A specialized tissue type, the keratinizing epithelium, protects terrestrial mammals from water loss and noxious physical, chemical and mechanical insults. This barrier between the body and the environment is constantly maintained by reproduction of inner living epidermal keratinocytes which undergo a process of terminal differentiation and then migrate to the surface as interlocking layers of dead stratum corneum cells. These cells provide the bulwark of mechanical and chemical protection, and together with their intercellular lipid surroundings, confer water-impermeability. Much of this barrier function is provided by the cornified cell envelope (CE), an extremely tough protein/lipid polymer structure formed just below the cytoplasmic membrane and subsequently resides on the exterior of the dead cornified cells. It consists of two parts: a protein envelope and a lipid envelope. The protein envelope is thought to contribute to the biomechanical properties of the CE as a result of cross-linking of specialized CE structural proteins by both disulfide bonds and N(epsilon)-(gamma-glutamyl)lysine isopeptide bonds formed by transglutaminases. Some of the structural proteins involved include involucrin, loricrin, small proline rich proteins, keratin intermediate filaments, elafin, cystatin A, and desmosomal proteins. The lipid envelope is located on the exterior of and covalently attached by ester bonds to the protein envelope and consists of a monomolecular layer of omega-hydroxyceramides. These not only serve of provide a Teflon-like coating to the cell, but also interdigitate with the intercellular lipid lamellae perhaps in a Velcro-like fashion. In fact the CE is a common feature of all stratified squamous epithelia, although its precise composition, structure and barrier function requirements vary widely between epithelia. Recent work has shown that a number of diseases which display defective epidermal barrier function, generically known as ichthyoses, are the result of genetic defects of the synthesis of either CE proteins, the transglutaminase 1 cross-linking enzyme, or defective metabolism of skin lipids.
Animal ; Cell Membrane/metabolism ; Epidermis/metabolism* ; Epidermis/chemistry* ; Human ; Ichthyosis/metabolism ; Ichthyosis/genetics ; Keratinocytes/metabolism* ; Keratinocytes/chemistry ; Membrane Lipids/metabolism* ; Membrane Proteins/metabolism* ; Protein-Glutamine gamma-Glutamyltransferase/metabolism

Objective: To fabricate TiO₂ nanotube material functionalized by antimicrobial peptide LL-37, and to explore its effects on biological behaviors such as adhesion and migration of human keratinocytes (HaCaT) and its antibacterial properties. Methods: The TiO₂ nanotube array (NT) was constructed on the surface of polished titanium (PT) by anodization, and the antimicrobial peptide LL-37 was loaded on the surface of TiO₂ nanotube (LL-37/NT) by physical adsorption. Three samples were selected by simple random sampling in each group. Surface morphology, roughness, hydrophilicity and release characteristics of LL-37 of the samples were analyzed with a field emission scanning electron microscope, an atomic force microscope, a contact angle measuring device and a microplate absorbance reader. HaCaT cells were respectively cultured on the surface of three groups of titanium samples. Each group had 3 replicates. The morphology of cell was observed by field emission scanning electron microscope. The number of cell adhesion was observed by cellular immunofluorescence staining. Cell counting kit-8 (CCK-8) assay was used to detect cell proliferation. Wound scratch assay was used to observe the migration of HaCaT. The above experiments were used to evaluate the effect of each group on the biological behavior of HaCaT cells. To evaluate their antibacterial effects, Porphyromonas gingivalis (Pg) was respectively inoculated on the surface of three groups of titanium samples. Each group had 3 replicates. The morphology of bacteria was observed by field emission scanning electron microscope. Bacterial viability was determined by live/dead bacterial staining. Results: A uniform array of nanotubes could be seen on the surface of titanium samples in LL-37/NT group, and the top of the tube was covered with granular LL-37. Compared with PT group [the roughness was (2.30±0.18) nm, the contact angle was 71.8°±1.7°], the roughness [(20.40±3.10) and (19.10±4.11) nm] and hydrophilicity (the contact angles were 22.4°±3.1° and 25.3°±2.2°, respectively) of titanium samples increased in NT and LL-37/NT group (P<0.001). The results of in vitro release test showed that the release of antimicrobial peptide LL-37 was characterized by early sudden release (1-4 h) and long-term (1-7 d) slow release. With the immunofluorescence, more cell attachment was found on NT and LL-37/NT than that on PT at the first 0.5 and 2.0 h of culture (P<0.05). The results of CCK-8 showed that there was no significant difference in the proliferation of cells among groups at 1, 3 and 5 days after culture. Wound scratch assay showed that compared with PT and NT group, the cell moved fastest on the surface of titanium samples in LL-37/NT group at 24 h of culture [(96.4±4.9)%] (F=35.55, P<0.001). A monolayer cells could be formed and filled with the scratch in 24 h at LL-37/NT group. The results of bacterial test in vitro showed that compared with the PT group, the bacterial morphology in the NT and LL-37/NT groups was significantly wrinkled, and obvious bacterial rupture could be seen on the surface of titanium samples in LL-37/NT group. The results of bacteria staining showed that the green fluorescence intensity of titanium samples in LL-37/NT group was the lowest in all groups (F=66.54,P<0.001). Conclusions: LL-37/NT is beneficial to the adhesion and migration of HaCaT cells and has excellent antibacterial properties, this provides a new strategy for the optimal design of implant neck materials.
Humans ; Titanium/chemistry* ; Antimicrobial Peptides ; Cathelicidins ; Sincalide ; Anti-Bacterial Agents/pharmacology* ; Nanotubes/chemistry* ; Dental Materials ; Bacteria ; Keratinocytes ; Surface Properties

Granular cell ameloblastoma is a rare, benign neoplasm of the odontogenic epithelium. A case of massive granular cell ameloblastoma in a 44-year-old Thai female is reported. Histopathological features displayed a follicular type of ameloblastoma with an accumulation of granular cells residing within the tumor follicles. After treatment by partial mandibulectomy, the patient showed a good prognosis without recurrence in a 2-year follow-up. To characterize the granular cells in ameloblastoma, we examined the expression of basement membrane (BM) proteins, including collagen type IV, laminins 1 and 5 and fibronectin using immunohistochemistry. Except for the granular cells, the tumor cells demonstrated a similar expression of BM proteins compared to follicular and plexiform ameloblastomas in our previous study, whereas the granular cells showed strong positivity to laminins 1 and 5 and fibronectin. The increased fibronectin expression in granular cells suggests a possibility of age-related transformation of granular cells in ameloblastoma.
Adult ; Ameloblastoma ; chemistry ; pathology ; Basement Membrane ; chemistry ; Cell Adhesion Molecules ; analysis ; Collagen Type IV ; analysis ; Female ; Fibronectins ; analysis ; Humans ; Keratinocytes ; chemistry ; pathology ; Laminin ; analysis ; Mandibular Neoplasms ; chemistry ; pathology ; Membrane Proteins ; analysis

OBJECTIVETo investigate the effects of Liuwei Dihuang (LWDH) decoction on serotonine (5-HTs), melatonin and the activity of the rate-limiting enzymes ANNAT and HIOMT in cultured human melanocytes and in melanocytes co-cultured with keratinocytes.

METHODSCCK-8 assay was used to assess the proliferation of melanocytes and melanocytes co-cultured with keratinocytes after treatment with the serum from rabbits fed with LWDH decoction. High-performance liquid chromatography was used to determine 5-HT and melatonin contents, and real-time fluorescent PCR was employed to evaluate the ANNAT and HIOMT activities in the cell cultures.

RESULTSThe serum from rabbits fed with LWDH Decoction at low doses did not affect the proliferation of melanocytes co-cultured with keratinocytes, but at the concentrations of 20%-40%, the serum significantly inhibited the proliferation of melanocytes, and the effect was optimal with a concentration of 40% (P<0.05). 5-HT and melatonin contents in the cell culture decreased as the serum concentration increased (P<0.05), which was the most obvious with a serum concentration of 40% (P<0.01). Exposure of the cells to low and moderate doses of the serum caused a dose-dependent decrease in AANAT activity (P<0.05), but the serum produced no significant changes in the level of HIOMT mRNA expression in the cells.

COUCLUSIONSThe serotoninergic/melatoninergic system mediate the regulation of melanin metabolism by LWDH Decoction, the mechanism of which may involve 5-HTs, melatonin and ANNAT.

Animals ; Cells, Cultured ; Coculture Techniques ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Keratinocytes ; Melanins ; metabolism ; Melanocytes ; drug effects ; metabolism ; Melatonin ; metabolism ; Rabbits ; Serotonin ; metabolism ; Serum ; chemistry

BACKGROUNDHaptoglobin (Hp) is one of the acute-phase proteins. Recent studies have demonstrated that Hp exerts immunoregulatory and anti-inflammatory actions and may be one of the inhibitory factors of immune reactions in the skin. In this study we investigated the regulation of Hp expression in a human keratinocyte cell line HaCaT by various cytokines and glucocorticoid.

METHODSHaCaT cells were cultured with IL-6 (50 ng/ml), TNF-alpha (20 ng/ml), IFN-gamma (20 ng/ml) or IL-4 (20 ng/ml) with or without 1 micromol/L dexamethasone in 6-well plates for 12, 24 and 48 hours. Both the cells and the supernatants were collected to detect the changes of Hp expression by reverse-transcription PCR, ELISA and immunohistochemistry.

RESULTSThe results showed that Hp expression were elevated at both the mRNA and protein level by the combination of IL-6, TNF-alpha or IL-4 with dexamethasone, whereas the three cytokines alone did not upregulate the Hp expression. IFN-gamma showed no effect on the Hp expression in HaCaT cells.

CONCLUSIONSThese findings suggest that different inflammatory cytokines as well as glucocorticoid may be involved in the regulation of Hp expression in keratinocytes, and this may be one of the negative feedback mechanisms in inflammatory skin diseases.

Cell Line ; Dexamethasone ; pharmacology ; Glucocorticoids ; pharmacology ; Haptoglobins ; analysis ; Humans ; Interferon-gamma ; pharmacology ; Interleukin-4 ; pharmacology ; Interleukin-6 ; pharmacology ; Keratinocytes ; chemistry ; drug effects ; Tumor Necrosis Factor-alpha ; pharmacology

An extracellular matrix protein plays an important role in skin wound healing. In the present study, we engineered a recombinant protein encompassing the 9th and 10th type III domains of fibronectin, and 4th FAS1 domain of beta ig-h3. This recombinant protein, in total, harbors four known-cell adhesion motifs for integrins: Pro-His-Ser-Arg-Asn (PHSRN) and Arg-Gly-Asp (RGD) in 9th and 10th type III domains of fibronectin, respectively, and Glu-Pro-Asp-Ile-Met (EPDIM) and Try-His (YH) in 4th FAS1 domain of big-h3, were designated to tetra-cell adhesion motifs (T-CAM). In vitro studies showed T-CAM supporting adhesion, migration and proliferation of different cell types including keratinocytes and fibroblasts. In an animal model of full-thickness skin wound, T-CAM exhibited excellent wound healing effects, superior to both 4th FAS1 domain of beta ig-h3 or 9th and 10th type III domains of fibronectin. Based on these results, T-CAM can be applied where enhancement of cell adhesion, migration and proliferation are desired, and it could be developed into novel wound healing drug.
Amino Acid Motifs ; Animals ; Cell Adhesion/*drug effects ; Cell Line ; Cell Movement/*drug effects ; Cell Proliferation/*drug effects ; Extracellular Matrix Proteins/chemistry/genetics/pharmacology ; Fibroblasts/cytology/drug effects/physiology ; Fibronectins/chemistry/genetics/*pharmacology ; Humans ; Keratinocytes/cytology/drug effects/physiology ; Mice ; NIH 3T3 Cells ; Rabbits ; Recombinant Fusion Proteins/chemistry/genetics/pharmacology ; Transforming Growth Factor beta/chemistry/genetics/pharmacology ; Wound Healing/*drug effects/physiology

PURPOSE: Acute side effects of radiation such as oral mucositis are observed in most patients. Although several potential radioprotective agents have been proposed, no effective agent has yet been identified. In this study, we investigated the effectiveness of synthetic compound 3-amino-3-(4-fluoro-phenyl)-1H-quinoline-2,4-dione (KR22332) as a radioprotective agent. MATERIALS AND METHODS: Cell viability, apoptosis, the generation of reactive oxygen species (ROS), mitochondrial membrane potential changes, and changes in apoptosis-related signaling were examined in human keratinocyte (HaCaT). RESULTS: KR22332 inhibited irradiation-induced apoptosis and intracellular ROS generation, and it markedly attenuated the changes in mitochondrial membrane potential in primary human keratinocytes. Moreover, KR22332 significantly reduced the protein expression levels of ataxia telangiectasia mutated protein, p53, and tumor necrosis factor (TNF)-alpha compared to significant increases observed after radiation treatment. CONCLUSION: KR22332 significantly inhibited radiation-induced apoptosis in human keratinocytes in vitro, indicating that it might be a safe and effective treatment for the prevention of radiation-induced mucositis.
Apoptosis/drug effects/physiology ; Cell Line, Tumor ; Cell Survival/drug effects/physiology ; Humans ; Keratinocytes/metabolism ; Membrane Potential, Mitochondrial/drug effects/physiology ; Radiation-Protective Agents/chemistry/*pharmacology ; Reactive Oxygen Species/metabolism

PURPOSE: Acute side effects of radiation such as oral mucositis are observed in most patients. Although several potential radioprotective agents have been proposed, no effective agent has yet been identified. In this study, we investigated the effectiveness of synthetic compound 3-amino-3-(4-fluoro-phenyl)-1H-quinoline-2,4-dione (KR22332) as a radioprotective agent. MATERIALS AND METHODS: Cell viability, apoptosis, the generation of reactive oxygen species (ROS), mitochondrial membrane potential changes, and changes in apoptosis-related signaling were examined in human keratinocyte (HaCaT). RESULTS: KR22332 inhibited irradiation-induced apoptosis and intracellular ROS generation, and it markedly attenuated the changes in mitochondrial membrane potential in primary human keratinocytes. Moreover, KR22332 significantly reduced the protein expression levels of ataxia telangiectasia mutated protein, p53, and tumor necrosis factor (TNF)-alpha compared to significant increases observed after radiation treatment. CONCLUSION: KR22332 significantly inhibited radiation-induced apoptosis in human keratinocytes in vitro, indicating that it might be a safe and effective treatment for the prevention of radiation-induced mucositis.
Apoptosis/drug effects/physiology ; Cell Line, Tumor ; Cell Survival/drug effects/physiology ; Humans ; Keratinocytes/metabolism ; Membrane Potential, Mitochondrial/drug effects/physiology ; Radiation-Protective Agents/chemistry/*pharmacology ; Reactive Oxygen Species/metabolism

Caesalpinia sappan L., belonging to the family Leguminosae, is a medicinal plant that is distributed in Southeast Asia. The dried heartwood of this plant is used as a traditional ingredient of food, red dyes, and folk medicines in the treatment of diarrhea, dysentery, tuberculosis, skin infections, and inflammation. Brazilin is the major active compound, which has exhibited various pharmacological effects, including anti-platelet activity, anti-hepatotoxicity, induction of immunological tolerance, and anti-inflammatory and antioxidant activities. The present study aimed to evaluate the antioxidant activity and expression of antioxidant enzymes of C. sappan L. extract and its major compound, brazilin, in human epidermal keratinocytes exposed to UVA irradiation. Our results indicated that C. sappan L. extract reduced UVA-induced HO production via GPX7 activation. Moreover, brazilin exhibited antioxidant effects that were similar to those of C. sappan L. via glutathione peroxidase 7 (GPX7), suggesting that C. sappan L. extract and its natural compound represent potential treatments for oxidative stress-induced photoaging of skin.
Antioxidants ; pharmacology ; Benzopyrans ; pharmacology ; Caesalpinia ; chemistry ; Humans ; Hydrogen Peroxide ; toxicity ; Keratinocytes ; cytology ; drug effects ; enzymology ; radiation effects ; Oxidative Stress ; drug effects ; radiation effects ; Peroxidases ; genetics ; metabolism ; Plant Extracts ; pharmacology ; Protective Agents ; pharmacology ; Ultraviolet Rays

The purpose of this study is to investigate the penetration effects and mechanism of N-arginine chitosan (ACS). This novel transdermal enhancer with a mimetic structure of cell-penetration peptides was synthesized by introducing hydrophilic arginine groups to the amino-group on chitosan's side chain. The structure of ACS was confirmed by FT-IR, 1H NMR and element analysis. In addition, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was used to study the protein conformation and the water content of stratum corneum, and the result suggested that ACS can change the orderly arrangement of the molecules in the stratum corneum, making the stack structure of keratin become loose. And ACS can increase the water content of the stratum corneurn. Inverted fluorescence microscope and flow cytometry were used to examine penetration effect of ACS on Hacat cell. The result confirmed that the uptake of ACS was enhanced with increased substitution degree of arginine by 4-8 folds compared to chitosan. In vitro penetration studies on three electrical types of drugs were carried out using three model drugs of negatively charged aspirin, positively charged terazosin and neutral drug isosorbide mononitrate by the method of Franz diffusion cells. The results showed that ACS has obviously penetration of the negatively charged drug aspirin, and certain penetration of neutral drug issorbide mononitrate, but inhibition of positively charged terazosin. In vivo imaging technology research results show that the ACS can significantly enhance the fluorescence intensity of morin, which is the auto-fluorescence anionic drug. These obtained results suggested that ACS, as a promising transdermal enhancer, can change the structure of the keratinocytes and analog penetrating peptides promote absorption, but have certain selectivity for the drug.
Administration, Cutaneous ; Animals ; Arginine ; chemical synthesis ; chemistry ; pharmacology ; Aspirin ; administration & dosage ; pharmacokinetics ; Cell Line ; Cell Survival ; drug effects ; Cell-Penetrating Peptides ; chemical synthesis ; chemistry ; pharmacology ; Chitosan ; chemical synthesis ; chemistry ; pharmacology ; Drug Carriers ; Humans ; Isosorbide Dinitrate ; administration & dosage ; analogs & derivatives ; pharmacokinetics ; Keratinocytes ; cytology ; Male ; Mice ; Prazosin ; administration & dosage ; analogs & derivatives ; pharmacokinetics ; Skin Absorption ; drug effects