Colostrum-Derived Exosomal Lactoferrin Promotes Skin Fibroblast Regeneration by Suppressing Inflammatory Responses

Abstract

Lactoferrin (LF), a multifunctional glycoprotein found abundantly in bovine colostrum, is known for its regenerative and anti-inflammatory properties. In this study, we investigated the wound healing and immunomodulatory effects of colostrum-derived exosome-encapsulated lactoferrin (EV-exoLF) on dermal fibroblasts. EV-exoLF was isolated and characterized via nanoparticle tracking analysis and flow cytometry. Functional assays demonstrated that EV-exoLF significantly promoted fibroblast viability and migration in both mouse NIH/3T3 and human HS-68 cell lines. Furthermore, EV-exoLF reduced the expression of pro-inflammatory cytokines (IL-1 and IL-6) and phosphorylated JNK in lipopolysaccharide (LPS)-treated fibroblasts. These findings suggest that EV-exoLF not only enhances fibroblast-mediated wound closure but also mitigates inflammation, highlighting its therapeutic potential in skin regeneration. Colostrum-derived exosomal lactoferrin may serve as a promising natural, cell-free strategy for managing inflammatory skin conditions and improving wound healing outcomes.

Keywords: colostrum; cytokines; exosomes; fibroblasts; inflammation; lactoferrin; skin regeneration; wound healing.

Figure 1

Characterization of exosome-encapsulated lactoferrin (EV-exoLF) derived from colostrum. (A) Size detected via nanoparticle tracking analysis. (B) Flow cytometry scatter plots of the expression levels of representative EV-exoLF surface biomarkers (CD9, CD63 and CD81).

Figure 2

CCK-8 assay results showing the dose-dependent effects of EV-exoLF on the viability of mouse NIH/3T3 (A) and human HS-68 (B) cell lines following 24, 48, and 72 h of exposure. Untreated cells served as the control group. Data are presented as mean ± SEM from three independent experiments (n = 3). Statistical significance was determined using Student’s t-test; p < 0.05 (*), p < 0.01 (**) versus untreated controls.

Figure 3

EV-exoLF enhances the rate of wound closure in the mouse NIH/3T3 fibroblast cell line. (A) Representative light micrographs of the scratch wound assays used to evaluate migration rate at 24 and 48 h. A significant increase in the extent of wound closure was observed in the EV-exoLF group compared with that in the control Dulbecco’s Modified Eagle’s Medium (DMEM)/serum-free groups at 24 and 48 h. Scale bar: 200 µm. (B) Statistical analysis of the wound closure area performed using ImageJ software (1.54p) (n = 3 per group; ** p < 0.01, *** p < 0.001, one-way ANOVA and Tukey’s post hoc test). Error bars denote the standard error of the mean (SEM).

Figure 4

EV-exoLF enhances the rate of wound closure in the human new foreskin fibroblast cell line, HS-68 (CRL-1635). (A) Representative light micrographs of the scratch wound assays used to evaluate migration rate at 24 and 48 h. A significant increase in the extent of wound closure was observed in the EV-exoLF group compared with that in the control DMEM/serum-free groups at 24 h, but no significant difference observed between the EV-exoLF and control groups at 48 h. Scale bar: 200 µm. (B) Statistical analysis of the wound closure area performed using ImageJ software. (n = 3 per group; * p < 0.05, one-way ANOVA and Tukey’s post hoc test). Error bars denote the SEM.

Figure 5

The effect of EV-exoLF on the inflammatory response in the lipopolysaccharide (LPS)-treated HS-68 cell line. (A,B) Interleukin (IL)-1 and IL-6 results of HS-68 cells conditioned after treatment with or without EV-exoLF. (C) Changes in the expression of pJNK. Actin was used as an internal control. (D) Quantitative results showing the pJNK protein levels assessed using ImageJ. All data are presented as the mean ± standard deviation (SD) (n = 3. ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA and Tukey’s post hoc test). Error bars denote the SD.