Syringaresinol derived from Panax ginseng berry attenuates oxidative stress-induced skin aging via autophagy

Abstract

Background: In aged skin, reactive oxygen species (ROS) induces degradation of the extracellular matrix (ECM), leading to visible aging signs. Collagens in the ECM are cleaved by matrix metalloproteinases (MMPs). Syringaresinol (SYR), isolated from Panax ginseng berry, has various physiological activities, including anti-inflammatory action. However, the anti-aging effects of SYR via antioxidant and autophagy regulation have not been elucidated.

Methods: The preventive effect of SYR on skin aging was investigated in human HaCaT keratinocytes in the presence of H₂O₂, and the keratinocyte cells were treated with SYR (0-200 μg/mL). mRNA and protein levels of MMP-2 and -9 were determined by real-time PCR and Western blotting, respectively. Radical scavenging activity was researched by 2,2 diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays. LC3B level was assessed by Western blotting and confocal microscopy.

Results: SYR significantly reduced gene expression and protein levels of MMP-9 and -2 in both H₂O₂-treated and untreated HaCaT cells. SYR did not show cytotoxicity to HaCaT cells. SYR exhibited DPPH and ABTS radical scavenging activities with an EC₅₀ value of 10.77 and 10.35 μg/mL, respectively. SYR elevated total levels of endogenous and exogenous LC3B in H₂O₂-stimulated HaCaT cells. 3-Methyladenine (3-MA), an autophagy inhibitor, counteracted the inhibitory effect of SYR on MMP-2 expression.

Conclusion: SYR showed antioxidant activity and up-regulated autophagy activity in H₂O₂-stimulated HaCaT cells, lowering the expression of MMP-2 and MMP-9 associated with skin aging. Our results suggest that SYR has potential value as a cosmetic additive for prevention of skin aging.

Keywords: 3-MA, 3-Methyladenine; ABTS, 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid; Anti-aging; Antioxidant; Autophagy; CMA, chaperone-mediated autophagy; DMEM, Dulbecco's Modified Eagle's medium; DMSO, dimethyl sulfoxide; DPPH, 2,2 diphenyl-1-picrylhydrazyl; ECM, extracellular matrix; FBS, fetal bovine serum; FoxO3a, Forkhead box O3; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; Panax ginseng berry; ROS, reactive oxygen species; SYR, Syringaresinol; Syringaresinol; UV, ultraviolet.

Graphical abstract

Fig. 1

Anti-aging activity of SYR in HaCaT cells. (A) Chemical structure of syringaresinol and 3-Methyladenin (Left panel) and analysis profile of syringaresinol by HPLC (Right panel). (B, C, and D) Following SYR (100 and 200 μg/mL) treatment, HaCaT cells were incubated with H₂O₂ (500 μM) for 24 h mRNA levels of MMP-2 (B) and MMP-9 (C) were determined by real-time PCR. Protein levels of MMP-2 and MMP-9 were measured by Western blotting (D). (E) HaCaT cells were treated with various concentrations (0–200 μg/mL) of SYR and then incubated for 24 h. Protein levels of MMP-2 and MMP-9 were determined by immunoblotting analysis. (F) Viability of SYR-treated HaCaT cells was measured by MTT assay. ^#p < 0.05 and ^##p < 0.01 compared with the normal group; ∗p < 0.05 and ∗∗p < 0.01 compared with the control group.

Fig. 2

Antioxidant activity of SYR. (A) DPPH and SYR (0–200 μg/mL) were incubated together at 37 °C for 30 min. Absorbance at 517 nm was measured by spectrophotometry. (B) SYR (0–200 μg/mL) was mixed with ABST in the dark at 37 °C for 30 min. Absorbance at 730 nm was observed by spectrophotometry. ∗p < 0.05 and ∗∗p < 0.01 compared with the control group.

Fig. 3

Effect of SYR on autophagy activation. (A) SYR (100 and 200 μg/mL)-treated HaCaT cells were incubated with H₂O₂ for 12 h. Then, the total level of LC3B was determined by Western blotting. β-Actin was used as a loading control. (B) HaCaT cells were treated with SYR (50, 100, and 200 μg/mL) for 24 h, and then the total levels of LC3B and β -actin were determined by immunoblotting analysis. (C and D) HaCaT cells were transfected with a plasmid expressing GFP-LC3B and incubated with 200 μg/mL SYR and H₂O₂ for 12 h (C). HaCaT cells were transfected with plasmid GFP-LC3B and incubated with 200 μg/mL SYR for 24 h (D). Confocal microscopy images were observed using a laser-scanning confocal microscope (Zeiss LSM 710 META). ^#p < 0.05 and ^##p < 0.01 compared with the normal group; ∗p < 0.05 and ∗∗p < 0.01 compared with the control group.

Fig. 4

Relevance of autophagy activation to the anti-aging activity of SYR. (A and B) HaCaT cells were incubated with H₂O₂ (500 μM) in the presence or absence of SYR (200 μg/mL) or 3-MA (5 mM) for 24 h (A). HaCaT cells were treated with SYR (200 μg/mL) in the presence or absence of 3-MA (5 mM) for 24 h (B). The expression of MMP-2 and β-actin was determined by Western blotting. ^#p < 0.05 and ^##p < 0.01 compared with the normal group; ∗p < 0.05 and ∗∗p < 0.01 compared with the control group; ns, not significant.

Fig. 5

Schematic summary of the anti-aging effects of SYR and its mechanisms.