In Vitro Evaluation of the Photoreactivity and Phototoxicity of Natural Polyphenol Antioxidants

Polyphenols are a large family of natural compounds widely used in cosmetic products due to their antioxidant and anti-inflammatory beneficial properties and their ability to prevent UV radiation-induced oxidative stress. Since these compounds present chromophores and are applied directly to the skin, they can react with sunlight and exert phototoxic effects. The available scientific information on the phototoxic potential of these natural compounds is scarce, and thus the aim of this study was to evaluate the photoreactivity and phototoxicity of five phenolic antioxidants with documented use in cosmetic products. A standard ROS assay was validated and applied to screen the photoreactivity of the natural phenolic antioxidants caffeic acid, ferulic acid, p-coumaric acid, 3,4-dihydroxyphenylacetic acid (DOPAC), and rutin. The phototoxicity potential was determined by using a human keratinocyte cell line (HaCaT), based on the 3T3 Neutral Red Uptake phototoxicity test. Although all studied phenolic antioxidants absorbed UV/Vis radiation in the range of 290 to 700 nm, only DOPAC was able to generate singlet oxygen. The generation of reactive oxygen species is an early-stage chemical reaction as part of the phototoxicity mechanism. Yet, none of the studied compounds decreased the viability of keratinocytes after irradiation, leading to the conclusion that they do not have phototoxic potential. The data obtained with this work suggests that these compounds are safe when incorporated in cosmetic products.

Phenolic Thiazoles with Antioxidant and Antiradical Activity. Synthesis, In Vitro Evaluation, Toxicity, Electrochemical Behavior, Quantum Studies and Antimicrobial Screening

Oxidative stress represents the underlying cause of many chronic diseases in human; therefore, the development of potent antioxidant compounds for preventing or treating such conditions is useful. Starting from the good antioxidant and antiradical properties identified for the previously reported Dihydroxy-Phenyl-Thiazol-Hydrazinium chloride (DPTH), we synthesized a congeneric series of phenolic thiazoles. The radical scavenging activity, and the antioxidant and chelation potential were assessed in vitro, a series of quantum descriptors were calculated, and the electrochemical behavior of the synthesized compounds was studied to evaluate the impact on the antioxidant and antiradical activities. In addition, their antibacterial and antifungal properties were evaluated against seven aerobic bacterial strains and a strain of C. albicans, and their cytotoxicity was assessed in vitro. Compounds 5a-b, 7a-b and 8a-b presented remarkable antioxidant and antiradical properties, and compounds 5a-b, 7a and 8a displayed good Cu+2 chelating activity. Compounds 7a and 8a were very active against P. aeruginosa ATCC 27853 compared to norfloxacin, and proved less cytotoxic than ascorbic acid against the human keratinocyte cell line (HaCaT cells, CLS-300493). Several phenolic compounds from the synthesized series presented excellent antioxidant activity and notable anti-Pseudomonas potential.

Effect of 1-MHz ultrasound on the proinflammatory interleukin-6 secretion in human keratinocytes

Keratinocytes, the main cell type of the skin, are one of the most exposed cells to environmental factors, providing a first defence barrier for the host and actively participating in immune response. In fact, keratinocytes express pattern recognition receptors that interact with pathogen associated molecular patterns and damage associated molecular patterns, leading to the production of cytokines and chemokines, including interleukin (IL)-6. Herein, we investigated whether mechanical energy transported by low intensity ultrasound (US) could generate a mechanical stress able to induce the release of inflammatory cytokine such IL-6 in the human keratinocyte cell line, HaCaT. The extensive clinical application of US in both diagnosis and therapy suggests the need to better understand the related biological effects. Our results point out that US promotes the overexpression and secretion of IL-6, associated with the activation of nuclear factor-κB (NF-κB). Furthermore, we observed a reduced cell viability dependent on exposure parameters together with alterations in membrane permeability, paving the way for further investigating the molecular mechanisms related to US exposure.

KCNQ1OT1 promotes the proliferation and migration of psoriatic keratinocytes by regulating miR-183-3p/GAB1

Background: Differentially expressed lncRNAs have been reported to be involved in keratinocyte proliferation and migration, and participate in the development of psoriasis. Potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (KCNQ1OT1) was implicated in the pathogenesis of various diseases, including cancer, sepsis, diabetic cardiomyopathy, and atherosclerosis. The influence of KCNQ1OT1 on proliferation and migration of psoriatic keratinocytes was unfolded in this study. Methods: Human keratinocyte cell line (HaCaT) was incubated with TNF-α to establish in vitro cell model of psoriasis. Cell viability and migration were assessed by MTT and wound healing, respectively. Target miRNA of KCNQ1OT1 was identified by luciferase activity and RNA immunoprecipitation (RIP) assays. Results: KCNQ1OT1 was up-regulated in TNF-α-induced HaCaT, and knockdown of KCNQ1OT1 reduced cell viability and suppressed migration of TNF-α-induced HaCaT. KCNQ1OT1 bind to miR-183-3p and negatively regulated expression of miR-183-3p. Over-expression of GAB1 (growth factor receptor binding 2-associated binding protein 1) counteracted with the suppressive effects of KCNQ1OT1 silence on cell viability and migration of TNF-α-induced HaCaT. Conclusion: Silence of KCNQ1OT1 suppressed proliferation and migration of TNF-α-induced HaCaT through regulation of miR-183-3p/GAB1, providing potential strategy for psoriasis.

Bolaamphiphilic Bis-Dehydropeptide Hydrogels as Potential Drug Release Systems

The self-assembly of nanometric structures from molecular building blocks is an effective way to make new functional materials for biological and technological applications. In this work, four symmetrical bolaamphiphiles based on dehydrodipeptides (phenylalanyldehydrophenylalanine and tyrosyldehydrophenylalanine) linked through phenyl or naphthyl linkers (terephthalic acid and 2,6-naphthalenedicarboxylic acid) were prepared, and their self-assembly properties were studied. The results showed that all compounds, with the exception of the bolaamphiphile of tyrosyldehydrophenylalanine and 2,6-naphthalene dicarboxylic acid, gave self-standing hydrogels with critical gelation concentrations of 0.3 wt % and 0.4 wt %, using a pH trigger. The self-assembly of these hydrogelators was investigated using STEM microscopy, which revealed a network of entangled fibers. According to rheology, the dehydrodipeptide bolaamphiphilic hydrogelators are viscoelastic materials with an elastic modulus G′ that falls in the range of native tissue (0.37 kPa brain–4.5 kPa cartilage). In viability and proliferation studies, it was found that these compounds were non-toxic toward the human keratinocyte cell line, HaCaT. In sustained release assays, we studied the effects of the charge present on model drug compounds on the rate of cargo release from the hydrogel networks. Methylene blue (MB), methyl orange (MO), and ciprofloxacin were chosen as cationic, anionic, and overall neutral cargo, respectively. These studies have shown that the hydrogels provide a sustained release of methyl orange and ciprofloxacin, while methylene blue is retained by the hydrogel network.

Identification of Differentially Expressed Genes and Elucidation of Pathophysiological Relevance of ABCA1 in HaCaT Cells Induced by PM2.5

Objective. In order to investigate the effects of PM2.5 on proliferation, cell cycle, apoptosis, and potential mechanism of human keratinocyte cell line HaCaT. Methods. HaCaT cells were treated with different concentrations of PM2.5 suspension for 24 hours. Cell viability was detected by the CCK-8 method. Cell cycle distribution and apoptosis were detected by flow cytometry. Microarray analyses were used to find out the microarray gene expression profiling; data processing included gene enrichment and pathway analysis. Western blot was conducted to validate the key pathways and regulators in the microarray analysis. Results. The cell activity decreased, and the cell cycle was significantly inhibited with the increase in PM2.5 concentration. Also, by conducting the gene expression microarray assay, we identified 541 upregulated genes and 935 downregulated genes in PM2.5-treated HaCaT cells. Real-time qPCR and western blot confirmed that PM2.5 treatment could induce the expression of ABCA1 while inhibiting that of END1 and CLDN1. Conclusion. Our results showed that PM2.5 could potentially regulate cell apoptosis and cell cycle arrest via ABCA1-, END1-, ID1-, and CLDN1-mediated pathways in human HaCaT cells, which laid a good foundation for follow-up drug intervention and drug development against skin damage caused by PM2.5 exposure.

Hemidesmosome-Related Keratin Filament Bundling and Nucleation

The epithelial cytoskeleton encompasses actin filaments, microtubules, and keratin intermediate filaments. They are interconnected and attached to the extracellular matrix via focal adhesions and hemidesmosomes. To study their interplay, we inhibited actin and tubulin polymerization in the human keratinocyte cell line HaCaT by latrunculin B and nocodazole, respectively. Using immunocytochemistry and time-lapse imaging of living cells, we found that inhibition of actin and tubulin polymerization alone or in combination induced keratin network re-organization albeit differently in each situation. Keratin filament network retraction towards the nucleus and formation of bundled and radial keratin filaments was most pronounced in latrunculin-B treated cells but less in doubly-treated cells and not detectable in the presence of nocodazole alone. Hemidesmosomal keratin filament anchorage was maintained in each instance, whereas focal adhesions were disassembled in the absence of actin filaments. Simultaneous inhibition of actin and tubulin polymerization, therefore, allowed us to dissect hemidesmosome-specific functions for keratin network properties. These included not only anchorage of keratin filament bundles but also nucleation of keratin filaments, which was also observed in migrating cells. The findings highlight the fundamental role of hemidesmosomal adhesion for keratin network formation and organization independent of other cytoskeletal filaments pointing to a unique mechanobiological function.

Citrus sudachi Peel Extract Suppresses Cell Proliferation and Promotes the Differentiation of Keratinocytes through Inhibition of the EGFR–ERK Signaling Pathway

Citrus sudachi is a well-known fruit in Tokushima Prefecture, Japan, and its peels are rich in phytochemicals, including phenolic compounds. Although it is expected that the extract of the C. sudachi peel elicits various beneficial physiological activities, the effect on the skin has not been investigated. In this study, we report that the aqueous extract from the peel of C. sudachi suppresses cell proliferation of the immortalized human keratinocyte cell line, HaCaT, and primary normal human epidermal keratinocytes. The extract of C. sudachi peel suppressed epidermal growth factor (EGF)-induced EGF receptor activation and tumor necrosis factor (TNF)-α-induced extracellular regulated kinase (ERK) 1/2 activation, which suggests that the extract exerts its inhibitory effect through inhibition of both the EGF receptor (EGFR) and its downstream molecules. Additionally, the extract of C. sudachi peel potentiated calcium-induced keratinocyte differentiation. These results suggest that the extract of C. sudachi peel may have beneficial effects against skin diseases that are characterized by hyperproliferation of epidermal keratinocytes, such as those seen in psoriasis and in cutaneous squamous cell carcinoma.