Skin acts as the outer barrier that is challenged by a series of external stress factors, resulting in frequent cell and barrier damage. After injury, wound healing is essential to restore the integrity of the skin barrier. Although the restoration of a functional epidermal barrier is highly efficient in normal physiological conditions, the normal repair response will go awry when the injured skin does not repair in a timely manner, and in turn results in delayed healing, chronic wounds or abnormal scar formation. A neglected fact is that traumatic injury has been one of the leading causes of mortality in many countries. In addition to trauma, there are millions of surgical wounds created in the course of routine medical care every year. In fact, the number of patients who are suffering from impaired healing conditions and chronic wounds is reaching epidemic proportions and will become even more burdensome in both human health and economic terms. Although a central concern of clinical care has focused on facilitating the healing process in clinical injuries, minimizing the aesthetic impact on the patient and maximal restoration of tissue function, we still lack efficient therapies for treating non-healing wounds, speeding up the repair of non-healing wounds, and speeding up the repair of acute wounds. Hence, there is a strong medical and social need to improve therapeutic approaches for enhancing the endogenous tissue regenerative capacity.
Cutaneous wound healing is a highly orchestrated biological process, requiring the collaborative efforts of many different cell types and cellular processes to achieve restoration of tissue integrity. Normal cutaneous wound repair is characterized by distinct, yet overlapping phases of wound healing, termed hemostasis, inflammation, proliferation, and remodeling. Molecular and cellular mechanisms investigation indicates that the spatiotemporal process of wound healing can be arbitrarily divided into three stages, including early stage, intermediate stage and late stage. Early stage includes hemostasis, activation of keratinocytes and recruitment of inflammatory cells. Intermediate stage involves proliferation and migration of keratinocytes, proliferation of fibroblasts, matrix deposition and angiogenesis. The late stage contains remodeling of extracellular matrix, scar formation and restoration of barrier. The spatiotemporal process of wound healing is tightly controlled by multiple cell types that secrete numerous signaling molecules, such as cytokines, chemokines, and growth factors, to achieve wound closure and functional restoration of the skin barrier. All of these phases and signaling molecules are potential therapeutic targets for modulating wound healing progression. The past decade has witnessed some new developments of various biological active therapeutic attempts, including epidermal growth factor (EGF), fibroblast growth factor 2 (FGF-2), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), keratinocyte growth factor-1 (KGF-1), granulocyte macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF) and so on. However, therapies based on growth factors have not yet been proven to be broadly effective in clinical application. Notably, these growth factors are large sizes that correspond to higher production cost which limit their widespread use in clinical. Recently, immunomodulatory peptides (also called wound healing-promoting peptides) with small size and potent wound-healing-promoting activities are becoming attractive candidates for treatment of wounds.
Amphibian skins are effective natural barriers between the organism and the environment. They play key roles in defense, respiration, and water regulation. As an outer covering of the body, amphibian skins are susceptible to biotic or abiotic injuries, such as predation, parasitization, microorganism infection, and physical harm, including aseptic wounds and radiation. However, amphibians have a strong capacity to restore their skin injuries with no post-operative care. They have evolved an effective wound healing system including a variety of wound healing peptides. Although several amphibian-derived wound healing-promoting peptides have been most recently investigated, the mechanism of action of these wound healing peptides, such as their effects on neutrophils, their effects on the cross-talk between effector cells, and their direct chemotactic effects on neutrophils and macrophages, remains to be further elucidated.
The Chinese concave-eared frog Odorrana tormota (formerly Amolops tormotus) is an arboreal, nocturnal frog that lives near noisy streams in Huangshan Hot Springs, China. Previous research primarily focused on the ultrasonic communication between male and female O. tormota during reproduction, but no O. tormota-derived bioactive peptides were investigated up until now. To find more amphibian-derived wound healing peptides and further understand their mechanism of action, the peptidomics of the skin secretions of O. tormota and their effects on wound healing were assayed in vivo and in vitro, and a O. tormota-derived wound healing peptide, designated as Ot-WHP, was identified from the skin of concave-eared frog. Ot-WHP showed potential wound-healing-promoting activity in a mouse model of dermal full-thickness wound. The effector cell types of Ot-WHP, the direct or indirect effects of Ot-WHP on effector cells and the effects of Ot-WHP on the crosstalk between different effector cells were investigated. Our results suggest that Ot-WHP acts as an efficient wound healing immunomodulator.
Adult O. tormota (both sexes, n = 30) were collected from Huangshan Hot Springs in Tangkou town (30°30′ N, 118°13′ E), China. Frogs were housed in a plastic box (56.3 cm × 42.5 cm × 32.3 cm), supplemented with a little water, and fed with mealworm larvae Tenebrio molitor. BALB/c mice (female, 18–20 g) were purchased from Shanghai Slac Animal Co. Inc. and housed in a pathogen-free facility. Animal experiments were performed in accordance with the Guide for the Care and Use of Medical Laboratory Animals (Ministry of Health, People's Republic of China, 1998), and were approved by the Animal Care and Use Committee as well as the Ethical Committee of Soochow University (SYXK2017-0043).
Bone marrow-derived macrophages (BMDMs) were prepared according to our previous method. BMDMs isolated from the tibia and femur of BABL/c mice were cultured in RPMI 1640 medium with 2 mM glutamine, 10 ng/ml M-CSF (PeproTech, NJ, USA) for 5 days. Differentiated BMDMs were harvested and re-plated for the experiment. Bone marrow-derived neutrophils were prepared as described previously. Briefly, bone marrow from BABL/c mice was harvested, rinsed with 5 ml PBS, filtered through a cell strainer (70 micron), and centrifuged at 500 × g for 5 min. A PBS diluted Percoll gradient with 72, 64, and 54% layers was created, and the bone marrow-derived neutrophils pellet was re-suspended in PBS and over-layered onto this gradient. The Percoll gradient was centrifuged for 25 min at 950 × g. Neutrophils were collected from the 72%/64% interface, and washed with ACK lysing buffer for 5 min, followed by suspension in PBS and centrifuged at 500 × g for 5 min. Neutrophils were re-suspended in 5 ml RPMI 1640 supplemented 10% FBS for cell counts and experiment. THP-1 cells were cultured in RPMI 1640 containing 5 nM phorbol myristate acetate (PMA, Sigma, USA) for 24 h, and cells were washed three times with PBS after differentiation into macrophage-like cells. Keratinocytes (HaCaT) were cultured in DMEM (Hyclone, UT, USA). Fibroblasts from newborn BABL/c mouse skin were isolated and preserved according to our previous paper, and were cultured in DMEM (Hyclone, UT, USA). All cells were supplemented with 10% FBS (Hyclone, UT, USA) and 100 U-100 μg/ml penicillin-streptomycin (GIBCO, USA), and were cultured in a humidified incubator under 5% CO 2 at 37°C.
Synthetic peptides, including Ot-WHP, scrambled Ot-WHP and AH90, were purchased from Synpeptide Co. Ltd. (Shanghai, China), and analyzed by RP-HPLC and MALDI-TOF MS to ensure that the purity was higher than 98%.
Frog skin secretions were collected as previously described. Briefly, frogs were stimulated with anhydrous ether, and skin secretions were collected, centrifuged, and lyophilized. Lyophilized skin secretion sample was dissolved in 10 ml PBS (0.1 M, pH 6.0, total absorption of 10 ml skin secretion solution at OD280 is 520), and was centrifuged at 5,000 × g for 10 min. The supernatant was applied to a Sephadex G-50 (Superfine, Amersham Biosciences, 2.6 cm × 100 cm) gel filtration column, and eluted with PBS (0.1 M, pH 6.0) at a flow rate of 3.0 ml/10 min. Absorbance of the eluted fractions was monitored at 280 nm. Fractions with wound healing-promoting effect on full-thickness wounds in mice were pooled, and applied to a C18 reversed-phase high-performance liquid chromatography column (RP-HPLC, 5 μm particle size, 110 Å pore size, 250 mm × 4.6 mm, Gemini, CA, USA) twice, using a linear gradient of 0–60% acetonitrile supplemented with 0.1% (v/v) trifluoroacetic acid/water over 80 min. The eluted peptide (0.5 μl) was spotted onto a matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) plate with 0.5 μl α-cyano-4-hydroxycinnamic acid matrix (10 mg/ml in 60% acetonitrile) to confirm its purity. The purified peptide candidate was subjected to a pulsed liquid-phase Shimadzu protein sequencer (PPSQ-31A; Shimadzu, Kyoto, Japan), according to the manufacturer's instruction.
Total RNA extraction from the frog skin was performed using RNeasy Protect Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instruction. A SMART TM PCR cDNA synthesis kit purchased from Clontech (Palo Alto, CA) was then used to construct the skin cDNA library, producing a library containing approximately 3.1 × 10 5 independent colonies. A PCR-based method was used to clone and isolate the nucleotide sequence encoding Ot-WHP from the cDNA library. A sense primer (5' PCR primer, 5′-AAGCAGTGGTATCAACGCAGAGT-3′, provided by the cDNA library construction kit), and an antisense primer (S1(5′- CC(A/G)TG(A/C/G/T)GG(A/C/G/T)CC(A/C/G/T)A(A/G) (A/G)TCCCA-3′, designed from the amino acid sequence of Ot-WHP determined by Edman degradation) were used in the first step PCR reaction. The full length nucleotide sequence was cloned using another sense primer (S2, 5′-ATGTTCACCTTGAAGAAATTC-3′, designed from the nucleotide sequence obtained by the first step PCR reaction), and another antisense primer (3′ PCR primer, 5′-ATTCTAGAGGCCGAGGCGGCCGACATG-3′, provided by the cDNA library construction kit). PCR conditions were 2 min at 95°C,
