Glycomacropeptide Protects against Inflammation and Oxidative Stress, and Promotes Wound Healing in an Atopic Dermatitis Model of Human Keratinocytes

Abstract

Keratinocytes are actively implicated in the physiopathology of atopic dermatitis (AD), a skin allergy condition widely distributed worldwide. Glycomacropeptide (GMP) is a milk-derived bioactive peptide generated during cheese making processes or gastric digestion. It has antiallergic and skin barrier restoring properties when it is orally administered in experimental AD. This study aimed to evaluate the effect of GMP on the inflammatory, oxidative, proliferative, and migratory responses of HaCaT keratinocytes in an in vitro AD model. GMP protected keratinocytes from death and apoptosis in a dose dependent manner. GMP at 6.3 and 25 mg/mL, respectively, reduced nitric oxide by 50% and 83.2% as well as lipid hydroperoxides by 27.5% and 45.18% in activated HaCaT cells. The gene expression of TSLP, IL33, TARC, MDC, and NGF was significantly downregulated comparably to control by GMP treatment in activated keratinocytes, while that of cGRP was enhanced. Finally, in an AD microenvironment, GMP at 25 mg/mL stimulated HaCaT cell proliferation, while concentrations of 0.01 and 0.1 mg/mL promoted the HaCaT cell migration. Therefore, we demonstrate that GMP has anti-inflammatory and antioxidative properties and stimulates wound closure on an AD model of keratinocytes, which could support its reported bioactivity in vivo.

Keywords: atopic dermatitis; bioactive peptides; cytoprotection; glycomacropeptide; immunomodulation; keratinocytes; oxidative stress; wound healing.

Figure 1

Glycomacropeptide (GMP) has no toxic effect on keratinocytes. HaCaT cells were treated with GMP for 24 h and the percentage of cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazole bromide (MTT) assay. n = 12, 3 independent experiments in quadruplicate. + p < 0.05, ++ p < 0.01, +++ p < 0.001 vs. control.

Figure 2

GMP protects keratinocytes against cell death and apoptosis. HaCaT cells were treated with GMP and stimulated with (A,C) 50 µM 2,4-dinitrochlorobenzene (DNCB) or (B,D) 200 µM hydrogen peroxide (H₂O₂) to determine: (A,B) the percentage of cell viability by the MTT assay and (C,D) apoptosis by ELISA. (A) n = 9, 3 independent experiments in triplicate; (B,D) n = 4 independent experiments; (C) n = 3 independent experiments. + p < 0.05, ++ p < 0.001, +++ p < 0.0001 vs. control; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. DNCB or H₂O₂.

Figure 3

GMP protects keratinocyte against oxidative damage. HaCaT cells were treated with GMP and stimulated with DNCB 50 µM to measure: (A) Nitrite (NO₂⁻) production and (B) Lipid hydroperoxide (LOOH) levels; (C) HaCaT cells were treated with GMP and stimulated with DNCB 15 µM to analyze HMOX1 gene expression. (A,B) n = 9, 3 independent experiments in triplicate; (C) n = 4 independent experiments. + p < 0.0001 vs. control; * p < 0.01, ** p < 0.0001 vs. DNCB.

Figure 4

GMP regulates keratinocyte gene expression associated with triggering inflammation in atopic dermatitis (AD). (A) In vitro development of an AD model of keratinocytes. HaCaT cells were stimulated with tumor necrosis factor (TNF)-α (10 ng/mL), interferon (IFN)-γ (10 ng/mL) or interleukin (IL)-4 (50 ng/mL) for 24 h. (B–E) HaCaT cells were incubated with GMP and stimulated with TNF-α/IFN-γ (10 ng/mL). (B) TSLP, (C) IL33, (D) TARC and (E) MDC gene expression was analyzed by qPCR. n = 3 independent experiments. + p < 0.05, ++ p < 0.001, +++ p < 0.0001 vs. control; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. TNF-α/IFN-γ.

Figure 5

GMP regulates induced NGF and cGRP expression in keratinocytes. HaCaT cells were incubated with GMP and stimulated with: (A,C) DNCB 15 µM or (B,D) TNF-α/IFN-γ 10 ng/mL. Gene expression of (A,B) NGF and (C,D) cGRP was analyzed by qPCR. (A,C) n = 4 independent experiments; (B,D) n = 3 independent experiments. + p < 0.01, ++ p < 0.0001 vs. control; * p < 0.05, ** p < 0.0001 vs. DNCB or TNF-α/IFN-γ.

Figure 6

Effect of GMP on proliferation and migration of keratinocytes in an in vitro AD model. (A) HaCaT cells were incubated with epidermal growth factor (EGF, positive control) or GMP and stimulated with TNF-α/IFN-γ (10 ng/mL) and the proliferation index was determined. (B–D) HaCaT cells were cultured until confluent, incubated with mitomycin C, scratched with a pipette tip, re-coated with fibronectin, and incubated with EGF or GMP plus TNF-α/IFN-γ. (B) Representative images at 72 h are shown. (C) Distance between wound edges was measured and the wound closure percentage at each indicated time was calculated. (D) The gene expression of TGFB1 was analyzed by qPCR at 48 h. (A) n = 6, two independent experiments in duplicate; (C) n = 12, 4 randomly selected areas per condition in 3 independent experiments; (D) n = 3 independent experiments. + p < 0.05, ++ p < 0.01, +++ p < 0.0001 vs. control.

Figure 7

Schematic representation of GMP effects on viability, inflammatory and oxidative response, and the re-epithelization capacity of keratinocytes in an in vitro AD model. Created with BioRender.com (accessed on 3 May 2023).