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Background: Streptococcus canis is a commensal bacterium in companion animals. This microorganism can infect humans who have been in deep contact with or bitten by pet dogs, suggesting that the skin/soft tissue is one of infection entry sites. To understand pathological process in human cells, we aimed to determine S. canis transcriptomic changes in invasive environments of human keratinocytes. Methods: We selected one isolate from candidates with whole-genome sequences, based on re-obtained cell invasion ability (CIA) data into human keratinocytes along with bacterial cytotoxicity. RNA-sequencing was conducted for the samples at baselines and 2 h/5 hr post-inoculation using NovaSeq 6000. Global/differential gene expression analyses [principal component analysis (PCA)/k-means clustering analysis/differentially expressed gene (DEG) analyses] were performed. We classified DEGs into their functional categories. To validate transcriptomic results, we did quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays. Results: FU1 isolate was selected from seven candidates, based on re-obtained CIA data with less cytotoxicity. Total read bases of 6.17–9.02 Gbp were obtained by RNA-sequencing. PCA and k-means clustering analysis indicated clustering according to their inoculation times. Volcano plots and Venn diagrams revealed that S. canis invasion into keratinocytes produced altered distributions of many genes. Gene ontology enrichment analysis showed most of the gene expressions were downregulated. DEG functional analysis showed the downregulated DEGs belonging to energy production and conversion/carbohydrate transport and metabolism/amino acid transport and metabolism/nucleotide transport and metabolism, with the upregulated DEGs belonging to transcription. qRT-PCR assays for downregulated/upregulated expressions of four genes (pgk–slo/opuAA–kdpB) validated transcriptomic results. Conclusion: Our observations suggest that S. canis can downregulate its metabolism-associated gene expressions in human keratinocyte environments. The observed gene expression changes can imply the latent infection in human cells. Further investigation is needed to elucidate the underlying mechanisms for the latent infection. [ABSTRACT FROM AUTHOR]

Jackfruit leaf protein hydrolysates obtained from the enzymatic hydrolysis of leaf protein concentrate with gastrointestinal enzymes have shown good techno-functional properties and high antioxidant capacity. However, molecular weight, antiproliferative activity, cytotoxicity and the ability to reduce reactive oxygen species (ROS) are still unknown. Therefore, this study aimed to evaluate the effect of jackfruit leaf protein hydrolysates obtained by enzymatic hydrolysis with pepsin and pancreatin at different hydrolysis times (30–240 min) on molecular weights, cytotoxicity, antiproliferation of cancer cells, and the reduction of reactive oxygen species in H2O2-induced HaCaT cells. The electrophoretic profile indicated that H-Pep contains peptides with molecular weights between 25 − 20 kDa. Meanwhile, H-Pan is composed of molecular weight products between 25 − 20 kDa and < 20 kDa. H-Pan and H-Pep (125–500 µg/mL) did not show significant cytotoxicity on HaCaT (human keratinocytes) and J774A.1 (murine macrophage cells). Antiproliferative activity was achieved in human cervical, ovarian, and liver cancer cells. H-Pan-240 min (1000 µg/mL) reduced the cell viability of cervical cancer cells by 23% while H-Pan-60 min significantly reduced cell viability of ovarian and liver cancer cells by 14.5 (500 µg/mL) and 17% (1000 µg/mL), respectively (P < 0.05). The protective effect against oxidative stress on H2O2-stressed HaCaT cells was obtained with H-Pep-60 min, which reduced 25% of ROS at 250 µg/mL (P < 0.05). The findings demonstrate the safe use of green biomass as a source of plant protein hydrolysates. [ABSTRACT FROM AUTHOR]

Objective To study the effect of cordycepin on proliferation, apoptosis and inflammatory reaction of human keratinocyte cell line HaCaT induced by TNF-α. Methods HaCaT cells were divided into control group, model group and intervention groups including low, middle, high dose (cordycepin 25 μmol/L, 50 μmol/L and 100 μmol/L, respectively). HaCaT cells were incubated with by 40 μg/L TNF-α in model group and cordycepin intervention group for 24 hours. Then, the cordycepin intervention groups were incubated with corresponding cordycepin for 24 hours. Cell proliferation was detected by CCK-8 method. Apoptosis was detected by flow cytometry. And mRNA expression of interleukin (IL)-8, IL-1β was detected by RT-qPCR. Results After 24 hours of cordycepin incubation, cell proliferation rate decreased (P＜0.05), cell apoptosis rate increased(P＜0.05)and mRNA expression of IL-8 and IL-1β decreased (P＜0.05). Conclusions Cordycepin can inhibit TNF-α-induced proliferation and inflammatory reaction of HaCaT cells and promote apoptosis of these cells.

This study aimed to explore the mechanism of naringin (Nar) in alleviating ultraviolet B (UVB)‐induced HaCaT cell senescence and damage. Human keratinocytes (HaCaT cells) were divided into control, UVB, UVB + Nar, UVB + Cap, and UVB + Nar + Cap groups. Analysis was performed using the MTT assay to assess cell viability, flow cytometry to measure the apoptosis level, SA‐β‐Gal staining to observe cellular senescence, and Western blot to assess protein levels of TRPV1, p16, p53, p21, matrix metalloproteinase (MMP)‐1, and MMP‐9. Both UVB irradiation and capsaicin (Cap) treatment upregulated the expression of TRPV1 in HaCaT cells, inhibited cell proliferation, promoted apoptosis, and increased the expression of p16, p53, p21, MMP‐1, and MMP‐9. Nar treatment reversed the above effects via inhibition of TRPV1 expression, thereby relieving senescence and cell damage induced by UVB irradiation. Taken together, these findings suggest that Nar can reduce UVB‐induced senescence and damage in HaCaT cells by acting as an antagonist of TRPV1. [ABSTRACT FROM AUTHOR]

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The Wnt/β-catenin signaling pathway plays important roles in the multi-phases of wound healing: homeostasis, inflammation, proliferative, and remodeling phases. However, there are no clinically available therapeutic agents targeting the Wnt/β-catenin pathway. In this study, we tested the effect of 5, 6-dichloroindirubin-3′-methoxime (KY19382), a small molecule that activates the Wnt/β-catenin pathway via interference with the function of the negative feedback regulator CXXC5, on cutaneous wound healing. KY19382 significantly enhanced cell migration of human keratinocytes and dermal fibroblasts with increased levels of β-catenin, phalloidin, Keratin 14, proliferating cell nuclear antigen (PCNA), Collagen I, and alpha-smooth muscle actin (α-SMA) by activating the Wnt/β-catenin signaling pathway without causing significant cytotoxicity. In addition, levels of Collagen I, Keratin 14, PCNA, and stem cell markers were significantly increased by KY19382 in a cutaneous murine wound healing model. Moreover, KY19382 treatment accelerated re-epithelialization and neo-epidermis formation with collagen deposition and stem cell activation at an early stage of cutaneous wound healing. Overall, KY19382 accelerates wound healing via activating the Wnt/β-catenin pathway, and may have the potential to be used for the development of a new wound healing agent. [ABSTRACT FROM AUTHOR]

The epidermis is one of the largest tissues in the human body, serving as a protective barrier. The basal layer of the epidermis, which consists of epithelial stem cells and transient amplifying progenitors, represents its proliferative compartment. As keratinocytes migrate from the basal layer to the skin surface, they exit the cell cycle and initiate terminal differentiation, ultimately generating the suprabasal epidermal layers. A deeper understanding of the molecular mechanisms and pathways driving keratinocytes' organization and regeneration is essential for successful therapeutic approaches. Single-cell techniques are valuable tools for studying molecular heterogeneity. The high-resolution characterization obtained with these technologies has identified disease-specific drivers and new therapeutic targets, further promoting the advancement of personalized therapies. This review summarizes the latest findings on the transcriptomic and epigenetic profiling of human epidermal cells, analyzed from human biopsy or after in vitro cultivation, focusing on physiological, wound healing, and inflammatory skin conditions. [ABSTRACT FROM AUTHOR]

An appropriate scaffold made of both synthetic and natural polymers can simultaneously supply desirable mechanical and biological characteristics such as tensile strength, elasticity, biocompatibility and bioactivity. Here, we investigated the synergistic effect of electrospun polyurethane (PU), gelatin and human amniotic polymers in the presence of mesenchymal stem cells (MSCs) and human keratinocyte (H-keratino) as cell sources for the regeneration of skin lesions. The in vitro biocompatibility was examined by the assay of (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and the epithelial differentiation was verified by real-time PCR after co-culture of MSCs and H-keratino on the proposed nanofibrous scaffold. A significant improvement in cell proliferation was found after cell culture on the electrospun scaffold containing the human amniotic membrane. In addition, gene expression after 14 days of co-culture process confirmed that both PU/gelatin and PU/gelatin-amnion electrospun scaffolds promote epithelial proliferation as well as dermal differentiation compared to H-keratino single-cell culture with regard to various markers such as Cytokeratin 10, Cytokeratin 14 and Involucrin. Also, the in vivo examination in mice indicated the considerable influence of the scaffold on the co-culturing of both cell types. Taking into account the results, the electrospun PU/gelatin-amnion nanofibrous scaffold in conjugation with the H-keratino/MSC co-culture could be considered as an exceptional nanofibrous substrate for applications in skin tissue engineering. [ABSTRACT FROM AUTHOR]

The activation of the Wnt/β-catenin signaling pathway plays a key role in the wound-healing process through tissue regeneration. The extract of Euodia daniellii Hemsl. (E. daniellii), a member of the Rutaceae family, activates the Wnt/β-catenin signaling pathway. However, the function of E. daniellii in wound healing has not yet been elucidated. We performed a migration assay to determine the wound-healing effect of E. daniellii extract in vitro using human keratinocytes and dermal fibroblast. In addition, a mouse acute wound model was used to investigate the cutaneous wound-healing effect of E. daniellii extract in vivo and confirm the potential mechanism. E. daniellii extract enhanced the migration of human keratinocytes and dermal fibroblasts via the activation of the Wnt/β-catenin pathway. Moreover, the E. daniellii extract increased the levels of keratin 14, PCNA, collagen I, and α-SMA, with nuclei accumulation of β-catenin in vitro. E. daniellii extract also efficiently accelerated re-epithelialization and stimulated wound healing in vivo. Furthermore, we confirmed that hesperidin, one of the components of E. daniellii, efficiently accelerated the migration of human keratinocytes and dermal fibroblasts, as well as wound healing in vivo via the activation of the Wnt/β-catenin pathway. Overall, E. daniellii extract and its active component, hesperidin, have potential to be used as therapeutic agents for wound healing. [ABSTRACT FROM AUTHOR]

Numerous epidemiological studies have reported that particulate matter 2.5 (PM2.5) causes skin aging and skin inflammation and impairs skin homeostasis. Hesperidin, a bioflavonoid that is abundant in citrus species, reportedly has anti-inflammatory properties. In this study, we evaluated the cytoprotective effect of hesperidin against PM2.5-mediated damage in a human skin cell line (HaCaT). Hesperidin reduced PM2.5-induced intracellular reactive oxygen species (ROS) generation and oxidative cellular/organelle damage. PM2.5 increased the proportion of acridine orange-positive cells, levels of autophagy-related proteins, beclin-1 and microtubule-associated protein light chain 3, and apoptosis-related proteins, B-cell lymphoma-2-associated X protein, cleaved caspase-3, and cleaved caspase-9. However, hesperidin ameliorated PM2.5-induced autophagy and apoptosis. PM2.5 promoted cellular apoptosis via mitogen-activated protein kinase (MAPK) activation by promoting the phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38. The MAPK inhibitors U0126, SP600125, and SB203580 along with hesperidin exerted a protective effect against PM2.5-induced cellular apoptosis. Furthermore, hesperidin restored PM2.5-mediated reduction in cell viability via Akt activation; this was also confirmed using LY294002 (a phosphoinositide 3-kinase inhibitor). Overall, hesperidin shows therapeutic potential against PM2.5-induced skin damage by mitigating excessive ROS accumulation, autophagy, and apoptosis. [ABSTRACT FROM AUTHOR]

The lipophilic yeast Malassezia furfur, is a member of the cutaneous commensal microbiota and is associated with several chronic diseases such as dandruff, pityriasis versicolor, folliculitis, and seborrheic dermatitis, that are often difficult to treat with current therapies. The development of alternatively effective antifungal therapies is therefore of paramount importance. In this study, we investigated the treatment effect of citral on M. furfur. The minimal inhibitory concentration of citral for M. furfur was 200 μg/mL, and the minimal fungicidal concentration was 300 μg/mL. Citral significantly increased the proportion of yeast cells to mycelial forms 2.6-fold. Phosphatidylserine externalization, DNA fragmentation, and metacaspase activation supported a citral-induced apoptosis in M. furfur. Moreover, citral at sub-minimum inhibitory concentrations reduced the invasion of M. furfur in HaCaT keratinocytes. Finally, we demonstrated that citral inhibited IL-6 and TLR-2 expression and enhanced HBD-2 and TSLP expression in M. furfur-infected HaCaT keratinocytes. These results showed that citral has antifungal activity at high concentrations and can decrease the infection of M. furfur by modulating the keratinocyte immune responses at low concentrations. Our results suggest that citral is a potential candidate for topical therapeutic application for M. furfur-associated human skin diseases. [ABSTRACT FROM AUTHOR]

Background and aim: The leaves of AnnonareticulataLinn (牛心果niú x ınguǒ; Bullock’s heart), a member of Annonaceae family, have been used extensively in folk medicine; however, its wound healing potential is yet to be explored. Our aim was to investigate the wound healing ability of A. reticulataleaf extract in vitro and in streptozotocin induced diabetic mice model. Material and methods: We observed the plant extract induced proliferation and migration of primary human dermal fibroblast (HDF), human skin fibroblast cell line (GM00637) and human keratinocyte cell line (HACAT). The expression of transforming growth factor beta (TGF-β), connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), alpha smooth muscle actin (α-SMA), matrix metalloproteinases (MMP-2, MMP-9), collagen-1, collagen-3, focal adhesion kinase (FAK) were evaluated by Western blot and gelatin zymography. Excisional diabetic wound model was used for in vivo wound healing assay. Furthermore, we processed wound tissue for histological and immunohistochemical study. Result: A. reticulata L. leaf extract stimulates proliferation and migration of HDF, skin fibroblast and keratinocyte significantly in a dose dependent manner; expression of TGF-β, CTGF, VEGF, α-SMA, MMP-2, MMP-9, collagen-1, collagen-3, FAK increased. Additionally, an enhanced expression of phospho-SMAD2, phospho-SMAD3 in the treated cells indicated the activation of TGF-β signal transduction pathway, similarly increased expression of phospho-AkT suggested activation of PI3/AkT pathway. Expression of CTGF and α-SMA was also increased significantly in wound tissue. Mass spectrometric analysis revealed that mainly two compounds to be present in the extract: quercetin and β-sitosterol. Conclusion: Collective data suggest that A.reticulata leaf extract may have a stimulatory effect in diabetic wound healing.

Eugenol is a phenolic compound isolated from clove essential oil. It is used in dentistry, fragrance, cosmetic, and food industries. According to the fragrance ingredient safety assessment report, eugenol does not cause phototoxic reactions and genotoxicity. However, its effect on UV-induced cytotoxicity and genotoxicity has not been well examined. Here in this study, cytotoxic and genotoxic effects of eugenol are investigated on UVA-induced damage using human keratinocyte cells (HaCaT). HaCaT cells were treated with increasing concentrations of eugenol (10-500 µM) for 1 hour and irradiated with 5-10-15 J/cm2 UVA. 24 hours later the neutral red uptake (NRU) assay was used to evaluate cytotoxicity. For genotoxicity assay cells were exposed to 1-10 µM eugenol for one hour and non-cytotoxic UVA irradiation doses (1, 2.5 J/cm2) were used. The alkaline comet assay was carried out immediately after the UVA irradiation to measure the genotoxic potential of eugenol. The cytotoxicity assay results indicate that eugenol caused a cytotoxic effect in a dose-dependent manner in HaCaT cells and increasing doses of UVA-irradiation enhanced the cytotoxic effect of eugenol. The alkaline comet assay results showed that eugenol causes DNA single-strand breaks and increasing doses of UVA-irradiation aggravates the genotoxic potential of eugenol. These data demonstrate that eugenol has cytotoxic and genotoxic potential and eugenol aggravates UVA-induced cytotoxic and genotoxic response in HaCaT human keratinocytes. [ABSTRACT FROM AUTHOR]

Numerous epidemiological studies have reported that particulate matter 2.5 (PM2.5) causes skin aging and skin inflammation and impairs skin homeostasis. Hesperidin, a bioflavonoid that is abundant in citrus species, reportedly has anti-inflammatory properties. In this study, we evaluated the cytoprotective effect of hesperidin against PM2.5-mediated damage in a human skin cell line (HaCaT). Hesperidin reduced PM2.5-induced intracellular reactive oxygen species (ROS) generation and oxidative cellular/organelle damage. PM2.5 increased the proportion of acridine orange-positive cells, levels of autophagy-related proteins, beclin-1 and microtubule-associated protein light chain 3, and apoptosis-related proteins, B-cell lymphoma-2-associated X protein, cleaved caspase-3, and cleaved caspase-9. However, hesperidin ameliorated PM2.5-induced autophagy and apoptosis. PM2.5 promoted cellular apoptosis via mitogen-activated protein kinase (MAPK) activation by promoting the phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38. The MAPK inhibitors U0126, SP600125, and SB203580 along with hesperidin exerted a protective effect against PM2.5-induced cellular apoptosis. Furthermore, hesperidin restored PM2.5-mediated reduction in cell viability via Akt activation; this was also confirmed using LY294002 (a phosphoinositide 3-kinase inhibitor). Overall, hesperidin shows therapeutic potential against PM2.5-induced skin damage by mitigating excessive ROS accumulation, autophagy, and apoptosis.

Skin is exposed to several harmful environmental effects including ultraviolet B (UVB) and air pollution, the most harmful component of which is particulate matter (PM). Damaging effects of UVB and PM include the generation of cellular reactive oxygen species (ROS), lipid peroxidation, protein carbonylation, DNA damage, and apoptosis. A compound with the potential to protect the skin against environmental oxidative damage is 3,4-dicaffeoylquinic acid (DQA), an antioxidant found in plant matter, including the coffee bean. In this study, we investigated the protective effects of DQA against UVB- and PM-induced oxidative cell damage in cultured human keratinocytes (HaCaT). We demonstrated that UVB, and PM with a diameter

Objective To construct the human pigment epithelium-derived factor (hPEDF) lentiviral vector, package lentivirus overexpressing PEDF and construct the human keratinocyte (HaCaT) cell line overexpressing PEDF. Methods The PEDF gene was inserted to lentiviral vector pLVX-IRES-puro to construct pLVX-IRES-puro-PEDF lentivirus recombinant plasmid. The pLVX-IRESpuro-PEDF lentivirus recombinant plasmid was transfected into 293T cells. The virus was packaged and amplificated. The virus supernatant was collected and the titer after amplification was authenticated. In the experimental group, HaCaT cells were infected with the constructed pLVX-IRES-puro-PEDF; whereas in the control group, HaCaT cells were infected with lentivirus containing empty vector. After amplification of 72 h, the HaCaT cells in both groups and conditioned medium were collected. The expression of PEDF mRNA in HaCaT cells of each group was detected by real-time PCR (RT-PCR) and the expression of PEDF protein was detected by western blot (WB). The PEDF protein secretion of HaCaT cells in both groups was detected by enzyme-linked immunosorbent assay (ELISA). Results It was confirmed that the recombinant lentivirus vector carrying PEDF gene was successfully constructed by using restriction enzyme digestion and Sanger sequencing. The titer of packaged lentivirus was up to 1×107 pfu/ml. The RT-PCR and WB results revealed that the expression of PEDF mRNA and protein in experimental group was significantly higher than that in the control group (P<0.05). Conclusion The recombinant lentiviral vector carrying PEDF gene was successfully constructed, and the HaCaT cell line infected with packaged lentivirus could over-express PEDF in vitro. [ABSTRACT FROM AUTHOR]

Background: In deep partial thickness dermal burns (DDB) where greater than 50% of the dermis is lost, severe pain, scarring and contractures occur. Therefore, skin grafting may be required. In children, scar contracture occurs because scarred skin does not stretch with growth creating the need for additional scar-releasing or skin-grafting surgeries. In order to resolve this problem, we used cryopreserved cultured epithelial allograft (cryopreserved allo-CEG), which can be grafted shortly after sustaining a wound. We reevaluated the promotion of early wound closure of burns and suppression of scarring by this treatment. Methods: Cryopreserved allo-CEGs were used to treat 50 cases of pediatric DDB from 1992 to 2000. These cases were reviewed with regard to the time until epithelialization, take percentage, and pain level. Also, in order to examine why cryopreserved allo-CEG promotes healing of burns and suppresses scarring, growth factors and cytokines in the cryopreserved allo-CEG were measured. Cryopreserved allo-CEG sheets were solubilized and concentrations of TGF-α, TGF-β1, IL-1α, IL-1β, PDGF-AA, VEGF, KGF, IL-6, b-FGF, as well as metalloprotease-1 (MMP-1) and HGF, which are noted to have scarring suppression effects, were measured before grafting. Results: Grafting of cryopreserved allo-CEGs in 50 cases of childhood DDB resulted in early epithelialization (9.32 ± 3.63 days on the average) and an almost 100% take rate. Also, pain relief (pain reduction or elimination, reduced need for anesthetics) was seen in all cases. Although 15–23 years have now elapsed, adverse events have not been observed. Cryopreserved allo-CEG contains IL-1α, IL-1β, PDGF-AA, TGF-α, TGF-β1, VEGF, and IL-6 have wound healing effects. The concentration of IL-1α was higher than the concentrations of other components, and this was followed by TGF-α, TGF-β1, b-FGF and VEGF. Although the concentration of MMP-1, which has a scarring suppression effect, was high, HGF was not detected. Conclusion: Cryopreserved allo-CEG contains growth factors that promote wound healing and factors that suppress scarring. Three effects, namely (1) early wound closure, (2) scarring suppression, and (3) pain relief were seen with grafts of cryopreserved allo-CEG in cases of childhood DDB. These observations show that cryopreserved allo-CEG is clinically useful and effective for the treatment of childhood DDB.

Hydrogels from bacterial, algal, and animal cells—bacterial cellulose (BC), alginate, and gelatin, respectively—were combined to fabricate a biocomposite film (BCAGG) via an eco-friendly casting technique. In addition, glycerol was added as a plasticizer to improve the elasticity and water absorption capacity of the film. In this study, BC pellicles were simply deconstructed into fibrils suspension and then reconstructed into films with a supplement of alginate, gelatin and glycerol. The physical appearance of fabricated films resembled native BC but possessed improved ductility, enhanced flexibility, higher water uptake ability and better biocompatibility. The film was found to resist tearing under suture pullout strength in a hydrated state. In vitro cytotoxicity tests showed that the film was cytocompatible. A cell study using a human keratinocyte culture demonstrated enhanced cell adhesion, spreading, and proliferation on the BCAGG film compared with BC/alginate film. The BCAGG film therefore has significant potential for use in biomedical applications, particularly in dermal treatment, skin tissue regeneration, and wound healing. [ABSTRACT FROM AUTHOR]

• Arsenite-induced oxidative stress increases WTAP and METTL14-catalyzed m6A level. • N -acetylcysteine inhibits oxidative stress and increased m6A level in arsenite exposure. • Bioinformatics analysis predicts m6A regulations in arsenite-induced oxidative stress. N6-methyladenosine (m6A) modification is affected by oxidative stress and gets involved in arsenite toxicity. However, whether oxidative stress is one factor in arsenite-induced alteration of m6A levels remains unclear. Here, reactive oxygen species (ROS), product of lipid peroxidation (MDA), antioxidants (GSH and SOD), m6A levels, m6A methyltransferases (METTL3, METTL14, and WTAP) and demethylases (FTO and ALKBH5) were detected in human keratinocytes exposed to different concentrations of arsenite. Antioxidant N -acetylcysteine was used to assess the influence of arsenite-induced oxidative stress on m6A modification. Possible regulations of m6A modification induced by arsenite were explored using bioinformatic analysis. Our results demonstrated that arsenite-induced oxidative stress increased the levels of m6A methylation possibly by mediating m6A methyltransferases and demethylases, especially elevated expressions of WTAP and METTL14, in human keratinocytes. Whereas N -acetylcysteine suppressed the elevated m6A level and its methyltransferases in human keratinocytes exposed to arsenite. Furthermore, arsenite-induced oxidative stress might mediate m6A methyltransferases and demethylases by reducing transcription of 4 genes (HECTD4 , ABCA5 , SLC22 A17 and KCNQ5) according to our bioinformatic analysis and experiments. Additionally, GO and Pathway analysis further suggested that the increase of m6A modification in arsenite-induced oxidative stress might be involved in some biological processes such as positive regulation of GTPase activity, apoptotic process, and platelet activation. Taken together, our study revealed the significant role of oxidative stress in m6A modification induced by arsenite. [ABSTRACT FROM AUTHOR]

The skin is the largest and a remarkably plastic organ that serves as a protective barrier against environmental stimuli and injuries throughout life. Skin injuries are serious health problems, and wound healing is a critical process to replace devitalized cellular and tissue structures. Although some endogenous opioids are known to be involved in the modulation of wound healing, it remains to be determined whether the β-neoendorphin (β-NEP), an endogenous opioid, has beneficial effects on wound repair in human keratinocyte. In this study, we found that β-NEP accelerated wound repair through activation of mitogen-activated protein kinase (MAPK)/Erk1/2 signaling pathways in human keratinocytes. Moreover, the wound healing effect of β-NEP is mainly through the acceleration of keratinocyte migration without affecting cell proliferation. Therefore, our studies reveal that β-NEP plays an important role in the regulation of wound repair and suggest a therapeutic strategy to promote wound healing using β-NEP.

We evaluated the cell invasion ability (CIA) of non-invasive Streptococcus dysgalactiae subsp. equisimilis using human keratinocytes and determined the association of CIA populations with their hosts and microbiological traits. Forty-two isolates from humans and companion animals were selected with host information. In addition to CIA, virulence-associated gene (VAG, spegg-ska-scpA-inlA-sicG-brpA-prtF1-prtF2-lmb-cbp-srtp1-srtp2) profiling, emm genotyping, multilocus sequence typing, and antimicrobial resistance (AMR) phenotyping/genotyping were performed. We designated CIA values higher than the mean of all isolates as high-frequency and those lower than the mean as low-frequency. Differences in the CIA between the different sources and Lancefield groups were assessed. We analyzed the association between high- and low-frequency CIA and VAG, emm genotype, sequence type/clonal complex, and AMR phenotype/genotype. Based on the mean (19.368 colony-forming units/100 cells) of 42 isolates, eight isolates had high-frequency CIA, whereas 34 had low-frequency CIA. We found an association between low-frequency CIA population and group G isolates, as well as a link between high-frequency CIA population and group C isolates. We also observed associations between low-frequency CIA population and oral/respiratory tract origin, ska, scpA, and lmb detection, and the AMR phenotype. Our observations suggest potential associations between high-/low-frequency CIA and the group, source, VAG, and AMR phenotypes.

Hepatocyte growth factor-like protein (HGFl) and its receptor, Recepteur d'Origine Nantais (RON), have been implicated in the development of wound chronicity. HGFl and RON expression was detected in acute wound tissue, chronic wound tissue and in normal skin using quantitative polymerase chain reaction (Q-PCR). HGFl and RON expression was also assessed in chronic healing and chronic non-healing wound tissues using Q-PCR and immunohistochemical staining. Expression was similarly detected in the HaCaT immortalized human keratinocyte cell line using reverse transcription polymerase chain reaction (RT-PCR). rhHGFl was used to assess the impact of this molecule on HaCaT cell functionality using in vitro growth assays and electric cell-substrate impendence sensing (ECIS) migration assays. HGFl and RON transcript expression were significantly increased in acute wound tissue compared to chronic wound tissue and were also elevated, though non-significantly, in comparison to normal skin. Minimal expression was seen in both healing and non-healing chronic wounds. Treatment of HaCaT cells with rhHGFl had no effect on growth rates but did enhance cell migration. This effect was abolished by the addition of a phospholipase C gamma (PLCγ) small molecule inhibitor. The increased expression of HGFl and RON in acute, healing wounds and the pro-migratory effect of HGFl in an in vitro human keratinocyte model, may indicate a role for HGFl in active wound healing.

Graphical abstract Exposure of UVB or PM 2.5 increased intracellular ROS and induced oxidative stress in HaCaT cells. DQA protects against UVB or PM 2.5 -indeced oxidative damage. In addition, it protects against UVB-induced apoptosis through inhibiting activation MAPKs. Highlights • DQA scavenged environmental UVB and PM 2.5 -induced intracellular ROS. • DQA treatment attenuated environmental oxidative stressed-skin cellular modifications and apoptotic cell death. • FFO may be developed as a functional food against environmental oxidative stress-induced skin damage. Abstract Skin is exposed to several harmful environmental effects including ultraviolet B (UVB) and air pollution, the most harmful component of which is particulate matter (PM). Damaging effects of UVB and PM include the generation of cellular reactive oxygen species (ROS), lipid peroxidation, protein carbonylation, DNA damage, and apoptosis. A compound with the potential to protect the skin against environmental oxidative damage is 3,4-dicaffeoylquinic acid (DQA), an antioxidant found in plant matter, including the coffee bean. In this study, we investigated the protective effects of DQA against UVB- and PM-induced oxidative cell damage in cultured human keratinocytes (HaCaT). We demonstrated that UVB, and PM with a diameter <2.5 µm (PM 2.5), induced cellular damage via oxidative stress, and this was mitigated by the antioxidative action of DQA. [ABSTRACT FROM AUTHOR]

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 10-5 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. [ABSTRACT FROM AUTHOR]

Grape extract rich in polyphenols (∼129 ± 32 mg of gallic acid equivalents per g of dry extract) was obtained from the pomaces of Cannonau grapes by homogenization in an ethanol/water mixture. The efficacy of ultrasounds in speeding up the extraction kinetics of polyphenols was demonstrated. The extract was incorporated in liposomes and PEVs (penetration enhancer containing vesicles) with Labrasol ® or Labrasol ® /ethanol. All the vesicles were spherical and predominantly unilamellar: liposomes were large (∼927 nm) and polydispersed (PI ∼0.56), while PEVs were small (∼140 nm) and fairly homogeneous (PI ∼0.3). Moreover, PEVs were able to incorporate a high amount of the extract (∼98% of the extract used for their preparation, 50 mg/ml). The formulations were highly cytocompatible and were able to promote the proliferation of keratinocytes and fibroblasts. In addition, thanks to their antioxidant activity, grape extract formulations provided a cytoprotective effect against oxidative stress damage. Therefore, an efficient, environmentally-friendly extraction strategy is proposed to obtain an extract with high phenolic content from waste grape pomaces, which was incorporated in vesicular systems to maximize the antioxidant power in a cell model of oxidative stress. [ABSTRACT FROM AUTHOR]

To investigate the biological effects of electronic cigarette (e-cigarette) and heated tobacco product extracts respect to tobacco smoke extract on human gingival fibroblasts and human oral keratinocytes analysing cell viability, morphology, migration, apoptosis, cell cycle and epithelial-mesenchymal transition (EMT). Human gingival fibroblasts and human oral keratinocytes viability was analysed by MTT assay, cell morphology using scanning electron microscope (SEM) and cell migration by Scratch assay, a method that mimics the cell migration during wound healing in vivo. Apoptosis and cell cycle were analysed with flow cytometry and the related-gene expression of TP53, BCL2, CDKN2A and CDKN1A was indagated using real-time polymerase chain reaction. EMT process was analysed through expression of specific markers: CDH1, SNAI2, TWIST1, MMP2, FN1 and VIM. All investigations were evaluated after 24 h an in vitro exposure. Undiluted tobacco smoke extract induced significant inhibition of cell viability and cell migration, caused morphological alterations and induced an increase in cell death. No alterations or damage were observed after treatment with e-cigarette extracts. Heated tobacco product extract induced proliferation as highlighted by an increase of cell viability, cell migration and alterations of cycle analysis. Comparing the different cigarette extracts, tobacco smoke turns out to be the most harmful, e-cigarette did not determine morphological and functional alterations and heated tobacco product must be carefully investigated for its possible clinical effects on oral cell populations. • The study compares biological profiles of tobacco smoke, e-cigarette, heated tobacco. • Tobacco smoke induced significant damage. • E-cigarette wasn't associated with significant morphological and functional changes. • E-cigarette emerged as less harmful smoking device to oral cell populations. • Heated tobacco product induced a complex pattern of functional changes. [ABSTRACT FROM AUTHOR]

Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid (ω-3 PUFA) that protects against photodamage and photocarcinogenesis in mammals. Aquaporin-3 (AQP3) is a water/glycerol transport protein that is found in basal layer keratinocytes. In this study, we have investigated the protective effect of EPA against ultraviolet B (UVB)-induced AQP3 downregulation in human keratinocytes. EPA treatment was found to increase AQP3 gene and protein expression in human epidermal keratinocytes (HaCaT). Using a specific inhibitor, we observed that the effect of EPA on AQP3 expression was mediated by extracellular signal-regulated kinase (ERK) activation. UVB radiation induced AQP3 downregulation in HaCaT cells, and it was found that EPA treatment attenuated UVB-induced AQP3 reduction and the associated cell death. UVB-induced downregulation of AQP3 was blocked by EPA and p38 inhibitor SB203580. Collectively, the present results show that EPA increased AQP3 expression and that this led to a reduction UVB-induced photodamage. [ABSTRACT FROM AUTHOR]

Desmosomes provide strong cell–cell adhesion which is crucial for the integrity of tissues such as the epidermis. However, nothing is known about the distribution and binding properties of desmosomal adhesion molecules on keratinocytes. Here we used atomic force microscopy (AFM) to simultaneously visualize the topography of living human keratinocytes and the distribution and binding properties of the desmosomal adhesion molecule desmoglein 3 (Dsg3). Using recombinant Dsg3 as sensor, binding events were detectable diffusely and in clusters on the cell surface and at areas of cell–cell contact. This was blocked by removing Ca 2 + and by addition of Dsg3-specific antibodies indicating homophilic Dsg3 binding. Binding forces of Dsg3 molecules were lower on the cell surface compared to areas of cell–cell contact. Our data for the first time directly demonstrate the occurrence of Dsg3 molecules outside of desmosomes and show that Dsg3 adhesive properties differ depending on their localization. From the Clinical Editor Using atomic force microscopy in the study of keratinocytes, this study directly demonstrates the occurrence of desmoglein 3 molecules outside of desmosomes and reveales that the adhesive properties of these molecules do differ depending on their localization. [ABSTRACT FROM AUTHOR]

Trans-resveratrol, a polyphenol extracted from Vitis vinifera, has different beneficial effects following its administration on the skin. Here the potential use of binary systems to enhance in vitro and in vivo activity of trans-resveratrol was investigated. Thus the aqueous solubility of trans-resveratrol was investigated in the presence of growing concentrations of polyethylene glycol (PEG) or β-cyclodextrin (βCD) as solubilizing excipients. Then, the solid dispersion of trans-resveratrol with PEG or inclusion complexes trans-resveratrol/βCD were prepared and characterised by different methods. Cytotoxicity and inhibition of reactive oxygen species (ROS) following H2O2 challenge in the presence of trans-resveratrol, alone or associated to the excipients, was evaluated on human keratinocyte HaCaT cell line. Both the trans-resveratrol-containing binary systems induced significant reduction of H2O2-induced ROS production, especially in the case of βCD that was selected for the following phase of the study. Thus, the effect of a cream containing trans-resveratrol, alone or associated to βCD, on different skin parameters such as corneometry, colorimetry and elastometry, was evaluated on human volunteers. All patients showed a visible improvement of clinical conditions with a remarkable decrease of aging signs, but this effect was higher of the hemi face treated with the βCD-containing formulation versus formulation containing trans-resveratrol alone. [ABSTRACT FROM AUTHOR]