta Thunb. fruit extract against UVB-induced photoaging through ERK/AP-1 signaling modulation in human keratinocytes

Abstract

Background: Quercus acuta Thunb. (Fagaceae) or Japanese evergreen oak is cultivated as an ornamental plant in South Korea, China, Japan, and Taiwan and used in traditional medicine. The acorn or fruit of Quercus acuta Thunb. (QAF) is the main ingredient of acorn jelly, a traditional food in Korea. Its leaf was recently shown to have potent xanthine oxidase inhibitory and anti-hyperuricemic activities; however, there have been no studies on the biological activity of QAF extracts. Solar ultraviolet light triggers photoaging of the skin, which increases the production of reactive oxygen species (ROS) and expression of matrix metalloproteinase (MMPs), and destroys collagen fibers, consequently inducing wrinkle formation. The aim of this study was to investigate the effect of water extracts of QAF against UVB-induced skin photoaging and to elucidate the underlying molecular mechanisms in human keratinocytes (HaCaT).

Methods: In this study, we used HPLC to identify the major active components of QAF water extracts. Anti-photoaging effects of QAF extracts were evaluated by analyzing ROS procollagen type I in UVB-irradiated HaCaT keratinocytes. Antiradical activity was determined using 2,2-diphenyl-1-picrylhydrazyl and 2,20-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) assays. The expression of MMP-1 was tested by western blotting and ELISA kits. QAF effects on phosphorylation of the MAPK (p38, JNK, and ERK) pathway and transcription factor AP-1, which enhances the expression of MMPs, were analyzed by western blots.

Results: We identified two major active components in QAF water extracts, gallotannic acid and ellagic acid. The QAF aqueous extracts recovered UVB-induced cell toxicity and reduced oxidative stress by inhibiting intracellular ROS generation in HaCaT cells. QAF rescued UVB-induced collagen degradation by suppressing MMP-1 expression. The anti-photoaging activities of QAF were associated with the inhibition of UVB-induced phosphorylation of extracellular signal-regulated kinase (ERK) and activator protein 1 (AP-1). Our findings indicated that QAF prevents UVB-induced skin damage due to collagen degradation and MMP-1 activation via inactivation of the ERK/AP-1 signaling pathway. Overall, this study strongly suggests that QAF exerts anti-skin-aging effects and is a potential natural biomaterial that inhibits UVB-induced photoaging.

Conclusion: These results show that QAF water extract effectively prevents skin photoaging by enhancing collagen deposition and inhibiting MMP-1 via the ERK/AP-1 signaling pathway.

Keywords: Activator protein 1; Extracellular signal-regulated kinases; Matrix metalloproteinase-1; Mitogen-activated protein kinase; Photoaging; Quercus acuta Thunb. fruit; Ultraviolet B.

Fig. 1

Classic features of Quercus acuta Thunb. fruit (QAF) and representative high-performance liquid chromatography (HPLC) chromatogram of a water extract obtained from QAF. (A) Image of QAF, (B) Powder form of QAF aqueous extract, (C) Structural formula of gallotannic acid and ellagic acid, (D) Mobile phase A was methanol and mobile phase B was water (containing 0.1% formic acid) with the following elution profile: initial, 15% A; 5–10 min, 15%–30% A; 10–17 min, 30%–40% A; 17–22 min, 40%–50% A; 22–35 min, 50% A; 35–43 min, 50%–100% A; 43–47 min 100% A; 47–50 min, 100%–15% A; and 50–55 min, 15% A. The flow rate was 1 mL/min, the injection volume was 10 μL, and the detection wavelength was 254 nm. Gallotannic acid and ellagic acid were detected at approximately 6.7 min and 27.3 min, respectively

Fig. 2

Effects of Quercus acuta Thunb. fruit (QAF) on cell proliferation, ultraviolet B (UVB)-reduced cell proliferation, and procollagen type I production in UVB-induced HaCaT keratinocytes. (A) HaCaT cells treated with a range of concentrations of QAF (5, 10, 20, 50, and 100 μg/mL) for 24 h. Cell viability was estimated using MTT assay by measuring the absorbance at 450 nm. (B) Cells were exposed to UVB irradiation (30 mJ/cm²) and treated with a range of concentrations of QAF (5, 10, 20, and 50 μg/mL) for 24 h. (C) Cell culture media were collected to determine the levels of procollagen type I. Data are expressed as the mean ± SD. #p < 0.05, ##p < 0.01, ###p < 0.001 versus control group; *p < 0.05, **p < 0.01, ***p < 0.001 versus UVB-treated group

Fig. 3

Free radical scavenging activity analysis and intracellular reactive oxygen species (ROS) production in Quercus acuta Thunb. fruit (QAF)-treated HaCaT cells. (A) Effects of QAF on ROS production following ultraviolet B (UVB) irradiation. HaCaT cells were pretreated with a range of concentrations of QAF (5, 10, 20, and 50 μg/mL) or vitamin C (ascorbic acid) at 200 μM for 24 h, H₂O₂ (100 μM) for 2 h, followed by exposure to 30 mJ/cm2 UVB. After incubation, cells were stained with DCFH-DA (20 μM) for 30 min. Fluorescence was then measured using a fluorescence spectrophotometer. (B) 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and (C) 2,20-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging activities. DPPH was examined with different concentrations of QAF, and ascorbic acid was used as a standard. Data are expressed as the mean ± SD. #p < 0.05, ##p < 0.01, ###p < 0.001 versus control group; *p < 0.05, **p < 0.01, ***p < 0.001 versus UVB-treated group

Fig. 4

Effects of QAF on matrix metalloproteinases (MMP)-1 expression in UVB-stimulated HaCaT cells. The cells were pretreated with Quercus acuta Thunb. fruit (QAF) for 24 h, followed by UVB-irradiation. (A) Cells were seeded and pretreated with a range of concentrations of QAF (5, 10, 20, and 50 µg/mL) and 30 ng/ml EGF, followed by UVB irradiation and cultured for an additional 24 h. The level of MMP-1 released in the cell culture medium was measured using ELISA. (B) Protein expression of MMP-1 was analyzed using western blotting and band intensities were quantified. Cells were seeded and pretreated with a range of concentrations of QAF (5, 10, 20, and 50 μg/mL), followed by UVB irradiation and cultured for an additional 24 h. Data are expressed as the mean ± SD. #p < 0.05, ##p < 0.01, ###p < 0.001 versus control group; *p < 0.05, **p < 0.01, ***p < 0.001 versus UVB-treated group

Fig. 5

Effects of Quercus acuta Thunb. fruit (QAF) on activator protein 1 (AP-1) and mitogen-activated protein kinase (MAPK) signaling pathways in UVB-induced HaCaT keratinocytes. (A) The effects of QAF on the phosphorylation of MAPK activated by UVB. Cells were seeded and treated with a range of concentrations of QAF (5, 10, 20, and 50 μg/mL) for 24 h. Cells were irradiated with UVB at a dose of 30 mJ/cm² and harvested 30 min later. Protein expression was evaluated using western blotting, and (B) the band intensities were quantified. (C) The effects of QAF on the phosphorylation of AP-1 activated by UVB. Cells were treated with a range of concentrations of QAF (5, 10, 20, and 50 μg/mL) for 24 h, irradiated with UVB at a dose of 30 mJ/cm², and harvested 10 h later. Protein expression was evaluated using western blotting, and (D) the band intensities were quantified. Data are expressed as the mean ± SD. #p < 0.05, ##p < 0.01, ###p < 0.001 versus control group; *p < 0.05, **p < 0.01, ***p < 0.001 versus UVB-treated group

Fig. 6

A proposed mechanism of action of Quercus acuta Thunb. fruit on ultraviolet B-induced photoaging in HaCaT cells