OA-GL21, a novel bioactive peptide from Odorrana andersonii, accelerated the healing of skin wounds

Abstract

Nowadays, the number of chronic trauma cases caused by a variety of factors such as the world's population-ageing and chronic diseases is increasing steadily, and thus effective treatment for chronic wounds has become a severe clinical challenge, which also burdens the patient both physically and financially. Therefore, it is urgent to develop new drugs to accelerate the healing of wounds. Bioactive peptides, which are relatively low cost, easy to produce, store and transport, have become an excellent choice. In this research, we identified a novel peptide OA-GL21, with an amino acid sequence of 'GLLSGHYGRVVSTQSGHYGRG', from the skin secretions of Odorrana andersonii Our results showed that OA-GL21 exerted the ability to promote wound healing of human keratinocytes (HaCaT) and human fibroblasts in a dose- and time-denpendent manner. However, OA-GL21 had no significant effect on the proliferation of these two cells. Significantly, OA-GL21 showed obvious ability to promote wound healing in the full-thickness skin wound model in dose- and scar-free manners. Further studies showed that OA-GL21 had no direct antibacterial, hemolytic, and acute toxic activity; it had weak antioxidant activities but high stability. In conclusion, this research proved the promoting effects of OA-GL21 on cellular and animal wounds, and thus provided a new peptide template for the development of wound-repairing drugs.

Keywords: Odorrana andersonii; skin secretions; wound healing promoting peptide.

Figure 1. Peptide purification procedures from skin secretions of O. andersonii

Skin secretions of O. andersonii were separated by a Sephadex G-75 column (shown as Figure 1A in our recent report [18]) and samples exhibiting wound healing activity were purified by a round of RP-HPLC. The sample with wound-healing-promoting activity was indicated by an arrow (A) and then further purified by another round of RP-HPLC by identical procedure with the first round of RP-HPLC. Finally, a peptide with wound-healing-promoting activity was obtained (indicated by an arrow in (B)) and is awaiting further research.

Figure 2. Primary structure of OA-GL21

Sequence of OA-GL21. The complete sequence of mature OA-GL21 is ‘GLLSGHYGRVVSTQSGHYGRG’, which has 21-amino acid residues in length (shown in red) and produced by post-translational modification of a 67-residue prepropeptide. (A) Observed molecular mass of native OA-GL21 and purity of peak, indicated by an arrow in Figure 1B. (B) The sequence alignment of OA-GL21 with other bioactive peptides from amphibian skins. (C) The sequence alignment of OA-GL21 with other bioactive peptides from amphibian skins.

Figure 3. The hemolytic, antioxidant activities and stability of OA-GL21

(A) At the same concentrations of cellular wound healing activity assay, OA-GL21 showed no hemolytic activity against human blood cells. (B) OA-GL21 showed scavenging activity against free radical ABTS⁺ in a dose-dependent manner when at the concentrations ranging from 10 to 500 µM. (C) OA-GL21 showed no obvious scavenging activity against free radical DPPH. (D) OA-GL21 was highly stable at 4°C and began to decrease after 4 days but also maintained above 60% within 10 days at a higher temperature (37°C). Data are means ± S.E.M. of six independent experiments. **P<0.01 and ***P<0.0001 indicate significant difference from the control or H₂O (Student’s ttests).

Figure 4. Effect of OA-GL21 on the healing rate of HaCaT cell scratch

(A) OA-GL21 (50 µM) accelerated HaCaT cell scratch healing significantly. (B) OA-GL21 (50 µM) accelerated HaCaT cell scratch healing while proliferation was inhibited by mitomycin-C. (C) OA-GL21 showed time- and dose-dependent HaCaT cell wound-healing-promoting activity. (D) OA-GL21 showed time- and dose-dependent HaCaT cell wound-healing-promoting activity while proliferation was inhibited by mitomycin-C. Data are means ± S.E.M. of six independent experiments. *P<0.05, **P<0.01, and ***P<0.0001 indicate significant difference from the negative control (Student’s ttests).

Figure 5. Effect of OA-GL21 on healing rate of HSF cell scratch

(A) OA-GL21 (50 µM) accelerated HSF cell scratch healing significantly. (B) OA-GL21 (50 µM) accelerated HSF cell scratch healing while proliferation was inhibited by mitomycin-C. (C) OA-GL21 showed obviously time- and dose-dependent HSF cell wound-healing-promoting activity from concentration 0.5 to 50 µM. (D) OA-GL21 showed time- and dose-dependent HSF cell wound-healing-promoting activity while proliferation was inhibited by mitomycin-C. Data are means ± S.E.M. of six independent experiments. **P<0.01 and ***P<0.0001 indicate significant difference from the negative control (Student’s ttests).

Figure 6. Effects of OA-GL21 on the proliferation of HaCaT and HSF cells

(A) OA-GL21 showed no effect on the proliferation of HaCaT cells. (B) OA-GL21 showed no effect on the proliferation of HSF cells (P>0.05, Student’s ttests; ‘Control’ meant Negative control).

Figure 7. Effects of OA-GL21 on mice full-thickness skin wounds and histological analysis

(A) Macroscopic view representative mice wounds were taken on post-operative days 1, 5, and 9. (B) Wound closure was assessed by morphometrical analysis of wound areas. Wound residual areas were determined (n=5). OA-GL21 (100 µg/ml) accelerated mice wound healing obviously. (C) Histological analysis of mice normal skin, saline-, KFX- (10 mg/ml), and OA-GL21- (100 µg/ml) treated full-thickness wound tissues stained with H&E. (E, epidermal; ES, escar; NE, neoepidermal; GT, granulation tissue; D, dermis; HF, hair follicle; M, muscle layer; LCT, loose connective tissue. Bars indicate 50 µm, arrow, wound edge.) (D) Thickness of neoepidermis and granulation tissues was measured and quantitated, and OA-GL21 induced thinner neoepidermis and granulation tissue than both saline and KFX group. **P<0.01 and ***P<0.0001 indicate significant difference from the negative control (Student’s ttests).