Effects of Collagen Hydrolysate From Large Hybrid Sturgeon on Mitigating Ultraviolet B-Induced Photodamage

Abstract

Ultraviolet B (UVB) radiation leads to the excessive accumulation of reactive oxygen species (ROS), which subsequently promote inflammation, degradation of the extracellular matrix, and photoaging in skin. Thus antioxidant activity is particularly important when screening for active substances to prevent or repair photodamage. Marine fish-derived bioactive peptides have become a trend in cosmetics and functional food industries owing to their potential dermatological benefits. In this study, 1-diphenyl- 2-pycryl-hydrazyl (DPPH) scavenging activity was selected to optimize the hydrolysis conditions of sturgeon skin collagen peptides with antioxidant activity. The optimal hydrolysis conditions for sturgeon skin collagen hydrolysate (SSCH) were determined by response surface methodology, which comprised an enzyme dosage of flavorzyme at 6,068.4 U/g, temperature of 35.5°C, pH of 7, and hydrolysis time of 6 h. SSCH showed good radical-scavenging capacities with a DPPH scavenging efficiency of 95%. Then, the effect of low-molecular-weight SSCH fraction (SSCH-L) on UVB irradiation-induced photodamage was evaluated in mouse fibroblast L929 cells and zebrafish. SSCH-L reduced intracellular ROS levels and the malondialdehyde content, thereby alleviating the oxidative damage caused by UVB radiation. Moreover SSCH-L inhibited the mRNA expression of genes encoding the pro-inflammatory cytokines IL-1β, IL-6, TNF-α, and Cox-2. SSCH-L treatment further increased the collagen Ⅰα1 content and had a significant inhibitory effect on matrix metalloproteinase expression. The phosphorylation level of JNK and the expression of c-Jun protein were significantly reduced by SSCH-L. Additionally, SSCH-L increased the tail fin area at 0.125 and 0.25 mg/ml in a zebrafish UVB radiation model, which highlighted the potential of SSCH-L to repair UVB-irradiated zebrafish skin damage. Peptide sequences of SSCH-L were identified by liquid chromatography-tandem mass spectrometry. Based on the 3D-QSAR modeling prediction, six total peptides were selected to test the UVB-protective activity. Among these peptides, DPFRHY showed good UVB-repair activity, ROS-scavenging activity, DNA damage-protective activity and apoptosis inhibition activity. These results suggested that DPFRHY has potential applications as a natural anti-photodamage material in cosmetic and functional food industries.

Keywords: UVB; anti-apoptosis activity; anti-photoaging; antioxidant; collagen hydrolysate; large hybrid sturgeon.

FIGURE 1

DPPH scavenging ability of seven proteases after hydrolysis.

FIGURE 2

DPPH clearance rate and degree of hydrolysis (DH) vary with hydrolysis temperature (A), pH (B), enzyme dosage (C), and time (D).

FIGURE 3

Interactive effect of extraction variables on DPPH scavenging activity.

FIGURE 4

The effects of SSCH-L at different concentrations (A) on the viability and (B) the migration of L929 cells. (C) The wound closure rate was calculated at 0, 24, and 48 h after scratching. (D) The protective effects of SSCH-L on L929 cells damaged by UVB radiation. The error bars refer to standard deviations obtained from the triplicate sample analysis.

FIGURE 5

Effect of SSCH-L on intracellular reactive oxygen species (ROS) generation (A) and contents of malondialdehyde (MDA) (B) after L929 exposured to UVB.

FIGURE 6

Effect of SSCH-L on the expression of pro-inflammation cytokines and Cox-2 in the L929 cells exposed to 40 mJ/cm² UVB.

FIGURE 7

The protective effect of SSCH-L on procollagen degradation in UVB exposed L929. The protein levels of procollagen I alpha (A) and MMP-1 (B) in L929 cells and the levels of MMP-2 (C) and MMP-3 (D) in cell culture supernatant were detected by ELISA.

FIGURE 8

Effects of SSCH-L on MAPK and AP-1 signaling pathway in UVB-irradiated l929 cells. The intensities for phosphorylation levels of ERK, JNK, and p38 were measured by Western blotting. The results are shown as the mean ± SD of three independent experiments.

FIGURE 9

The potential of SSCH-L to repair skin damage in zebrafish exposed to UVB. (A). UVB-induced malformed fin phenotypes can be attenuated by SSCH-L. (B). Quantification of fin phenotypes. (C). Schematic representation of the zebrafish experimental protocols performed in this study.

FIGURE 10

Screening and photo-protective effect of DPFRHY. (A). Schematic representation of the virtual screening of antioxidant peptide from SSCH-L. (B). The protective effects of 6 SSCH-L-screened peptides on L929 cells damaged by UVB radiation by cell viability assay. (C). ROS scavenging effect of DPFRHY in UVB exposed L929 cell. (D). Comet assay of L929 cells after UVB exposure and DPFRHY treatment. (E). Quantification of comet scores of D by Cometscore 2.0 software.

FIGURE 11

Evaluation of apoptosis protection in UVB-irradiated L929 fibroblasts treated with DPFRHY. (A) DNA condensation formation was observed under a fluorescence microscope following Hoechst 33342 staining. (B) The units statistics of DNA condensation in each field of microscope.