Migration Of Human Epidermal Keratinocytes Research Articles

Phloroglucinol Enhances Anagen Signaling and Alleviates H2O2-Induced Oxidative Stress in Human Dermal Papilla Cells.

Phloroglucinol (PG) is one of the abundant isomeric benzenetriols in brown algae. Due to its polyphenolic structure, PG exhibits various biological activities. However, the impact of PG on anagen signaling and oxidative stress in human dermal papilla cells (HDPCs) is unknown. In this study, we investigated the therapeutic potential of PG for improving hair loss. A non-cytotoxic concentration of PG increased anagen-inductive genes and transcriptional activities of β-Catenin. Since several anagen-inductive genes are regulated by β-Catenin, further experiments were performed to elucidate the molecular mechanism by which PG upregulates anagen signaling. Various biochemical analyses revealed that PG upregulated β-Catenin signaling without affecting the expression of Wnt. In particular, PG elevated the phosphorylation of protein kinase B (AKT), leading to an increase in the inhibitory phosphorylation of glycogen synthase kinase 3 beta (GSK3β) at serine 9. Treatment with the selective phosphoinositide 3-kinase/AKT inhibitor, LY294002, restored the increased AKT/GSK3β/β-Catenin signaling and anagen-inductive proteins induced by PG. Moreover, conditioned medium from PG-treated HDPCs promoted the proliferation and migration of human epidermal keratinocytes via the AKT signaling pathway. Subsequently, we assessed the antioxidant activities of PG. PG ameliorated the elevated oxidative stress markers and improved the decreased anagen signaling in hydrogen peroxide (H2O2)-induced HDPCs. The senescence-associated β-galactosidase staining assay also demonstrated that the antioxidant abilities of PG effectively mitigated H2O2-induced senescence. Overall, these results indicate that PG potentially enhances anagen signaling and improves oxidative stress-induced cellular damage in HDPCs. Therefore, PG can be employed as a novel therapeutic component to ameliorate hair loss symptoms.

Pyrogallol loaded chitosan-based polymeric hydrogel for controlling Acinetobacter baumannii wound infections: Synthesis, characterization, and topical application

Antibacterial hydrogels have emerged as a promising approach for wound healing, owing to their ability to integrate antibacterial agents into the hydrogel matrix. Benefiting from its remarkable antibacterial and wound-healing attributes, pyrogallol has been introduced into chitosan-gelatin for the inaugural development of an innovative antibacterial polymeric hydrogel tailored for applications in wound healing. Hence, we observed the effectiveness of pyrogallol in inhibiting the growth of A. baumannii, disrupting mature biofilms, and showcasing robust antioxidant activity both in vitro and in vivo. In addition, pyrogallol promoted the migration of human epidermal keratinocytes and exhibited wound healing activity in zebrafish. These findings suggest that pyrogallol holds promise as a therapeutic agent for wound healing. Interestingly, the pyrogallol-loaded chitosan-gelatin (Pyro-CG) hydrogel exhibited enhanced mechanical strength, stability, controlled drug release, biodegradability, antibacterial activity, and biocompatibility. In vivo results established that Pyro-CG hydrogel promotes wound closure and re-epithelialization in A. baumannii-induced wounds in molly fish. Therefore, the prepared Pyro-CG polymeric hydrogel stands poised as a potent and promising agent for wound healing with antibacterial properties. This holds considerable promise for the development of effective therapeutic interventions to address the increasing menace of A. baumannii-induced wound infections.

Autophagy-related LncRNA PRDM10-DT responds to UVB radiation in keratinocytes

Ultraviolet (UV)-B radiation is a major environmental risk factor that is responsible for the development and progression of many skin disorders. Autophagy is the process of degradation and recycling of damaged cytoplasmic organelles, macromolecular aggregates, and long-lived proteins. Previously, we found that the autophagy inducer apigenin restored UVB-impaired autophagy and the cellular response by downregulating the expression of autophagy-related genes such as ATG5. To explore long noncoding RNAs (lncRNAs) involved in regulating these autophagy-related genes, in this study, we assessed the expression profiles of lncRNAs and mRNAs using a microarray in human epidermal keratinocytes (HEKs) treated with or without apigenin after UVB radiation. The expression levels of 80 selected autophagy-related genes and related lncRNAs were confirmed by quantitative real-time polymerase chain reaction (qRT‒PCR). The lncRNA PRDM10-DT was proposed to regulate IRGM based on the ceRNA and coexpression pattern and was demonstrated to be involved in autophagy regulation, proliferation and migration of HEKs by qRT‒PCR, Western blotting, colony formation and scratch wound assays, respectively. These findings suggest an autophagy-related lncRNA in response to UVB radiation that promotes the proliferation and migration of HEKs through inducing autophagy by competing microRNAs for IRGM.

Bee Venom in Wound Healing.

Bee venom (BV), also known as api-toxin, is widely used in the treatment of different inflammatory diseases such as rheumatoid arthritis or multiple sclerosis. It is also known that BV can improve the wound healing process. BV plays a crucial role in the modulation of the different phases of wound repair. It possesses anti-inflammatory, antioxidant, antifungal, antiviral, antimicrobial and analgesic properties, all of which have a positive impact on the wound healing process. The mentioned process consists of four phases, i.e., hemostasis, inflammation, proliferation and remodeling. The impaired wound healing process constitutes a significant problem especially in diabetic patients, due to hypoxia state. It had been found that BV accelerated the wound healing in diabetic patients as well as in laboratory animals by impairing the caspase-3, caspase-8 and caspase-9 activity. Moreover, the activity of BV in wound healing is associated with regulating the expression of transforming growth factor (TGF-β1), vascular endothelial growth factor and increased collagen type I. BV stimulates the proliferation and migration of human epidermal keratinocytes and fibroblasts. In combination with polyvinyl alcohol and chitosan, BV significantly accelerates the wound healing process, increasing the hydroxyproline and glutathione and lowering the IL-6 level in wound tissues. The effect of BV on the wounds has been proved by numerous studies, which revealed that BV in the wound healing process brings about a curative effect and could be applied as a new potential treatment for wound repair. However, therapy with bee venom may induce allergic reactions, so it is necessary to assess the existence of the patient’s hypersensitivity to apitoxin before treatment.

Royal jelly-derived proteins enhance proliferation and migration of human epidermal keratinocytes in an in vitro scratch wound model

BackgroundSkin injury is inevitable in daily life. In recent years, with the increasing morbidity of diseases such as diabetes and metabolic disorders, chronic wounds have become a considerable challenge in clinical practice. Royal jelly, reported to have multifarious biological and physiological properties, has been used as a remedy for a variety of wounds since ancient times. However, the active components and mechanisms underlying the wound-healing properties of royal jelly are still largely unknown.MethodsWater-soluble proteins of royal jelly were fractionated and investigated for the proliferative and migratory effects on human epidermal keratinocytes (HaCaT) in an in vitro wound healing model. The proteins present in bioactive fractions were characterised and quantified using Label-free protein quantification method. The potential functions of these proteins in biological systems were further analysed using bioinformatic tools.ResultsA protein fraction, mainly containing major royal jelly proteins 2 (MRJP2), MRJP3 and MRJP7, stimulated proliferative and migratory activities in HaCaT cells without visible cytotoxicity. It exerted the greatest effects on the growth of HaCaT cells in the first 48 h. Furthermore, when treated with this protein fraction, the closure rates of the in vitro scratch wound were significantly increased. Functional analysis indicated that MRJP2, MRJP3 and MRJP7 were associated with carbohydrate transport and metabolism.ConclusionsWe fractionated the water-soluble proteins of royal jelly and identified one fraction (Fraction 2) that induced both proliferative and migratory effects on a human epidermal keratinocyte cell line. Major royal jelly proteins (MRJP2, MRJP3 and/or MRJP7) were speculated to possess potential wound-healing bioactivity. This is the first report that royal jelly may improve wound closure via MRJP-induced cellular proliferation and migration. These proteins may be valuable lead compounds for the development of novel wound healing medications. Our findings would facilitate better understanding of the wound repair mechanisms of royal jelly.

A Novel Substance P-Based Hydrogel for Increased Wound Healing Efficiency.

The neuropeptide substance P (SP) is known to stimulate wound healing by regulating the production of relevant cytokines as well as cell proliferation and migration. However, the therapeutic application of SP is limited by its low stability under biological conditions and oxidation during purification, formulation, and storage. To address this problem, we developed a novel formulation of SP as an SP gel, and investigated its wound healing activity both in vitro and in vivo. SP in SP gel was stable at various temperatures for up to 4 weeks. In vitro, SP gel exhibited more potential as a candidate wound-healing agent than SP alone, as evidenced by the observed increases in the proliferation and migration of human epidermal keratinocytes and human dermal fibroblasts. In vivo experiments showed that SP gel treatment enhanced the healing of full-thickness wounds in mice as compared to SP alone. These results demonstrate the benefits of SP gel as a promising topical agent for wound treatment.

Biosynthesis of D-Series Resolvins in Skin Provides Insights into their Role in Tissue Repair

Cutaneous injury causes underlying tissue damage that must be quickly repaired to minimize exposure to pathogens and to restore barrier function. While the role of growth factors in tissue repair is established, the role of lipid mediators in skin repair has not been investigated extensively. Using a mass spectrometry-based lipid mediator metabolomics approach, we identified D-series resolvins and related pro-resolving lipid mediators during skin injury in mice and pigs. Differentiation of human epidermal keratinocytes increased expression of 15-lipoxygenase and stereospecific production of 17S-hydroxydocosahexaenoic acid, the common upstream biosynthetic marker and precursor of D-series resolvins. In human and pig skin, specific receptors for D-series resolvins were expressed in the epidermal layer and mice deficient in RvD1 receptor Alx/Fpr2 showed an endogenous defect in re-epithelialization. Topical application of D-series resolvins expedited re-epithelialization during skin injury and they enhanced migration of human epidermal keratinocytes in a receptor-dependent manner. The enhancement of re-epithelialization by RvD2 was lost in mice genetically deficient in its receptor and migration of keratinocytes stimulated with RvD2 was associated with activation of the PI3K-AKT-mTOR-S6 pathway, blockade of which prevented its pro-migratory actions. Collectively, these results demonstrate that resolvins have direct roles in the tissue repair program.

Leptin promotes wound healing in the skin.

IntroductionLeptin, a 16 kDa anti-obesity hormone, exhibits various physiological properties. Interestingly, skin wound healing was proven to delay in leptin-deficient ob/ob mice. However, little is known on the mechanisms of this phenomenon. In this study, we attempted to elucidate a role of leptin in wound healing of skin.MethodsImmunohistochemical analysis was performed to confirm the expression of the leptin receptor (Ob-R) in human and mouse skin. Leptin was topically administered to chemical wounds created in mouse back skin along with sustained-release absorbable hydrogel. The process of wound repair was histologically observed and the area of ulceration was measured over time. The effect of leptin on the proliferation, differentiation and migration of human epidermal keratinocytes was investigated.ResultsOb-R was expressed in epidermal cells of human and mouse skin. Topical administration of leptin significantly promoted wound healing. Histological analysis showed more blood vessels in the dermal connective tissues in the leptin-treated group. The proliferation, differentiation/function and migration of human epidermal keratinocytes were enhanced by exogenous leptin.ConclusionTopically administered leptin was proven to promote wound healing in the skin by accelerating proliferation, differentiation/function and migration of epidermal keratinocytes and enhancing angiogenesis around the wounded area. These results strongly suggest that topical administration of leptin may be useful as a treatment to promote wound healing in the skin.

Protective Effect of Geranylgeranylacetone against Radiation-Induced Delayed Effects on Human Keratinocytes

Skin exposure to ionizing radiation affects the normal wound healing process. We investigated the beneficial effects of a pharmacological treatment with geranylgeranylacetone (GGA) on keratinocytes using in vitro scratch wound injury assay in nonirradiated and irradiated conditions. Irradiation affected the wound closure of keratinocytes 24 h after scratch injury, whereas re-epithelialization was markedly accelerated after GGA treatment when compared to nontreated keratinocytes. We demonstrated that GGA treatment increased migration of human epidermal keratinocytes and this migratory property was not related to RhoA signaling. Interestingly, Western blot analysis revealed that GGA treatment down-regulated caspase 3 active form expression and up-regulated the activated phenotype by inducing both keratin 6 (K6) expression and interleukin-1β (IL-1β) release without modification of the differentiate phenotype. Finally, the proteomic profiling was performed on keratinocytes, showing that global protein changes occurred after irradiation of keratinocytes treated or untreated with GGA.

PPARδ promotes wound healing by up‐regulating TGF‐β1‐dependent or ‐independent expression of extracellular matrix proteins

Although the peroxisome proliferator-activated receptor (PPAR) δ has been implicated in the wound healing process, its exact role and mechanism of action have not been fully elucidated. Our previous findings showed that PPARδ induces the expression of the transforming growth factor (TGF)-β1, which has been implicated in the deposit of extracellular matrix proteins. Here, we demonstrate that administration of GW501516, a specific PPARδ ligand, significantly promoted wound closure in the experimental mouse and had a profound effect on the expression of collagen types I and III, alpha-smooth muscle actin, pSmad3 and TGF-β1, which play a pivotal role in wound healing processes. Activation of PPARδ increased migration of human epidermal keratinocytes and dermal fibroblasts in in vitro scrape-wounding assays. Addition of a specific ALK5 receptor inhibitor SB431542 significantly suppressed GW501516-induced migration of human keratinocytes and fibroblasts. In these cells, activated PPARδ also induced the expression of collagen types I and III and fibronectin in a TGF-β1-dependent or -independent manner. The effect of PPARδ on the expression of type III collagen was dually regulated by the direct binding of PPARδ and Smad3 to a direct repeat-1 site and a Smad-binding element, respectively, of the type III gene promoter. Taken together, these results demonstrated that PPARδ plays an important role in skin wound healing in vivo and that it functions by accelerating extracellular matrix-mediated cellular interactions in a process mediated by the TGF-β1/Smad3 signaling-dependent or - independent pathway.

Acceleration of Cutaneous Wound Healing by Suppression of Large Conductance Ca2+-Activated K+ Channels

Many kinds of K+ channels are involved in the regulation of cell migration and proliferation, which are required for the processes of wound healing. However, the role of K+ channels on cutaneous wound healing has not yet been reported. Here, we demonstrate that inhibition of large conductance Ca2+-activated K+ (BKCa) channels expressed in human epidermal keratinocyte facilitate cutaneous wound healing by activating both cell migration and proliferation. In the group treated with 25 mM KCl, in vivo wound healing was facilitated more rapidly than that in control group. In vitro assay of wound healing showed that 25 mM KCl significantly increased wound closure in keratinocytes after creation of linear wound with ∼200 ∈1/4m wide defect. KCl (25 mM) promoted processes of cell migration and proliferation. BKCa and two-pore domain K+ channels were recorded in the keratinocytes by using patch-clamp technique. The BKCa channel, among these K+ channels, is the most frequently observed in cell-attached mode. NS1619, a BKCa channel opener, inhibited the proliferation and migration of keratinocytes in a dose- and time-dependent manner. Charybdotoxin and iberiotoxin, BKCa channel blockers, facilitated both cell proliferation and migration by 10±7% and 30±4%, respectively. Cutaneous wound healing was also facilitated by siRNA against BKCa (BKCa/siRNA). The migration and proliferation were more enhanced by cotransfection with BKCa/siRNA and TASK-1/siRNA. BKCa channel blockers activated PKC and ERK in a time-dependent manner. These results show that BKCa and TASK-1 channels regulate proliferation and migration of human epidermal keratinocytes by activation of PKC-ERK pathway and indicate that BKCa channel could be a molecular target for regulation of cell proliferation and migration.

Human dermal fibroblasts do not exhibit directional migration on collagen I in direct-current electric fields of physiological strength.

Endogenous electric fields are generated lateral to skin wounds, with the cathodal pole of the field residing in the center of the wound. These fields are thought to be an important mechanism in guiding the migration of keratinocytes and other cells into wounds to effect healing. In this work, human dermal fibroblasts were exposed to direct current electric fields of physiological strength, and their migrational behavior was quantitated. Only random migration of human dermal fibroblasts was observed in direct-current electric fields under conditions that support the directional migration of human epidermal keratinocytes. Additionally, neither the presence of serum nor serum plus additional Mg++ in the experimental medium supported directional migration. Migratory rates of fibroblasts varied depending on the experimental medium used: in serum-containing medium the average velocity was as low as 0.23 micro m/min, while in serum-free keratinocyte medium the average velocity was as high as 0.36 micro m/min. These studies suggest that dermal fibroblasts do not respond to the endogenous electric field of a wound, and use other migratory cues to direct their movement into the wound bed.

Simulation of Migration of Human Epidermal Keratinocytes over Three-Dimensional Collagen Gel

We developed a novel model for studying migration of keratinocyte colonies over 3D collagen gel. It was shown that keratinocytes previously cultured on plastic and then seeded on the surface of collagen gel proliferate more actively than the primary cells. The gel density could significantly affect the morphology and migration of keratinocyte colonies. Modification of collagen gel by fibronectin-like protein or poly-L-lysine altered the morphometric parameters of colonies without significant changes in the velocity of migration.

Insulin stimulates haptotactic migration of human epidermal keratinocytes through activation of NF-kappa B transcription factor.

Insulin-mediated cell motility as well as the role of transcription factors in insulin-activated intracellular signal events have not been extensively studied. In this report we have examined whether insulin could mediate haptotactic migration of cultured human epidermal keratinocytes through activation of transcription factor NF-kappa B. Insulin caused a dose-dependent stimulation of keratinocyte migration that maximally reached 2-fold at 2 x 10(-7) M hormone. This phenomenon was independent of the nature of the extracellular matrix component (collagen I or laminin5/nicein) on which the cells migrated, indicating that a specific integrin-ligand complex is not required. A 10(-7) M insulin treatment of keratinocytes resulted in activation of a major kappa B DNA binding complex within 15 to 30 minutes, which was identified as the p65/p50 NF-kappa B heterodimer by electrophoretic mobility shift assays. The activation induced nuclear translocation of cytosolic pools of NF-kappa B factor. Pyrrolidine dithiocarbamate and N-acetyl-leucinyl-leucinyl-norleucinal H (two compounds that differentially inhibit I kappa B alpha degradation and, thus, NF-kappa B activation) reversed the insulin-stimulated keratinocyte haptotactic migration without affecting insulin receptor activation. These compounds inhibited the insulin-induced nuclear translocation of NF-kappa B as detected by confocal laser scanning microscopy. Taken together our experiments demonstrate that insulin stimulates haptotactic migration of human epidermal keratinocytes through activation of NF-kappa B transcription factor. They emphasize the ability of insulin to stimulate keratinocyte movement and provide a first clue to the mechanism of insulin-induced haptotactic signaling.