Exosome-like nanovesicles derived from kale juice enhance collagen production by downregulating Smad7 in human skin fibroblasts

Abstract

Plant-derived exosome-like nanovesicles (ELNs) are critical mediators of cross-kingdom communication, modulating gene expression in animal cells despite their plant origin. In this study, we investigated the effects of glucoraphanin-enriched kale (GEK)-derived ELNs (GELNs) on collagen production in normal human dermal fibroblasts NB1RGB. The ELNs isolated from GEK juice powder had particle sizes similar to those of typical exosomes. GELNs increased type I collagen expression in NB1RGB cells significantly. Microarray analysis demonstrated that GELN-derived total RNA upregulated the expression of genes related to extracellular matrix formation, including those involved in collagen synthesis. Further investigation revealed that microRNA-enriched fraction of GELNs promoted collagen production by inhibiting the expression of Smad7. These findings suggest that GELNs and their microRNA content enhance collagen production through the downregulation of Smad7.

Keywords: collagen; exosome-like nanovesicles; exosomes; fibroblasts; kale; miRNA.

Figure 1

Characterization of glucoraphanin-enriched kale-derived exosome-like nanovesicles (GELNs). (A) Transmission electron microscopy image of GELNs. Scale bar = 200 nm. (B) Nanoparticle size analyzer measurement of the particle size of GELNs.

Figure 2

Effects of glucoraphanin-enriched kale-derived exosome-like nanovesicles (GELNs) on collagen I expression in NB1RGB cells. Values are presented as mean ± standard deviation (n = 4–5). Significant differences between groups were analyzed using one-way analysis of variance, followed by Dunnett’s test. *p < 0.05, ***p < 0.001 compared to the control.

Figure 3

Effects of glucoraphanin-enriched kale-derived exosome-like nanovesicles on COL1A1 gene expression in NB1RGB cells. Values are presented as mean ± standard deviation (n = 3–4). Significant differences between groups were analyzed using one-way analysis of variance, followed by Dunnett’s test. *p < 0.05, ****p < 0.0001 compared to 0 h.

Figure 4

Gene Ontology (GO) and pathway enrichment analysis of upregulated genes (n = 3). (A) Bar chart of top 15 GO enriched terms in biological processes, molecular function, and cellular component. (B) Bar chart of GO terms related to extracellular matrix formation-related GO terms. (C) Bar chart of pathways related to extracellular matrix formation.

Figure 5

Quantitative PCR validation of differentially expressed genes involved in extracellular matrix formation. Values are presented as mean ± standard deviation (n = 3). Significant differences between groups were analyzed using Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to the control.

Figure 6

Effects of glucoraphanin-enriched kale-derived exosome-like nanovesicles (GELNs) (A,C) and GELN-RNA (B,D) on collagen (A,B) and hyaluronic acid (C,D) production in NB1RGB cells. Values are presented as mean ± standard deviation (n = 3–4). Significant differences between groups were analyzed using Student’s t-test. *p < 0.05, ***p < 0.001, ****p < 0.0001 compared to the control.

Figure 7

Effects of total RNA, miRNA-enriched fraction, and novel_11 miRNAs on collagen I and Smad7 expression in NB1RGB cells. (A) The sequence of putative novel_11 binding site in Smad7, (B) collagen I expression, (C) Smad7 expression. Values are presented as mean ± standard deviation (n = 4). Significant differences between groups were analyzed using one-way analysis of variance, followed by Tukey’s multiple comparison test. Different letters denote significant differences between the groups (p < 0.05).