Stem-Cell-Regenerative and Protective Effects of Squid (Symplectoteuthis oualaniensis) Skin Collagen Peptides against H2O2-Induced Fibroblast Injury

Abstract

Recently, there has been a growing interest in collagen peptides derived from marine sources for their notable ability to protect skin cells against apoptosis induced by oxidants. Therefore, the current study aimed to investigate the fundamental properties of collagen peptides, including their physicochemical, thermal, structural, stem-cell-regenerative, and skin-cell-protective effects, in comparison to commercial collagen peptides. The acid-soluble (ASC) and pepsin-soluble (PSC) collagens exhibited three distinct bands on SDS-PAGE, namely α (α₁ and α₂), β, and γ chains, confirming a type I pattern. The thermal profiles obtained from TG and DSC analyses confirmed the denaturation of PSC and ASC at temperatures ranging from 51.94 to 56.4 °C and from 52.07 to 56.53 °C, respectively. The purified collagen peptides were analyzed using SDS-PAGE and MALDI-TOF mass spectrometry, revealing a mass range of 900-15,000 Da. Furthermore, the de novo peptide sequence analysis confirmed the presence of the Gly-X-Y repeating sequence in collagen peptides. Collagen peptide treatments significantly enhanced HFF-1 cell proliferation and migration compared to the control group. ELISA results confirmed the potential interactions between collagen peptides and HFF-1 cells through α₂β₁, α₁₀β₁, and α₁₁β₁ integrin receptors. Notably, collagen peptide treatment effectively restored the proliferation of HFF-1 cells damaged by H₂O₂. Consequently, the advantageous characteristics of squid skin collagen peptides highlight their promising role in regenerative medicine.

Keywords: H2O2 oxidative damage; regenerative medicine; skin cells; squid collagen peptides.

Figure 1

SDS-PAGE patterns (A) of PSC and ASC from squid skin on 7.5% gel. Lane 1, molecular weight standard marker; lane 2, PSC; lane 3, U-PSC; lane 4, ASC; lane 5, U-ASC. The elution curve of Sephadex G-50 (B) and SDS-PAGE protein pattern (C) of SCP on 12.5% gel. Lane 1, molecular weight standard marker; lane 2–5, four components collected after elution. MALDI-TOF mass spectrometry (D) of SCP; obtained mass range: 900–5000 amu.

Figure 2

Scanning electron microscopic (SEM) structure and atomic force microscopic (AFM) structure of PSC (A) and ASC (B) isolated from squid skin. Letters (i,ii) represent SEM images, and (iii) represents AFM images. SEM image with different magnifications: (i) 50 μm, (ii) 20 μm. AFM image with magnifications of 200 nm (iii).

Figure 3

TG (A) and DSC (B) thermal denaturation curves of PSC and ASC from squid skin.

Figure 4

UV-vis spectra (A), FTIR spectra (B), and CD spectra (C) of PSC, ASC, and SCP from squid skin. The secondary structure of PSC (D) and ASC (E) was determined by Gaussian deconvolution analysis of the amide I region. (F) Percentage of PSC and ASC secondary structure resolved by CDNN software version 2.1.

Figure 5

Cell proliferation rate of HFF-1 cells cultured with different concentrations of collagen and collagen peptide (A) for 24 h. Scanning electron microscopy (B) of HFF-1 cells treated by collagen and collagen peptide for 24 h. Effect of PMSM on migration ability of HFF-1 (scale bar: 10 μm). (C) Cell mobility and migration maps of wounds treated with collagen and collagen peptides in scratch tests, respectively. Images were taken at 0, 12, and 24 h. Scale: 200 μm. Data represent mean ± SD, n = 3. *, **, and *** represent p < 0.05, p < 0.01, and p < 0.001 compared with the control group. # and ### indicate p < 0.05 and p < 0.001; the difference between the two groups is statistically significant. Note: PSC: squid-derived pepsin-soluble collagen; MPSC: market-derived bovine collagen; SCP: squid-derived collagen peptides; MSCP: market-derived ichthyosis collagen tripeptide, the same below.

Figure 6

Relative protein expression of HFF-1 cells cultured with collagen and collagen peptide for 24 h. (A) Quantification of integrin receptors α2β1, α10β1, and α11β1 by ELISA. (B) Fluorescence microscopy images, scale: 50 μm, and quantification of COL-I by ELISA. Data are expressed as mean ± SD, n = 3. *, **, and *** mean p < 0.05, p < 0.01, and p < 0.001 compared with the control group. ## and ### indicate that p < 0.001 and p < 0.001 are statistically significant between the two groups.

Figure 7

Effects of different concentrations of collagen and collagen peptide on the cell viability (A) of HFF-1 cells damaged by H₂O₂ for 24 h. Expression of inflammatory factors IL-2 (B) and IL-6 (C) in HFF-1 cells damaged by H₂O₂ cultured with different concentrations of collagen and collagen peptide. *, **, ***, and **** indicate p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively, compared with the injury control group. # and ## mean p < 0.05 and p < 0.01 compared with the blank control group, respectively. #, ##, ###, and #### indicate that p < 0.05, p < 0.01, p < 0.001, and p < 0.0001 are statistically significant between the two groups. Values are mean ± SD, n = 3.3.

Figure 8

Schematic diagram of squid skin collagen extraction steps.

Figure 9

Schematic diagram of squid skin collagen peptide extraction steps.