Ginseng root-derived exosome-like nanoparticles protect skin from UV irradiation and oxidative stress by suppressing activator protein-1 signaling and limiting the generation of reactive oxygen species

Abstract

Background: Recently, plant-derived exosome-like nanoparticles (PDENs) have been isolated, and active research was focusing on understanding their properties and functions. In this study, the characteristics and molecular properties of ginseng root-derived exosome-like nanoparticles (GrDENs) were examined in terms of skin protection.

Methods: HPLC-MS protocols were used to analyze the ginsenoside contents in GrDENs. To investigate the beneficial effect of GrDENs on skin, HaCaT cells were pre-treated with GrDENs (0-2 × 10⁹ particles/mL), and followed by UVB irradiation or H₂O₂ exposure. In addition, the antioxidant activity of GrDENs was measured using a fluorescence microscope or flow cytometry. Finally, molecular mechanisms were examined with immunoblotting analysis.

Results: GrDENs contained detectable levels of ginsenosides (Re, Rg1, Rb1, Rf, Rg2 (S), Gyp17, Rd, C-Mc1, C-O, and F2). In UVB-irradiated HaCaT cells, GrDENs protected cells from death and reduced ROS production. GrDENs downregulated the mRNA expression of proapoptotic genes, including BAX, caspase-1, -3, -6, -7, and -8 and the ratio of cleaved caspase-8, -9, and -3 in a dose-dependent manner. In addition, GrDENs reduced the mRNA levels of aging-related genes (MMP2 and 3), proinflammatory genes (COX-2 and IL-6), and cellular senescence biomarker p21, possibly by suppressing activator protein-1 signaling.

Conclusions: This study demonstrates the protective effects of GrDENs against skin damage caused by UV and oxidative stress, providing new insights into beneficial uses of ginseng. In particular, our results suggest GrDENs as a potential active ingredient in cosmeceuticals to promote skin health.

Keywords: Aging; Ginseng-derived exosome-like nanoparticles; Ginsenosides; Oxidative stress; UV irradiation.

Graphical abstract

Fig. 1

Cell viability and intracellular ROS levels of HaCaT cells treated with GrDENs. (A and B) HaCaT cells were treated alone with GrDENs (0–2 × 10⁹ particles/mL) for 24 h in panel A. For panel B, HaCaT cells were pre-treated with GrDENs (0–2 × 10⁹ particles/mL) for 30 min and stimulated with UVB irradiation for 24 h. Cell viability was examined by MTT assay. (C and D) HaCaT cells were pre-treated with GrDENs (0–2 × 10⁹ particles/mL) for 30 h and irradiated by UVB for 24 h, and the cells were incubated with H2DCFDA (C) or DCFH-DA (D) for 20 min. Intracellular ROS levels were determined by fluorescent imaging (C) and flow cytometry (D). Fluorescent intensity was measured with ImageJ (C). Data in (A), (B), and (C) are presented as mean ± standard deviation of at least three independent experiments. Results in (D) are representative images from three independent experiments. ##p < 0.01 compared to the normal group (non-treatment), and *p < 0.05, **p < 0.01 compared to the control group (UV irradiation).

Fig. 2

Effect of GrDENs on the expression of apoptotic genes and activities of caspases. HaCaT cells were pre-treated with GrDENs for 30 min and irradiated by UVB for 24 h. (A–F) The mRNA levels of BAX (A), caspase-1 (B), caspase-3 (C), caspase-6 (D), caspase-7 (E), and caspase-8 (F) were determined by real-time PCR. (G–J) To examine the alterations of caspase activities when treated with GrDENs, cleaved caspase levels were analyzed by immunoblotting (G). Band intensity was measured with ImageJ, and the relative band intensities of cleaved caspase-8 (H), cleaved caspase-9 (I), and cleaved caspase-3 (J) were normalized to the corresponding total caspases. Data in (A), (B), (C), (D), (E), (F), (H), (I), and (J) are presented as mean ± standard deviation of three independent experiments. Results in (G) are representative images from three independent experiments. ##p < 0.01 compared to the normal group (non-treatment), and *p < 0.05, **p < 0.01 compared to the control group (UV irradiation).

Fig. 3

Effect of GrDENs in expression of skin aging- and inflammation-related genes. HaCaT cells were pre-treated with GrDENs (0–2 × 10⁹ particles/mL) for 30 min and exposed by H₂O₂ for 24 h. (A–C) The expression of the aging-related genes, such as MMP-2 (A), MMP-3 (B), and MMP-9 (C), was determined by real-time PCR. (D and E) The mRNA levels of the inflammation-associated genes, such as COX-2 (D) and IL-6 (E), were examined with real-time PCR. (F) The gene expression of p21, a cellular senescence biomarker, was determined with real-time PCR. All data are presented as mean ± standard deviation of three independent experiments. ##p < 0.01 compared to the normal group (non-treatment), and *p < 0.05, **p < 0.01 compared to the control group (H₂O₂ exposure group).

Fig. 4

Effect of GrDENs in AP-1 signaling. HaCaT cells were pre-treated with GrDENs (2 × 10⁹ particles/mL) for 30 min, and the cells were then exposed to H₂O₂ for the indicated time in the figures. (A–C) Phosphorescence and total levels of AP-1 subunits, including c-jun (A and B) and c-fos (A and C), were determined by immunoblotting. (D–G) Phosphorescence and total levels of AP-1 pathway-related molecules such as ERK (D and E), JNK (D and F), and p38 (D and G) were detected with immunoblotting analysis. (H and I) Phosphorescence and total levels of NF-kB pathway-related molecule, p65 were detected with immunoblotting analysis. ImageJ was used to measure the band intensity, and the relative band intensity of phospho-proteins was normalized to the corresponding total proteins. Data in (B), (C), (E), (F), (G), and (I) are shown as mean ± standard deviation of three independent experiments, and representative images are presented in (A), (D), and (H). ##p < 0.01, #p < 0.05 compared to the normal group (non-treatment), and *p < 0.05, **p < 0.01 compared to the control group (H₂O₂ exposure group).

Fig. 5

Effect of GrDENs on MEK1/2. (A and B) HaCaT cells were pre-treated with GrDENs (2 × 10⁹ particles/mL) for 30 min, and the cells were exposed to H₂O₂ for 5, 15, or 30 min. (C and D) The HaCaT cells were transfected with Myc-MEK2 for 24 h, and the cells were treated with GrDENs (0–2 × 10⁹ particles/mL) for an additional 24 h. Phosphorescence and total MEK1/2 levels were determined with immunoblotting, and β-actin was used as a loading control. ImageJ was used to measure the band intensity, and the relative band intensity of phospho-proteins was normalized to the corresponding total proteins. Data in (B) and (D) are presented as mean ± standard deviation of three independent experiments, and representative images are presented in (A) and (C). ##p < 0.01 compared to the normal group (non-treatment), and *p < 0.05, **p < 0.01 compared to the control group (H₂O₂ exposure or Myc-MEK2 overexpression group).

Fig. 6

The effect of each ginsenoside component upon H₂O₂ exposure. (A and B) HaCaT cells were pre-treated with ginsenoside Re (3.8 μg/mL), Rg1 (3.5 μg/mL), Rb1 (1.1 μg/mL), Rc (0.7 μg/mL), GrDENs (2 × 10⁹ particles/mL), or Ascorbic acid (500 μM) for 30 min and exposed by H₂O₂ for 24 h. The expression of the aging-related genes, such as MMP-2 (A), MMP-3 (B) was determined by real-time PCR. All data are presented as mean ± standard deviation of three independent experiments. ##p < 0.01 compared to the normal group (non-treatment), and *p < 0.05, **p < 0.01 compared to the control group (H₂O₂ exposure group).

Fig. 7

Summary of protective effect of GrDENs against UV irradiation and oxidative stress.