Apple Derived Exosomes Improve Collagen Type I Production and Decrease MMPs during Aging of the Skin through Downregulation of the NF-κB Pathway as Mode of Action

Abstract

Skin ageing is strictly related to chronic inflammation of the derma and the decay of structural proteins of the extracellular matrix. Indeed, it has become common practice to refer to this phenomenon as inflammageing. Biotech innovation is always in search of new active principles that induce a youthful appearance. In this paper, apple-derived nanovesicles (ADNVs) were investigated as novel anti-inflammatory compounds, which are able to alter the extracellular matrix production of dermal fibroblasts. Total RNA sequencing analysis revealed that ADNVs negatively influence the activity of Toll-like Receptor 4 (TLR4), and, thus, downregulate the NF-κB pro-inflammatory pathway. ADNVs also reduce extracellular matrix degradation by increasing collagen synthesis (COL3A1, COL1A2, COL8A1 and COL6A1) and downregulating metalloproteinase production (MMP1, MMP8 and MMP9). Topical applications for skin regeneration were evaluated by the association of ADNVs with hyaluronic-acid-based hydrogel and patches.

Keywords: apple; exosomes; extracellular matrix; inflammageing; wound healing.

Figure 1

(A) SEM Imaging of ADNVs (MAG, 5.0 KX; WD, 15 mm; EHT, 15 kV; High Vacuum); (B) Protein quantification of the ADNV fraction after isolation (μg/mL); and (C) TRPS analysis output.

Figure 2

(A,B) Release curve of ADNVs from hyaluronic-acid-based materials, MeHA patch and HY 2% gel, at room temperature (RT = 25 °C) and physiological temperature (37 °C). ADNV release is expressed in form of protein concentration (μg/mL) in time (hours). (C) Values corresponding to the area underneath each curve in (A,B) (SE). (D) Illustrative image of MeHA patch and HY 2% gel. **** p < 0.0001.

Figure 3

(A) MTT results for NCTC L929 cells grown in control medium (CTRL) and in ADNV-spiked medium (ADNVs); cell viability is expressed as absorbance. (B,C) Migration assay results are displayed as images taken in time, from 0 to 12 h (hours); the red line indicates the scratch’s middle, while black arrows point towards cells that have significantly moved since the previous image was taken.

Figure 4

(A,B) Volcano plot of microarray outputs for primary dermal fibroblasts grown w/ TNFα (A) or w/o TNFα. Both graphs represent control (w/o ADNVs) vs treated (w/ ADNVs) conditions. Ten most relevant genes in terms of fold change and significance are reported.

Figure 5

(A–C) Expression logarithmic ratios of differentially regulated genes from the w/ TNFα microarray involved in ECM maintenance. Expression is displayed as a bar, while significance, expressed as −log10(p-value), is reported in orange.

Figure 6

(A–C) Expression logarithmic ratios of differentially regulated genes from the w/ TNFα microarray involved in the inflammation response. Expression is displayed as a bar, while significance, expressed as −log10(p-value), is reported in orange. (D) KEGG pathway: IL-1b and NF-κB pathway, with color-coded protein slots corresponding to DEGs expression.

Figure 7

Canonical pathway analysis by IPA for w/ TNFα. (A) Ten first entries. (B–E) Genes involved in the first four pathways with expression log ratio values.

Figure 8

(A) Upstream miRNA regulators for w/ TNFα IPA analysis, z-Score and p-value are shown. (B) IPA regulator’s ranked by influenced functions, shown as percentage of the total regulator effects. (C) KEGG pathway: Toll-like receptors signaling pathway, with color-coded protein slots corresponding to DEGs expression.

Figure 9

(A) Canonical pathway analysis by IPA for w/o TNFα. (A) Ten first entries. (B) Gene expression of genes involved in cholesterol biosynthesis (w/o TNFα dataset); expression is displayed as a bar, while significance, expressed as −log10(p-value), is reported in orange. (C) RT-qPCR results for gene expression in w/ TNFα fibroblasts after 1× and 0.5× ADNV treatment (** p < 0.01, *** p < 0.001).

Figure 10

Mitochondrial stress under all conditions is expressed as percentage of cells with above mitochondrial ROS production. * p < 0.05, *** p < 0.001.