Exosomes derived from human dermal fibroblasts protect against UVB‑induced skin photoaging

Abstract

Exosomes are used as innovative treatment options for repairing skin defects, such as aging, atopic dermatitis and wounds. However, the effects of exosomes obtained from human foreskin fibroblasts BJ‑5ta (BJ‑5ta Exo) on ultraviolet B (UVB)‑mediated photoaging have not been previously reported, at least to the best of our knowledge. Therefore, the present study aimed to investigate the anti‑photoaging effects of BJ‑5ta Exo on UVB radiation in human skin fibroblasts and SKH‑1 hairless mice. The results revealed that BJ‑5ta Exo decreased the production of reactive oxygen species and inhibited the decrease in the expression levels of superoxide dismutase 1 and 2, glutathione peroxidase and catalase following UVB exposure. In addition, BJ‑5ta Exo attenuated the decrease in nuclear factor erythroid 2‑related factor 2 levels induced by UVB rays, indicating its scavenging activity against oxidative stress. Moreover, BJ‑5ta Exo inhibited the UVB‑induced increase in the levels of γH2AX, p53/21 and cleaved PARP, whereas it promoted DNA double‑strand break repair through radiation sensitive 52 and effectively activated the TGF‑β1/Smad pathway. BJ‑5ta Exo also protected against UVB‑induced senescence, as indicated by the downregulation in the levels of senescence‑associated β‑galactosidase and p16. In a mouse model of photoaging, BJ‑5ta Exo prevented the UVB‑induced increase in transepidermal water loss, wrinkle formation and MMP‑1 expression, while also suppressing the UVB‑mediated decrease in collagen type I and elastin levels in the dorsal skin. Overall, the findings of the present study suggest that BJ‑5ta Exo represent an effective anti‑photoaging agent, which can be used as a component in cosmetic products.

Keywords: BJ‑5ta fibroblast; DNA damage; ROS; UVB; exosome; oxidative stress; skin photoaging.

Figure 1

Characterization of BJ-5ta Exo and its effects on cell viability. (A) Representative image of BJ-5ta Exo obtained using a field emission scanning electron microscope (FE-SEM). Scale bar, 100 nm (left panel) and 300 nm (right panel). (B) Representative histogram illustrating the particle concentration and size distribution of BJ-5ta Exo measured by nanoparticle tracking analysis. (C) Expression of CD63, ALIX and calnexin in BJ-5ta-Exo as determined using western blot analysis. (D) BJ-5ta cells were treated with BJ-5ta-Exo (10³-10⁶ particles/ml) for 24 h, followed by cell viability analysis using the WST-8 cell viability assay. (E) Cell viability examined in UVB-irradiated cells at various concentrations. (F) The protective effect of BJ-5ta Exo (10⁴ particles/ml) on UVB-irradiated BJ-5ta cells (30 mJ/cm²) as assessed using the WST-8 cell viability assay. (G) Immunocytochemistry was performed using Ki-67 (in red) and DAPI (in blue) on BJ-5ta cells pre-treated with BJ-5ta Exo (10⁴ particles/ml) for 6 h, followed by irradiation with UVB (30 mJ/cm²) and 24 h of culture. Scale bar, 50 μm. ^****P<0.0001, compared with UVB-irradiated cells; ^###P<0.001 and ^####P<0.0001s compared with normal cells. BJ-5ta Exo, exosomes derived from BJ-5ta cells; UVB, ultraviolet B.

Figure 2

Effects of BJ-5ta Exo on intercellular ROS levels. (A) UVB-induced intracellular ROS generation was attenuated by BJ-5ta Exo (10³,10⁴ and 10⁵ particles/ml) in BJ-5ta cells. (B) Representative intracellular ROS images of cells pre-treated with BJ-5ta Exo (10⁴ particles/ml) for 6 h, followed by UVB (30 mJ/cm²) irradiation. Scale bar, 200 μm. (C) The mRNA expression levels of Nrf2 downstream antioxidant enzymes (SOD-1 or 2, CAT and GPX). (D) Total Nrf2 protein levels. (E) Nrf2 localization was determined using immunocytochemistry with an anti-Nrf2 antibody (green fluorescence) and DAPI staining (blue fluorescence). Scale bar, 20 μm. BJ-5ta cells were pre-treated with BJ-5ta Exo (10⁴ particles/ml) for 6 h, followed by exposure to UVB (30 mJ/cm²). Following 3 h of incubation (C-E), the cells were analyzed using RT-qPCR, western blot analysis, or immunocytochemistry. ^*P<0.05, ^**P<0.01, ^***P<0.001 and ^****P<0.0001, compared with UVB-irradiated cells; ^##P<0.01, ^####P<0.0001, compared with normal cells. BJ-5ta Exo, exosomes derived from BJ-5ta cells; UVB, ultraviolet B; ROS, reactive oxygen species; SOD, superoxide dismutase; CAT, catalase; GPX, glutathione peroxidase; Nrf2, nuclear factor erythroid 2-related factor 2.

Figure 3

Effects of BJ-5ta Exo on UVB-induced DNA damage, the p53/p21 pathway and senescence in BJ-5ta cells. (A) Cell lysates were analyzed using western blot analysis with antibodies against γH2AX. (B) The expression of γH2AX was determined using immunocytochemistry with an anti-γH2AX antibody (green fluorescence) and DAPI (blue fluorescence). Scale bar, 20 μm. Protein levels of (C) RAD51 and cleaved PARP were analyzed using western blot analysis. (D) Apoptosis and (E) cell cycle distribution was analyzed using flow cytometry. (F) The levels of p53, and its downstream effector p21, were assessed using western blot analysis. (G) SA-β-Gal-positive cells were observed in BJ-5ta cells pre-treated with and without BJ-5ta Exo (10⁴ particles/ml) and exposed to UVB (30 mJ/cm²). Scale bar, 200 μm. (H) The protein levels of p16, a marker of cellular senescence, were determined using western blot analysis. Cells were pre-treated with and without BJ-5ta Exo (10⁴ particles/ml) for 6 h and then exposed to UVB radiation (30 mJ/cm²). Following 24 h of incubation, the cells were analyzed using western blot analysis or immunocytochemistry. In western blot analysis, the protein levels were quantified and presented relative to the β-actin levels. The results are expressed as the mean ± standard deviation. ^*P<0.05, ^**P<0.01 and ^****P<0.0001, compared with UVB-irradiated cells; ^#P<0.05, ^##P<0.01, ^###P<0.001 and ^####P<0.0001, compared with normal cells. BJ-5ta Exo, exosomes derived from BJ-5ta cells; UVB, ultraviolet B; SA-β-Gal, senescence-associated β-galactosidase.

Figure 4

Inhibition of UVB-induced expression of p-NF-κB, iNOS and COX-2 by BJ-5ta Exo. BJ-5ta cells were treated with and without BJ-5ta-Exo for 6 h and then irradiated with UVB (30 mJ/cm²). After 24 h, cell lysates were examined using western blot analysis for the protein levels of (A) COX-2 and iNOS, and (B) p-NF-κB (Ser⁵³⁶). (C) NF-κB localization was determined using immunocytochemistry with an anti-NF-κB antibody (green fluorescence) and DAPI (blue fluorescence). The levels of the phosphorylated proteins were normalized to β-actin or total NF-κB proteins. The results are expressed as the mean ± standard error. P<0.05, ^**P<0.01, ^***P<0.001 compared with UVB-irradiated cells; ^##P<0.01, ^####P<0.0001 compared with normal cells. BJ-5ta Exo, exosomes derived from BJ-5ta cells; UVB, ultraviolet B; iNOS, inducible nitric oxide synthase; COX-2, cyclooxynase 2.

Figure 5

Effects of BJ-5ta Exo on the levels of collagen type I, MMP-I, TGF-β1/Smads and MAPK/AP-1 phosphorylation in UVB-irradiated BJ-5ta fibroblasts. Protein levels of (A) collagen type I and MMP-1; (B) TGF-β1, p-Smad2 (Ser^465/467), p-Smad3 (Ser^423/425) and Smad7; (C) p-JNK (Thr¹⁸³/Tyr¹⁸⁵), p-ERK (Thr²⁰²/Tyr²⁰⁴), and p-p38 (Thr¹⁸⁰/Tyr¹⁸²); (D) p-c-Fos (Ser³²) and p-c-Jun (Ser⁷³) were examined using western blot analysis. Cells were pre-treated with and without BJ-5ta Exo (10⁴ particles/ml) for 6 h and then exposed to UVB radiation (30 mJ/cm²). Following (A) 24 h or (B-D) 30 min of incubation, the cells were examined using western blot analysis. The levels of the phosphorylated proteins were normalized to β-actin or total proteins. The results are expressed as the mean ± standard error. ^*P<0.05, ^**P<0.01, ^***P<0.001 compared with UVB-irradiated cells; ^#P<0.05, ^##P<0.01, ^###P<0.001 and ^####P<0.0001 compared with normal cells. BJ-5ta Exo, exosomes derived from BJ-5ta cells; UVB, ultraviolet B; MMP, matrix metalloproteinase.

Figure 6

Subcutaneous injection of BJ-5ta Exo attenuates UVB-induced wrinkle formation in skin of mice. (A) To evaluate the changes in dorsal wrinkle formation, images were obtained using a DSLR and PRIMOS CR. (B) H&E staining. (C) MT staining. (D) VVG staining. (E) The quantification of wrinkles (roughness) revealed that the depth of the wrinkles was reduced by BJ-5ta Exo. RA, average roughness. (F) Skin hydration was evaluated using a Corneometer. (G) TEWL was evaluated using a Tewameter TM300. (H) Epidermal thickness was quantified using ImageJ software. (I) Procollagen type I, (J) collagen type I, (K) elastin, and (L) MMP-1 were analyzed using immunohistochemistry. Scale bar, 50 μm. (M) Immunohistochemistry arbitrary units (a.u.) of procollagen type I, collagen type I, MMP-1 and elastin in SKH-1 mice. The results are expressed as the mean ± standard error. ^*P<0.05, ^**P<0.01, ^***P<0.001 and ^****P<0.0001, compared with UVB irradiation; ^#P<0.05, ^##P<0.01, ^###P<0.001 and ^####P<0.0001, compared with the control. BJ-5ta Exo, exosomes derived from BJ-5ta cells; UVB, ultraviolet B; H&E, hematoxylin and eosin; MT, Masson's trichome; VVG, Verhoeff-van Gieson; TEWL, transepidermal water loss.

Figure 7

Protective effects of BJ-5ta Exo on UVB-induced photoaging was evaluated using a reconstructed human skin model, Neoderm^®-ED. The impact of BJ-5ta Exo on UVB-induced skin tissue damage was assessed. Following UVB irradiation, the skin tissues were subjected to staining with (A) H&E, (B) collagen type I, (C) elastin, and (D) MMP-1. (E) Epidermal thickness was quantified using ImageJ software. (F) PIP and (G) MMP-1 expression levels were analyzed using ELISA. The reconstructed human skin model was exposed to UVB (128 mJ/cm²), and the supernatants were collected after 48 h. BJ-5ta Exo, exosomes derived from BJ-5ta cells; UVB, ultraviolet B; H&E, hematoxylin and eosin; MMP, matrix metalloproteinase; PIP, procollagen type I C peptide. ^*P<0.05 and ^***P<0.001, compared with UVB irradiation; ^#P<0.05, ^##P<0.01 and ^###P<0.001, compared with the control.

Figure 8

BJ-5ta Exo exert a protective effect against UVB-induced photoaging. BJ-5ta Exo protect against UVB-mediated winkle formation by inhibiting the MAPK/AP-1 signaling pathway and subsequent MMP-1 release and activating collagen synthesis. Furthermore, BJ-5ta Exo inhibited inflammation, apoptosis, cell cycle arrest and DNA damage caused by UVB exposure. Overall, these findings suggest that BJ-5ta Exo may be a valuable anti-photoaging agent for cosmetic products. BJ-5ta Exo, exosomes derived from BJ-5ta cells; UVB, ultraviolet B; MMP, matrix metalloproteinase.