Effect of a Thermal Spring Water on Carbohydrate-Protein Interactions in In-Vitro Models Implicating Normal Human Keratinocytes and Recombinant Lectins

Background: Sugar moiety of macromolecules is today very well known for its implications in many biological recognition mechanisms including cell-cell, extracellular matrix-cell and/or bacteria-cell interactions. In this context lectins, which are carbohydrate-binding proteins displaying a high affinity for sugar groups of other molecules, are of a great importance, notably in immune response involving bacteria, viruses and fungi. As protein-carbohydrate interactions are often mediated by ions such as calcium, zinc or magnesium, we were prompted to study the effect of a thermal spring water (which contains this type of component) on interactions existing between: 1) osidic receptors of human normal keratinocytes and 2) two lectins greatly implicated in the immune response mechanisms (i.e. the dectin-1 and the langerin), and their ligands. Materials and Methods: In a first series of experiments, we studied the effect of increasing concentrations of a thermal spring water on interactions existing between glycosylated molecules and the osidic receptors expressed at the normal human keratinocytes surface. In a second step, and in order to better understand the putative effect of our thermal spring water on the immune response, we analyzed its effect on the interactions existing between the dectin-1 (implicated in the recognition of bacteria, viruses and fungi) and the langerin (expressed by Langerhans cells, the immune cells of the cutaneous tissue), and their ligands in a model using recombinant human lectins and appropriate binding molecules. Results: We showed here that our thermal spring water was able to reinforce interactions between keratinocytes osidic receptors and some of their ligands, in a dose-related manner: From 8% to 55% of increase with 10% to 30% (v/v) of thermal spring water. In the second part of our studies, we also showed that our thermal spring water was able to modulate interactions between dectin-1 and langerin and their ligands through a biphasic effect: Interactions were enhanced by more than 40% and 20% respectively with 10% of thermal spring water, and return to their basal level or lower for higher concentrations. Conclusion: The tested thermal spring water, probably due to its ionic composition, could significantly affect interactions of osidic receptors with their ligands. This property could be of a great interest to help immune system to maintain an appropriate “vigilance state” by using the thermal water at up to a concentration of 10%, and by avoiding any runaway reaction in case of aggression, by using concentrations higher than 10%. .

Exposure to heat-inactivated Trichophyton rubrum resulting in a limited immune response of human keratinocytes

Background Trichophyton rubrum （T. rubrum） represents the most important agent of dermatophytosis in humans. T. rubrum infection causes slight inflammation, and tends to be chronic and recurrent. It is suggested that it may result from the failure of epithelial cells to recognize T. rubrum effectively and initiate effective immune responses. The C-type lectin receptors （CLR） and toll-like receptors （TLR） are the two major pattern recognition receptors （PRRs） that recognize fungal components. Therefore, the purpose of the study was to analyze the expression of those PRRs and the cytokines in HaCaT cells stimulated with heat-inactivated T. rubrum conidia and hyphae, respectively. Methods HaCaT cells were unstimulated or stimulated with heat-inactivated T. rubrum conidia and hyphae （l×106 and 1.5×105 colony-forming unit （CFU） in 2 ml medium, respectively） for 6, 12 and 24 hours. The mRNA expression of PRRs involved in recognizing fungal pathogen-associated molecular patterns （PAMPs） and signaling molecules were measured by quantitative reverse transcription polymerase chain reaction （RT-PCR）. Meanwhile, surface toll-like receptor （TLR） 2, TLR4 and Dectin-1 were analyzed by fluorescence-activated cell sorter （FACS） 24 hours after treatment. The cytokines were detected in cell culture supernatants of HaCaT cells in 12 and 24 hours after treatment. Results HaCaT cells constitutively expressed mRNA of membrane-bound TLR1,2, 4 and 6, Dectinl and DC-SIGN, but not Dectin-2 or Mincle. Heat-killed T. rubrum did not significantly upregulate gene transcriptions of the PRRs of HaCaT cells. Heat-inactivated T. rubrum conidia significantly reduced the surface expression of TLR2 and Dectin-1, and suppressed the secretions of interferon-inducible protein-10 （IP-10） and monocyte chemotactic protein-1 （MCP-1） of HaCaT cells, while heat-killed T. rubrum hyphae significantly induced the secretions of IP-10 and MCP-I. Conclusion The cell-wall antigens of T. rubrum fail to activate transcriptional expression of PRRs and induce a lower immune response of HaCaT cells by limited cytokines secretion.

Sub-cytotoxic concentrations of ionic silver promote the proliferation of human keratinocytes by inducing the production of reactive oxygen species

Silver-containing preparations are widely used in the management of skin wounds, but the effects of silver ions on skin wound healing remain poorly understood. This study investigated the effects of silver ions （Ag^＋） on the proliferation of human skin keratinocytes （HaCaT） and the production of intracellular reactive oxygen species （ROS）. After treating HaCaT cells with Ag^＋ and/or the active oxygen scavenger N-acetyl cysteine （NAC）, cell proliferation and intracellular ROS generation were assessed using CCK-8 reagent and DCFH-DA fluorescent probe, respectively. In addition, 5-bromo-2-deoxyUridine （BrdU） incorporation assays, cell cycle flow cytometry, and proliferating cell nuclear antigen （PCNA） immunocytochemistry were conducted to further evaluate the effects of sub-cytotoxic Ag^＋ concentrations on HaCaT cells. The proliferation of HaCaT cells was promoted in the presence of 10^-6 and 10^-5s mol/L Ag^＋at 24, 48, and 72 h. Intracellular ROS generation also significantly increased for 5 60 min after exposure to Ag^＋ The number of BrdU-positive cells and the presence of PCNA in HaCaT cells increased 48 h after the addition of 10^-6 and 10^-5 mol/L Ag^＋, with 10^-5 mol/L Ag^＋ markedly increasing the cell proliferation index. These effects of sub-cytotoxic Ag^＋ concentrations were repressed by 5 mmol/L NAC. Our results suggest that sub-cytotoxic Ag^＋ concentrations promote the proliferation of human keratinocytes and might be associated with a moderate increase in intracellular ROS levels. This study provides important experimental evidence for developing novel silver-based wound agents or dressings with few or no cytotoxicity.

Establishment and Application of A Human Primary Keratinocyte Inflammation Model for Cosmetic Raw Material Screening

Using inducers to induce cells to produce inflammatory response is a common in vitro experimental method to study inflammation.However,there are relatively few inflammatory models developed for the cosmetic industry,and there are also great differences in the control of model induction,the selection of inflammatory indicators,and the concentration of inducers.Therefore,in this paper,by systematically studying the effects of Lipopolysaccharide(LPS)on the cell viability,the levels of IL-1α,IL-8 and ROS of human primary keratinocytes,a skin inflammation model based human primary keratinocyte was developed.The results showed that 0.01~100μg/mL LPS had no significant effect on the cell viability of human primary keratinocytes,while 100μg/mL LPS could simultaneously induce human primary keratinocytes to produce large amounts of IL-1αand IL-8,and 0.01μg/mL LPS could induce plentiful ROS.Therefore,a skin inflammation model for differential induction of different inflammatory indicators was established,and the sample OSM2021041301 was tested with this model,it was found that sample OSM2021041301 could significantly inhibit LPS-induced elevated IL-1αand IL-8 levels,the inhibitory effect on LPS-induced elevated ROS level was weak.The results indicated that OSM2021041301 has certain anti-inflammatory effect on inflammation caused by the increase of IL-1α,IL-8 and ROS induced by LPS and its analogues.

Development of an in Vitro Assay to Evaluate the Biological Impact of 5G Technology on Human Skin—Shield Effect of a Tannin-Rich Plant Extract

Background: The new 5G telecommunication technology has stirred concerns about potential negative effects on human health by radiofrequency electromagnetic fields. As to whether skin biology can be affected by 5G waves has remained an unsolved challenge despite recent studies dealing with this issue. In particular, a strategy for rational design of an assay allowing to 1) reproducibly evaluate and decipher the 5G effects on skin as well as 2) test the potential protective effects of cosmetic active ingredients, has yet to be found. Here we describe an in vitro model of human normal keratinocytes irradiated by 5G waves and show their impact on two biomarkers of inflammatory stress, i.e. interleukin-1β (IL-1β) and reactive oxygen species (ROS) production. In addition, the capacity of a tannin-rich plant extract to protect against 5G impact is evaluated. Materials and Methods: In the first series of experiments, monolayers of human normal keratinocytes were irradiated or not (control) by 5G waves (3.5 MHz) in an anechoic chamber and were incubated at 37˚C for 24 hours. At the end of the incubation period, extracellular IL-1β and intracellular ROS were quantified using specific ELISA and colorimetric assays, respectively. In the second series of experiments, the effect of an overnight pre-incubation with increasing concentrations of a tannin-rich plant extract was evaluated. Additionally, we studied in a prospective way the expression of a set of 88 genes selected for their relevance to keratinocyte homeostasis, in relation to the 5G challenge as well as the protective effect of a tannin-rich plant extract. Results: 5G waves significantly increased IL-1β production by 48.4% (p β and ROS production. Finally, the expression of 47 genes was modified by 5G waves and/or by the tannin-rich plant extract. Conclusion: This is to our knowledge the first evaluation of the impact of 5G technology on inflammatory biomarkers of human normal skin cells. Here we provide an innovative and pertinent tool to screen for natural compounds with protective effects against 5G waves to develop cosmetic products shielding against the potentially deleterious effects of electromagnetic waves on human skin.

Amphibian-derived peptide homodimer OA-GL17d promotes skin wound regeneration through the miR-663a/TGF-β1/Smad axis

Background:Amphibian-derived peptides exhibit considerable potential in the discovery and development of new therapeutic interventions for clinically challenging chronic skin wounds.MicroRNAs(miRNAs)are also considered promising targets for the development of effective therapies against skin wounds.However,further research in this field is anticipated.This study aims to identify and provide a new peptide drug candidate,as well as to explore the underlying miRNA mechanisms and possible miRNA drug target for skin wound healing.Methods:A combination of Edman degradation,mass spectrometry and cDNA cloning were adopted to determine the amino acid sequence of a peptide thatwas fractionated from the secretion of Odorrana andersonii frog skin using gel-filtration and reversed-phase high-performance liquid chromatography.The toxicity of the peptide was evaluated by Calcein-AM/propidium iodide(PI)double staining against human keratinocytes(HaCaT cells),hemolytic activity against mice blood cells and acute toxicity against mice.The stability of the peptide in plasma was also evaluated.The prohealing potency of the peptide was determined by MTS,scratch healing and a Transwell experiment against HaCaT cells,full-thickness injury wounds and scald wounds in the dorsal skin of mice.miRNA transcriptome sequencing analysis,enzyme-linked immunosorbent assay,real-time polymerase chain reaction and western blotting were performed to explore the molecular mechanisms.Results:A novel peptide homodimer(named OA-GL17d)that contains a disulfide bond between the 16th cysteine residue of the peptide monomer and the sequence‘GLFKWHPRCGEEQSMWT’was identified.Analysis showed that OA-GL17d exhibited no hemolytic activity or acute toxicity,but effectively promoted keratinocyte proliferation and migration and strongly stimulated the repair of full-thickness injury wounds and scald wounds in the dorsal skin of mice.Mechanistically,OA-GL17d decreased the level of miR-663a to increase the level of transforming growth factor-β1(TGF-β1)and activate the subsequent TGF-β1/Smad signaling pathway,thereby resulting in accelerated skin wound re-epithelialization and granular tissue formation.Conclusions:Our results suggest that OA-GL17d is a new peptide drug candidate for skin wound repair.This study emphasizes the importance of exogenous peptides as molecular probes for exploring competing endogenous RNA mechanisms and indicates that miR-663a may be an effective target for promoting skin repair.

Study on the effects of alternating capacitive electric fields with different frequencies on promoting wound healing

Some researches to facilitate wound healing by using electrical stimulation are based on electric current stimulation,which may cause secondary damage and the imbalance of the microenvironment in vivo.In this study,alternating capacitive electric field(ACEF)was applied via a self-designed system so as to avoid direct contact with cells and to maintain stable microenvironment for cell growth.The influences of 58 mV/mm ACEFs with various frequencies of 10,60 and 110 Hz on epidermal cells,fibroblasts and macrophages which involve in wound healing were comprehensively explored.The results suggested that ACEFs of 10,60 and 110 Hz all significantly promoted the proliferation of human dermal fibroblasts(HDFs)and human epidermal keratinocyte cell line(HaCaT)cells,and 60 Hz ACEF furtherly accelerated the migration of these two kinds of cells.Moreover,ACEFs of all different studied frequencies facilitated M2-type polarization of macrophages,and YAP/TAZ expression of macrophages were enhanced under the stimulations of 10 and 60 Hz ACEFs.The enhancements in cell activity,migration rate and M2-type polarizability indicated that 58 mV/mm ACEFs especially at 60 Hz possessing potentially affirmative applications for wound healing without the risks of secondary damage and microenvironment imbalance.