Human Hair Follicle Mesenchymal Stem Cell-Derived Exosomes Attenuate UVB-Induced Photoaging via the miR-125b-5p/TGF-β1/Smad Axis

Abstract

Cutaneous photoaging, induced by chronic exposure to ultraviolet (UV) radiation, typically manifests as alterations in both the physical appearance and functional properties of the skin and may predispose individuals to cancer development. Recent studies have demonstrated the reparative potential of exosomes derived from mesenchymal stem cells in addressing skin damage, while specific reports highlight their efficacy in ameliorating skin photoaging. However, the precise role of exosomes derived from human hair follicle mesenchymal stem cells (HFMSC-Exos) in the context of cutaneous photoaging remains largely unexplored. We successfully isolated HFMSC-Exos using the ultracentrifugation technique. In cellular experiments, we assessed the migration of human dermal fibroblasts (HDFs) through scratch and transwell assays, evaluated the angiogenesis of human umbilical vein endothelial cells through angiogenesis assays, and examined the expression levels of collagen and matrix metalloproteinase 1 (MMP-1) using Western blotting and quantitative reverse transcription polymerase chain reaction. Furthermore, we established a nude mouse model of photoaging to observe wrinkle formation on the dorsal surface of the animals, as well as to assess dermal thickness and collagen fiber generation through histological staining. Ultimately, we performed RNA sequencing on skin tissues from mice before and after treatment to elucidate the relevant underlying mechanisms. Our findings revealed that HFMSC-Exos effectively enhanced the migration and proliferation of HDFs and upregulated the expressions of transforming growth factor-β1 (TGF-β1), p-Smad2/p-Smad3, collagen type 1, and collagen type 3 while concurrently down-regulating MMP-1 levels in HDFs. Additionally, mice in the HFMSC-Exo group showed quicker wrinkle healing and increased collagen production. HFMSC-Exos miR-125b-5p was demonstrated to reduce skin photoaging by increasing profibrotic levels via TGF-β1 expression. UV-irradiated HDFs and photoaged nude mouse skin showed low TGF-β1 expressions, whereas overexpression of TGF-β1 in HDFs increased collagen type 1, collagen type 3, and p-Smad2/p-Smad3 expressions while decreasing MMP-1 expression. HDFs overexpressing TGF-β1 produced more collagen and altered the Smad pathway. This study demonstrated, both in vitro and in vivo, that HFMSC-Exos increased collagen formation, promoted HDF cell proliferation and migration, and reversed the senescence of UV-irradiated HDFs. TGF-β1 was identified as a target of HFMSC-Exos miR-125b-5p, which controls photoaging via regulating the Smad pathway. The antiphotoaging capabilities of HFMSC-Exos may occur via the miR-125b-5p/TGF-β1/Smad axis, suggesting a promising therapeutic approach for treating skin photoaging.

Fig. 1.

Identification of HFMSCs and HFMSC-Exo. (A) Representative flow cytometry analysis of HFMSCs, MSC surface markers (CD90, 99.8%; CD105, 99.6%; CD49d, 100%; and CD73, 99.9%) and HSC surface markers (CD14, 0.48%; CD34, 5.63%; HLA-DR, 0.048%; and CD45, 0.19%). (B) Image of osteogenic differentiation measured by Alizarin Red S staining (scale bar = 500 μm). (C) Image of adipogenic differentiation measured by Oil Red O staining (scale bar = 500 μm). (D) Image of chondrogenic differentiation measured by Alcian blue staining. (E) Schematic diagram of the HFMSC-Exo preparation process. (F) Transmission electron microscopy analysis of HFMSC-Exo morphology (scale bar = 500 nm). (G) Particle size distribution in HFMSC-Exos was quantified using NanoSight tracking analysis. (H) Western blotting examination of recognized positive indicators (CD9, CD63, and CD81) and negative indicators (calnexin). FITC, fluorescein isothiocyanate; PE, phycoerythrin; HFMSC, human hair follicle mesenchymal stem cells; HFMSC-Exos, exosomes derived from human hair follicle mesenchymal stem cells; MSCs, mesenchymal stem cells; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig. 2.

Impact of HFMSC-Exos on HDF proliferation and migration. (A) Representative image of DiR-labeled HFMSC-Exos internalized by HDFs (scale bar = 100 μm). (B) Analysis of HDF proliferation rate after exposure to HFMSC-Exos. (C) Representative image of HDF β-galactosidase expression after exposure to HFMSC-Exos (scale bar = 1,000 μm). (D) Proportion of senescent cells in each group in panel (C). (E and F) Representative images of the angiogenic capacity of human umbilical vein endothelial cells exposed to HFMSC-Exos analyzed by angiogenesis assay (scale bar = 1,000 μm). (G and H) The impact of HFMSC-Exos on HDF migration was evaluated using a scratch assay and a transwell migration assay (scale bar = 200 μm). (I and J) Statistical analyses were performed of the scratch migration rates of panels (G) and (H) and the number of cells crossed in the transwell assay (scale bar = 1,000 μm). Data are shown as mean ± standard error of the mean (SEM) (*P < 0.05; **P < 0.01). HDF, human dermal fibroblast; HFMSC, human hair follicle mesenchymal stem cells; HFMSC-Exos, exosomes derived from human hair follicle mesenchymal stem cells; MSCs, mesenchymal stem cells; DAPI, 4′,6-diamidino-2-phenylindole; UVB, ultraviolet B.

Fig. 3.

HFMSC-Exos accelerated extracellular matrix remodeling by inhibiting the generation of reactive oxygen species and upregulating collagen expression. (A) Representative images of dichlorodihydrofluorescein diacetate staining to detect the ability of HFMSC-Exos to scavenge intracellular reactive oxygen species from HDFs; scale bar = 25 μm. (B) Relative quantitative plots of fluorescence intensities for each group in panel (A). The fluorescence intensities of reactive oxygen species were markedly decreased in HDFs exposed to HFMSC-Exos compared to that of the ultraviolet B group, and this variance held statistical significance. (C to E) Quantitative real-time polymerase chain reaction plots of relative quantification of COL-1, COL-3, and MMP-1 messenger RNA (mRNA) expression in HDFs exposed to HFMSC-Exos, with statistically marked differences. (F) Bands of protein expression of COL-1, COL-3, MMP-1, and NF-E2-related factor 2 in HDFs exposed to HFMSC-Exos detected by Western blotting. (G to J) The relative quantitative statistics of COL-1, COL-3, MMP-1, and NF-E2-related factor 2 protein expression are plotted. Quantitative data were normalized against glyceraldehyde-3-phosphate dehydrogenase, and the differences were statistically marked. Data are shown as mean ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001). HDFs, human dermal fibroblasts; HFMSCs, human hair follicle mesenchymal stem cells; HFMSC-Exos, exosomes derived from human hair follicle mesenchymal stem cells; COL-1, collagen type 1; COL-3, collagen type 3; MMP-1, matrix metalloproteinase 1; MSCs, mesenchymal stem cells; CON, control; DCFH-DA, dichlorodihydrofluorescein diacetate; Nrf2, NF-E2-related factor 2; MFI, mean fluorescence intensity.

Fig. 4.

Effect of HFMSC-Exos on a photoaging model in nude mice. (A) Schematic diagram of the experimental procedure. (B) Digital photographs of the dorsal skin areas after ultraviolet B irradiation at weeks 0 and 8 and treatment with HFMSC-Exos. (C) Bar graphs showing the Wrinkle Severity Rating Scale scores of each group. (D) Representative images of H&E and Masson’s trichrome staining of dorsal skin after 2 weeks of treatment with HFMSC-Exos (scale bar = 100 μm). (E and F) Relative quantitative plots of dermal thickness and collagen fibers in sections. (G and H) Representative immunohistochemical images of CD31 in mouse skin tissue and graphs of the relative orientation of CD31. Data are shown as mean ± standard error (*P < 0.05; **P < 0.01; ***P < 0.001). H&E, hematoxylin and eosin; HFMSCs, human hair follicle mesenchymal stem cells; HFMSC-Exos, exosomes derived from human hair follicle mesenchymal stem cells; MMP-1, matrix metalloproteinase 1; MSCs, mesenchymal stem cells; WSRS, Wrinkle Severity Rating Scale; IH, immunohistochemical staining.

Fig. 5.

Antiphotoaging properties and safety assessment of HFMSC-Exos in vivo. (A) Representative Western blotting images of COL-1, COL-3, and MMP-1 in skin tissues. (B to D) Statistical analysis of histograms against the Western blotting images shown in panel (A). The differences were statistically marked. (E and F) Statistical histograms of the relative expressions of COL-1 and MMP-1 mRNA obtained by reverse transcription polymerase chain reaction; the differences were statistically marked. (G to I) Statistical representations of the levels of inflammatory agents interleukin-4, interleukin-6, and interleukin-10 in mice. (J to M) Biochemical analyses of the statistical images of ALT, AST, BUN, and Cr obtained from mice in vivo (*P < 0.05; **P < 0.01). ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; Cr, creatinine; HFMSC-Exos, exosomes derived from human hair follicle mesenchymal stem cells; IH, immunohistochemical staining; IL-4, interleukin-4; IL-6, interleukin-6; IL-10, interleukin-10; MMP-1, matrix metalloproteinase 1.

Fig. 6.

Analysis of RNA sequencing results. (A) Heatmap of differentially expressed genes of the TGF-β1/Smad pathway. (B) Gene Ontology plots for all differential genes. (C) Kyoto Encyclopedia of Genes and Genomes enrichment maps for all differential genes. (D) Heatmap of differentially expressed genes associated with the TGF-β1/Smad pathway Smad2/3. (E) Bioinformatics-predicted binding sites of miR-125b-5p to TGF-β1. (F and G) Reverse transcription polymerase chain reaction validation results of the dual-luciferase reporter (**P < 0.01). TGF-β1, transforming growth factor-β1; UTR, untranslated region; ECM, extracellular matrix; WT, wild type; MUT, mutant; luc, luciferase activity; NC, negative control.

Fig. 7.

Validation of miRNA-125b-5p in relation to TGF-β1 targeting and its effect on the Smad pathway. (A) Representative Western blotting images of microRNA (miRNA) regulation of TGF-β1 and downstream COL-1, COL-3, and MMP-1. (B to E) Statistical images of the proteins shown in panel (A). (F) Representative effects of miRNA-125b-5p on Smad2/3 and phosphorylated protein Western blotting images. (G and H) Relative quantitative statistical images of phosphorylated proteins shown in panel (F). (I) Schematic representation of experimental results (*P < 0.05; **P < 0.01). COL-1, collagen type 1; COL-3, collagen type 3; MMP-1, matrix metalloprotein 1; TGF-β1, transforming growth factor-β1; OE, overexpression.