A Novel Peptide from Polypedates megacephalus Promotes Wound Healing in Mice

Amphibian skin contains wound-healing peptides, antimicrobial peptides, and insulin-releasing peptides, which give their skin a strong regeneration ability to adapt to a complex and harsh living environment. In the current research, a novel wound-healing promoting peptide, PM-7, was identified from the skin secretions of Polypedates megacephalus, which has an amino acid sequence of FLNWRRILFLKVVR and shares no structural similarity with any peptides described before. It displays the activity of promoting wound healing in mice. Moreover, PM-7 exhibits the function of enhancing proliferation and migration in HUVEC and HSF cells by affecting the MAPK signaling pathway. Considering its favorable traits as a novel peptide that significantly promotes wound healing, PM-7 can be a potential candidate in the development of novel wound-repairing drugs.     

Discovery of two bombinin peptides with antimicrobial and anticancer activities from the skin secretion of Oriental fire‐bellied toad,Bombina orientalis

Amphibian skin secretions are known to contain numerous peptides with a large array of biological activities. Bombinins are a group of amphibian‐derived peptides with broad spectrum antimicrobial activities that have been only identified from the ancient toad species, Bombina. In this study, we described the identification and characterization of a novel bombinin precursor which encoded a bombinin‐like peptide (BLP‐7) and a novel bombinin H‐type peptide (named as Bombinin H‐BO) from the skin secretion of Oriental fire‐bellied toad, Bombina orientalis. The primary structures of both mature peptides were determined by combinations of molecular cloning of peptide precursor‐encoding cDNAs and mass spectrometry techniques. Secondary structure prediction revealed that both peptides had cationic amphipathic α‐helical structural features. The synthetic replicate of BLP‐7 displayed more potent antimicrobial activity than Bombinin H‐BO against Gram‐positive and Gram‐negative bacteria and yeast. Also, in vitro antitumour assay showed that both peptides possessed obvious antiproliferative activity on three human hepatoma cells (Hep G2/SK‐HEP‐1/Huh7) at the non‐toxic doses. These results indicate the peptide family of bombinins could be a potential source of drug candidates for anti‐infection and anticancer therapy.     

Thrombin peptide, TP508, stimulates angiogenic responses in animal models of dermal wound healing, in chick chorioallantoic membranes, and in cultured human aortic and microvascular endothelial cells

The alpha-thrombin peptide, TP508, accelerates the healing of full-thickness wounds in both normal and ischemic skin. In wounds treated with TP508, a pattern of increased vascularization is consistently observed both grossly and microscopically when compared to wounds treated with saline. One possible mechanism by which the peptide accelerates wound healing is by promoting revascularization of granulation tissue at the injured site. To evaluate the angiogenic potential of TP508, the peptide was tested in the chick embryo chorioallantoic membrane (CAM), where it increased the density and size of CAM blood vessels relative to controls. Additionally, TP508 stimulated chemokinesis and chemotaxis in a dose-dependent fashion in cultured human aortic and human microvascular endothelial cells. Taken together, these in vivo and in vitro data support an angiogenic role for TP508 in wound healing. A working model is presented to explain how this 23-amino-acid peptide, which lacks proteolytic activity, is generated during wound healing and contributes to the nonproteolytic functions associated with alpha-thrombin during tissue repair.     

Preliminary investigations into developing all‐D Omiganan for treating Mupirocin‐resistant MRSA skin infections

Staphylococcus aureus is the primary pathogen responsible for the majority of human skin infections, and meticillin‐resistant S. aureus (MRSA) currently presents a major clinical concern. The overuse of Mupirocin, the first‐line topical antibacterial drug over 30 years, has led to the emergence of Mupirocin‐resistant MRSA, creating a clinical concern. The antimicrobial peptide Omiganan was touted to be a promising antibacterial drug candidate due to its rapid membrane‐disrupting bactericidal mode of action, entering clinical trials in 2005 as a topical gel to prevent catheter site infections. However, drug development ceased in 2009 due to a lack of efficacy. We postulate this to be due to proteolytic degradation caused by endogenous human skin proteases. Herein, we tested our hypothesis using Omiganan and its all‐D enantiomer in a human skin protease stability assay, followed by anti‐MRSA activity assay against of a panel of clinical MRSA isolates, a bactericidal/static determination and a time‐kill assay to gauge all‐D Omiganan's potential for further topical antibacterial drug development.     

Wound healing acceleration of a novel transforming growth factor-beta inducer, SEK-1005

The studies were carried out to elucidate the effect of a novel cyclic peptide, SEK-1005 (C(45)H(70)N(8)O(13)), on wound healing. SEK-1005 (4-10 microg/wound) applied topically significantly accelerated the healing of a full-thickness wound on the dorsal skin of a rat. In a healing-impaired mouse, the peptide (2-10 microg/wound) had more potent activity, exerting an effect comparable to that of basic fibroblast growth factor (FGF). However, SEK-1005 (0.1-100 ng/ml) scarcely promoted the proliferation of cultured fibroblasts (NIH3T3 cells) while basic FGF (0.2-5 ng/ml) showed marked mitogenic activity. SEK-1005 (2-10 microg/wound) significantly increased the topical production of transforming growth factor (TGF)-beta1, a cytokine that is known to accelerate wound healing. This activity was closely correlated with the wound-repairing effect. From the above, SEK-1005 can be considered as a new type of wound healing agent with potent TGF-beta1-inducing activity.     

Isolation,functional characterization,and biological properties of MCh‐AMP1, a novel antifungal peptide from Matricaria chamomilla L.

The antimicrobial activities of natural products have attracted much attention due to the increasing incidence of pathogens that have become resistant to drugs. Thus, it has been attempted to promisingly manage infectious diseases via a new group of therapeutic agents called antimicrobial peptides. In this study, a novel antifungal peptide, MCh‐AMP1, was purified by reverse phase HPLC and sequenced by de novo sequencing and Edman degradation. The antifungal activity, safety, thermal, and pH stability of MCh‐AMP1 were determined. This peptide demonstrated an antifungal activity against the tested Candida and Aspergillus species with MIC values in the range of 3.33–6.66 μM and 6.66–13.32 μM, respectively. Further, physicochemical properties and molecular modeling of MCh‐AMP1 were evaluated. MCh‐AMP1 demonstrated 3.65% hemolytic activity at the concentration of 13.32 μM on human red blood cells and 10% toxicity after 48 hr at the same concentration on HEK293 cell lines. The antifungal activity of MCh‐AMP1 against Candida albicans was stable at a temperature range of 30–50°C and at the pH level of 7–11. The present study indicates that MCh‐AMP1 may be considered as a new antifungal agent with therapeutic potential against major human pathogenic fungi.     

Epigallocatechin‐3‐gallate augments therapeutic effects of mesenchymal stem cells in skin wound healing

In non‐healing wounds, mesenchymal stem cell (MSC)‐based therapies have the potential to activate a series of coordinated cellular processes, including angiogenesis, inflammation, cell migration, proliferation and epidermal terminal differentiation. As pro‐inflammatory reactions play indispensable roles in initiating wound repair, sustained and prolonged inflammation exhibit detrimental effects on skin wound closure. We investigated the feasibility of using an antioxidant agent epigallocatechin‐3‐gallate (EGCG), along with MSCs, to improve wound repair through their immunomodulatory actions. In a rat model of wound healing, a single dose of EGCG at 10 mg/kg increased the efficiency of MSC‐induced skin wound closure. Twenty days after the wound induction, MSC treatment significantly enhanced the epidermal thickness, which was further increased by EGCG administration. Consistently, the highest extent of growth factors upregulation for neovascularization induction was seen in the animals treated by both MSCs and EGCG, associated with a potent anti‐scarring effect throughout the healing process. Finally, expression levels of pro‐inflammatory cytokines, such as tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β) and IL‐6, in the wound area were reduced by MSCs, and this reduction was further potentiated by EGCG co‐administration. EGCG, together with MSCs, can promote skin wound healing likely through their combinational effects in modulating chronic inflammation.     

In vitro evaluation of Spirulina platensis extract incorporated skin cream with its wound healing and antioxidant activities

Context: Algae have gained importance in cosmeceutical product development due to their beneficial effects on skin health and therapeutical value with bioactive compounds. Spirulina platensis Parachas (Phormidiaceae) is renowned as a potential source of high-value chemicals and recently used in skincare products.

Objective: This study develops and evaluates skin creams incorporated with bioactive S. platensis extract.

Materials and methods: Spirulina platensis was cultivated, the aqueous crude extract was prepared and in vitro cytotoxicity of S. platensis extract in the range of 0.001–1% concentrations for 1, 3 and 7?d on HS2 keratinocyte cells was determined. Crude extracts were incorporated in skin cream formulation at 0.01% (w/w) concentration and in vitro wound healing and genotoxicity studies were performed. Immunohistochemical staining was performed to determine the collagen activity.

Results: 0.1% S. platensis extract exhibited higher proliferation activity compared with the control group with 198% of cell viability after 3?d. Skin cream including 1.125% S. platensis crude extract showed enhanced wound healing effect on HS2 keratinocyte cell line and the highest HS2 cell viability % was obtained with this concentration. The micronucleus (MN) assay results indicated that S. platensis extract incorporated creams had no genotoxic effect on human peripheral blood cells. Immunohistochemical analysis showed that collagen 1 immunoreactivity was improved by increased extract concentration and it was strongly positive in cells treated with 1.125% extract incorporated skin cream.

Conclusions: The cell viability, wound healing activity and genotoxicity results showed that S. platensis incorporated skin cream could be of potential value in cosmeceutical and biomedical applications.     

Molecular Cloning of a Novel Tryptophyllin Peptide from the Skin of the Orange‐Legged Monkey Frog,Phyllomedusa hypochondrialis

Tryptophyllins are a group of small (4–14 amino acids), heterogenous peptides, mostly from the skins of hylid frogs from the genera, Phyllomedusa and Litoria. To date, more than forty TPHs have been discovered in species from these two genera. Here, we describe the identification of a novel tryptophyllin type 3 peptide, PhT‐3, from the extracts of skin of the orange‐legged monkey frog, Phyllomedusa hypochondrialis, and molecular cloning of its precursor‐encoding cDNA from a cDNA library constructed from the same skin sample. Full primary structural characterization was achieved using a combination of direct Edman degradation, mass spectrometry and deduction from cloned skin‐derived cDNA. The open‐reading frame of the precursor cDNA was found to consist of 63 amino acid residues. The mature peptide arising from this precursor contains a post‐translationally modified N‐terminal pyroglutamate (pGlu) residue, formed from acid‐mediated cyclization of an N‐terminal Gln (Q) residue, and with the structure: pGlu‐Asp‐Lys‐Pro‐Phe‐Trp‐Pro‐Pro‐Pro‐Ile‐Tyr‐Pro‐Met. Pharmacological assessment of a synthetic replicate of this peptide on phenylephrine preconstricted rat tail artery segments, revealed a reduction in relaxation induced by bradykinin. PhT‐3 was also found to mediate antiproliferative effects on human prostate cancer cell lines.     

Novel bioactive peptides demonstrating anti‐dengue virus activity isolated from the Asian medicinal plant Acacia Catechu

The therapeutic activities of food‐derived bioactive proteins and peptides are attracting increased attention within the research community. Medicinal plants used in traditional medicines are an excellent source of bioactive proteins and peptides, especially those traditionally prepared by water extraction for use as tea or food supplement. In this study, novel bioactive peptides were isolated from enzymatic digests of 33 Thai medicinal plants. The inhibitory activity of each against dengue virus (DENV) infection was investigated. Of 33 plants, peptides from Acacia catechu extract demonstrated the most pronounced anti‐DENV activity. Half maximal inhibitory concentration of 0.18 μg/ml effectively inhibited DENV foci formation. Treatment with 1.25 μg/ml crude peptide extract could reduce virus production less than 100‐fold with no observable cell toxicity. Peptide sequences were determined by high‐performance liquid chromatography and liquid chromatography–tandem mass spectrometry. Two bioactive peptides isolated from Acacia catechu inhibited DENV foci formation >90% at the concentration of 50 μM; therefore, they are recommended for further investigation as antiviral peptides against DENV infection.     

伤口治疗用载药微针的研究进展

在伤口治疗过程中,因皮肤屏障的限制,药物在角质层积聚使得治疗效率低。微针是治疗伤口的一种新型药物递送系统,可刺穿皮肤的表皮层,将药物送入真皮层,从而发挥促进伤口愈合的作用。该文综述了近几年国内外用于伤口治疗的微针系统,根据微针负载的药物对研究进行分类,并讨论了微针系统在促进伤口愈合方面的优势,总结了微针用于治疗伤口的未来前景和挑战。     

Antibacterial biomaterials for skin wound dressing

Bacterial infection and the ever-increasing bacterial resistance have imposed severe threat to human health. And bacterial contamination could significantly menace the wound healing process. Considering the sophisticated wound healing process, novel strategies for skin tissue engineering are focused on the integration of bioactive ingredients,antibacterial agents included, into biomaterials with different morphologies to improve cell behaviors and promote wound healing. However, a comprehensive ...     

Spidroin in carbopol-based gel promotes wound healing in earthworm's skin model

Spider silk's regenerative, biocompatible, and antimicrobial properties render it a promising biomaterial for wound healing promotion. Spidroin as the main protein component of spider silks was used in this study to evaluate the potential effects on wound healing via topical application of novel spidroin-containing carbopol 934 (CP934) gel. Spidroin was extracted, formulated into CP934 gel, and characterized both in vitro and in vivo. Spidroin gel was translucent and brownish-yellow in color. An optimum viscosity was obtained at 0.6% CP934 at neutral pH. Optimized spidroin gel (0.6% CP934) effectively inhibited the growth of clinical bacterial isolates of methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA) and Escherichia coli at 440 μg/mL with MIC values of 0.98, 4.6, and 8.2 μg/mL, respectively. Optimized spidroin gel was evaluated for wound healing via topical application on wounds surgically induced in Allolobophora caliginosa earthworms used as a robust human skin model. After application for three consecutive days, dramatic reductions in wound closure and reepithelialization duration were observed macroscopically and via histological studies (light and electron microscopy) when compared with control. In conclusion, these results show that spidroin gel is a promising promoter for wound healing, and further studies would be directed toward investigating mechanisms underlying this effect.     

自组装纤维支架肽对深Ⅱ度烧伤创面收缩作用的实验研究

目的：探讨纳米短肽对深Ⅱ度烧伤创面的收缩及创面表皮的再生作用。方法：以电力机械烧伤法在雌性SD大鼠背部制造皮肤深Ⅱ度烫伤模型。烧伤后分别以不同生物辅料处理创面,以起到治疗作用。全程记录处理伤口的大体形态学变化。分别在手术后7、10、14、21 d以创面描记及数字图像处理软件系统描述创面生长情况。结果：与对照组生物敷料比较,这种自组装纤维支架肽可以加速缩短焦痂的的形成3～5 d时间,并加速伤口的收缩达20%～30%的水平。结论：自组装纤维支架肽对深度烧伤创面收缩具有促进作用。     

Novel topical formulation for ischemic chronic wounds. Technological design,quality control and safety evaluation

Ulceration of the foot in diabetes is common and disabling, and frequently leads to amputation of the leg. The pathogenesis of foot ulceration is complex, clinical presentation variable and management requires early expert assessment. Despite treatment, ulcers readily become chronic wounds. Chronic wounds are those that remain in a chronic inflammatory state failing a normal healing process patterns. This is partially caused by inefficient eradication of opportunistic pathogens like Pseudomonas aeruginosa. We propose its control or eradication will promote wound healing. Lactobacillus plantarum cultures supernatants (LAPS) shows antipathogenic and pro-healing properties. The main objective was to design two pharmaceutical dosage forms by using LAPS as active pharmaceutical ingredient and to perform its quality control, in vitro activity conservation tests and human trials (safety evaluation). Both selected formulations reach the technological quality expected for 120 days, shows adequate occlusive characteristics and proper adhesion to human skin. From the in vitro release assays were found that LAPS shows adequate release from matrix and maintain its antimicrobial and anti-biofilm activity. First human trials were developed and neither edema nor erythema on healthy skin voluntaries was found. We conclude that C80 and C100 are adequate for their use in future clinical trials to demonstrate a comprehensive therapeutic effectiveness in ischemic chronic wounds.     

In Vitro Evaluation of Novel Phenytoin-Loaded Alkyd Nanoemulsions Designed for Application in Topical Wound Healing

Phenytoin-loaded alkyd nanoemulsions were prepared spontaneously using the phase inversion method from a mixture of novel biosourced alkyds and Tween 80 surfactant. Exposure of human adult keratinocytes (HaCaT cells) for 48 h to alkyd nanoemulsions producing phenytoin concentrations of 3.125-200 μg/mL resulted in relative cell viability readings using tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide of 100% confirming nontoxicity and suggesting cell proliferation activity. Phenytoin-loaded alkyd nanoemulsions generally resulted in higher mean cell viability compared with equivalent concentration of phenytoin solutions, suggesting that the nanoemulsions provided a controlled-release property that maintained the optimum phenytoin level for keratinocyte growth. HaCaT cell proliferation, measured by 5-bromo-2-deoxyuridine uptake, was found to increase following exposure to increasing phenytoin concentration from 25 to 50 μg/mL in solution or encapsulated in nanoemulsions but declined at a drug concentration of 100 μg/mL. An in vitro cell monolayer wound scratch assay revealed that phenytoin solution or nanoemulsions producing 50 μg/mL phenytoin concentration resulted in 75%-82% “scratch closure” after 36 h, similar to medium containing 10% fetal bovine serum as a cell growth promoter. These findings indicate that phenytoin-loaded alkyd nanoemulsions show potential for promoting topical wound healing through enhanced proliferation of epidermal cells.     

In vitro wound healing activity of 1-hydroxy-5,7-dimethoxy-2-naphthalene-carboxaldehyde (HDNC) and other isolates of Aegle marmelos L.: Enhances keratinocytes motility via Wnt/β-catenin and RAS-ERK pathways

Wound healing is a complex process in which injured skin and tissues repaired by interaction of a complex cascade of cellular events that generates resurfacing, reconstitution and restoration of the tensile strength of injured skin. It follows β-catenin, extracellular signal regulated kinase (ERK) and Akt signaling pathways. Aegle marmelos L., generally known as bael is found to act as anti-inflammatory, antioxidant and anti-ulcer agent. Furthermore, studies have demonstrated that this Indian traditional medicinal plant, A. marmelos flower extract (AMF) was used for wound injury. Henceforth, the current study was investigated to ascertain the effect of its active constituents in vitro wound healing with mechanism involve in migration of cells and activation of β-catenin in keratinocytes, inhibition of PGE₂ in macrophages and production of collagen in fibroblasts. We have taken full thickness wound of rats and applied AMF for 2 weeks. Cutaneous wound healing activity was performed using HaCaT keratinocytes, Hs68 dermal fibroblasts and RAW264.7 macrophages to determine cell viability, nitric oxide production, collagen expression, cell migration and β-catenin activation. Results shows that AMF treated rats demonstrated reduced wound size and epithelisation was improved, involved in keratinocytes migration by regulation of Akt signaling, beta-catenin and extracellular signal-regulated kinase (ERK) pathways. AMF and its active constituent’s increased mRNA expression, inhibited nitric oxide, PGE₂ release, mRNA expression of mediators in RAW 264.7 macrophages and enhances the motility of HaCaT keratinocytes in vitro wound healing of rats.     

Rational design of peptides with enhanced antimicrobial and anti‐biofilm activities against cariogenic bacterium Streptococcus mutans

Streptococcus mutans (S. mutans) is known to be a leading cariogenic pathogen in the oral cavity. Antimicrobial peptides possess excellent properties to combat such pathogens. In this study, we compared the antimicrobial activity of novel linear reutericin 6‐ and/or gassericin A‐inspired peptides and identified LR‐10 as the leading peptide. Antibacterial assays demonstrate that LR‐10 is more active against S. mutans (3.3 μM) than many peptide‐based agents without resistance selection, capable of killing many oral pathogens, and tolerant of physiological conditions. LR‐10 also presented a faster killing rate than chlorhexidine and erythromycin, and appeared to display selective activity against S. mutans within 10 s. S. mutans is usually encased in plaque biofilms. Biofilm inhibitory assays indicated that LR‐10 had excellent inhibitory effect on the biofilm formation of S. mutans and biofilm‐encased cells in vitro at low concentrations (6.5 μM). Consistent with most peptides, LR‐10 kills S. mutans mainly by disrupting the cell membranes. Notably, both hemolytic activity assays and cytotoxicity tests indicated that LR‐10 could keep biocompatible at the effective concentrations. Hence, LR‐10 could be a good candidate for clinical treatment of dental caries.     

Discrimination of skin sensitizers from non‐sensitizers by interleukin‐1α and interleukin‐6 production on cultured human keratinocytes

In vitro testing methods for classifying sensitizers could be valuable alternatives to in vivo sensitization testing using animal models, such as the murine local lymph node assay (LLNA) and the guinea pig maximization test (GMT), but there remains a need for in vitro methods that are more accurate and simpler to distinguish skin sensitizers from non‐sensitizers. Thus, the aim of our study was to establish an in vitro assay as a screening tool for detecting skin sensitizers using the human keratinocyte cell line, HaCaT. HaCaT cells were exposed to 16 relevant skin sensitizers and 6 skin non‐sensitizers. The highest dose used was the dose causing 75% cell viability (CV75) that we determined by an MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] assay. The levels of extracellular production of interleukin‐1α (IL‐1α) and IL‐6 were measured. The sensitivity of IL‐1α was 63%, specificity was 83% and accuracy was 68%. In the case of IL‐6, sensitivity: 69%, specificity: 83% and accuracy: 73%. Thus, this study suggests that measuring extracellular production of pro‐inflammatory cytokines IL‐1α and IL‐6 by human HaCaT cells may potentially classify skin sensitizers from non‐sensitizers. Copyright © 2015 John Wiley & Sons, Ltd.