Inhibition of cyclin‐dependent kinase activity exacerbates H2O2‐induced DNA damage in Kindler syndrome keratinocytes

Kindler syndrome (KS) is an autosomal recessive skin disorder characterized by skin blistering and photosensitivity. KS is caused by loss of function mutations in FERMT1, which encodes Kindlin‐1. Kindlin‐1 is a FERM domain containing adaptor protein that is found predominantly at cell‐extracellular matrix adhesions where it binds to integrin β subunits and is required for efficient integrin activation. Using keratinocytes derived from a patient with KS, into which wild‐type Kindlin‐1 (Kin1WT) has been expressed, we show that Kindlin‐1 binds to cyclin‐dependent kinase (CDK)1 and CDK2. CDK1 and CDK2 are key regulators of cell cycle progression, however, cell cycle analysis showed only small differences between the KS and KS‐Kin1WT keratinocytes. In contrast, G2/M cell cycle arrest in response to oxidative stress induced by hydrogen peroxide (H₂O₂) was enhanced in KS keratinocytes but not KS‐Kin1WT cells, following inhibition of CDK activity. Furthermore, KS keratinocytes were more sensitive to DNA damage in response to H₂O₂ and this was exacerbated by treatment with the CDK inhibitor roscovitine. Thus, in Kindlin‐1 deficient keratinocytes, CDK activity can further regulate oxidative damage induced cell cycle arrest and DNA damage. This provides further insight into the key pathways that control sensitivity to oxidative stress in KS patients.     

17β‐estradiol protects human skin fibroblasts and keratinocytes against oxidative damage

Background Reactive oxygen species (ROS) cause severe damage to extracellular matrix and to molecular structure of DNA, proteins and lipids. Accumulation of these molecular changes apparently constitutes the basis of cell ageing. 17b‐estradiol (E2) has a key role in skin ageing homeostasis as evidenced by the accelerated decline in skin appearance seen in the perimenopausal years. Oestrogens improve many aspects of the skin such as skin thickness, vascularization, collagen content and quality. Despite these clinical evidences, the effects of oestrogens on skin at the cellular level need further clarification. Materials and Methods HaCaT and human fibroblasts were cultured under various conditions with E₂ and H₂O₂; then were subjected to immunofluorescence and western blot analysis. Lipoperoxidation was investigated using BODIPY. Results In human fibroblasts oxidative stress decreases procollagen‐I synthesis, while E₂ significantly increases it. Fibroblasts and HaCaT cells viability in the presence of E₂ demonstrates a notably increased resistance to H₂O₂ effects. Furthermore E₂ is able to counteract H₂O₂‐mediated lipoperoxidation and DNA oxidative damage in skin cells. Discussion In this study we highlight that the menopause‐associated oestrogens decline is involved in reduced collagen production and that E₂ could counteract the detrimental effects of oxidative stress on the dermal compartment during skin aging. Furthermore, our data show that physiological concentrations of oestrogens are able to interfere with ROS‐mediated cell viability reduction and to protect human skin cells against oxidative damage to cellular membranes and nucleic acids structure. Conclusion Our experimental data show that the presence of 17β‐estradiol may protect skin cells against oxidative damage and that the dramatic lowering of oestrogen levels during menopause, could render skin more susceptible to oxidative damage.     

Flavonolignans from Silybum marianum moderate UVA-induced oxidative damage to HaCaT keratinocytes

BACKGROUND: UV radiation from sunlight is a very potent environmental risk factor in the pathogenesis of skin cancer. Exposure to UV light, especially the UVA part, provokes the generation of reactive oxygen species (ROS), which induce oxidative stress in exposed cells. Topical application of antioxidants is a successful strategy for protecting the skin against UV-caused oxidative damage. OBJECTIVE: In this study, silybin (SB) and 2,3-dehydrosilybin (DS) (1-50 micromol/l), flavonolignan components of Silybum marianum, were tested for their ability to moderate UVA-induced damage. METHODS: Human keratinocytes HaCaT were used as an appropriate experimental in vitro model, to monitor the effects of SB and DS on cell viability, proliferation, intracellular ATP and GSH level, ROS generation, membrane lipid peroxidation, caspase-3 activation and DNA damage. RESULTS: Application of the flavonolignans (1-50 micromol/l) led to an increase in cell viability of irradiated (20 J/cm(2)) HaCaT keratinocytes. SB and DS also suppressed intracellular ATP and GSH depletion, ROS production and peroxidation of membrane lipids. UVA-induced caspases-3 activity/activation was suppressed by treatment with SB and DS. Lower concentrations of both compounds (10 micromol/l) significantly reduced cellular DNA single strand break formation. CONCLUSION: Taken together, the results suggest that these flavonolignans suppress UVA-caused oxidative stress and may be useful in the treatment of UVA-induced skin damage.     

Oxygen tension changes the rate of migration of human skin keratinocytes in an age-related manner

Migration of keratinocytes to re-epithelialize wounds is a key step in dermal wound healing. In aged human skin, wound healing rates decrease and cellular damage by reactive oxygen species (ROS) accumulates. The relationship between age, ROS and human skin keratinocyte migration is not clearly understood. In this study, 4% and 21% oxygen tensions were used to modify levels of ROS produced by metabolism to model low and high oxidative stress conditions. When migration of keratinocytes from young and old primary skin was compared using an in vitro scratch assay, old keratinocytes migrated faster in high oxygen tension than did young keratinocytes, whereas young keratinocytes migrated faster in low oxygen tension. Although all young and old cells at the scratch margins showed intense increases in dihydroethidium oxidation immediately after scratching, the old keratinocytes grown at 21% oxygen demonstrated a greater decrease in the DHE oxidation following scratching and migrated the fastest. These results show that old and young keratinocytes respond to oxygen tension differently and support the hypothesis that keratinocyte migration is affected by the capacity to remove ROS.     

Date seed oil inhibits Hydrogen peroxide‐induced oxidative stress in human epidermal keratinocytes

Background Oxidative stress has been implicated in various skin diseases through the generation of reactive oxygen species and the depletion of endogenous antioxidant systems. The administration of antioxidants is reportedly helpful, notably to enhance the healing process. To protect the skin against oxidative damages, we have studied the effect of new oil: “date seed oil” (DSO). This oil, may serve as a potential source of natural antioxidants such as phenols and tocopherols. Methods Here, we report the protective effect of DSO against hydrogen peroxide (H₂O₂)‐induced oxidative stress in terms of lipid peroxidation, depletion of endogenous antioxidant defense enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) using normal human epidermal keratinocytes (NHEK). Results In the investigated model system, DSO has significant chemoprotective effect, by inhibition of damage caused by H₂O₂ compared with cells without such addition endowing with a radical scavenging ability. Treatment of NHEK with DSO inhibited H₂O₂‐induced lipid peroxidation. In addition, this oil inhibited H₂O₂‐induced depletion of antioxidant defense components, such as SOD, CAT and GPx. Conclusions Our findings demonstrate that DSO is an efficient extract that is able to prevent keratinocytes oxidative damage induced by H₂O₂ exposure and may thus be a potential promising candidate, as a chemopreventive agent, in the development of keratinocytes‐related pathologies.     

Sesamol inhibits UVB-induced ROS generation and subsequent oxidative damage in cultured human skin dermal fibroblasts

The exposure of cells to ultraviolet B radiation (UVB) can induce the production of reactive oxygen species (ROS) which damage cellular components. Free radical scavengers and antioxidants can interfere with the production of ROS. We studied cytotoxicity, intracellular ROS levels, lipid peroxidation, antioxidant status and oxidative DNA damage in cultured human skin dermal fibroblast adult cells (HDFa) exposed to UVB in the presence of sesamol, a natural phenolic compound. The levels of cytotoxicity, intracellular ROS, lipid peroxidation, oxidative DNA damage and apoptotic morphological changes were significantly increased in UVB irradiated HDFa cells. We also observed that the activities of enzymatic antioxidants (superoxide dismutase, catalase and glutathione peroxidase) and the levels of non-enzymatic antioxidant status (GSH) were significantly decreased in UVB irradiated cells. On the other hand, sesamol pretreatment significantly decreased cytotoxicity, intracellular ROS, lipid peroxidation, oxidative DNA damage and apoptotic morphological changes in sesamol-pretreated and UVB-irradiated HDFa cells. We have also observed increased enzymatic and non-enzymatic antioxidants status in sesamol plus UVB-irradiated cells. Among the different doses tested, 80 μM of sesamol shows maximum protection for UVB-induced oxidative damage. In conclusion, UVB-induced ROS formation, cell fatality, lipid peroxidation, antioxidant depletion and oxidative DNA damage in HDFa cells is inhibited by sesamol, which, probably through its ROS scavenging activity.     

Phenolic‐enriched maple syrup extract protects human keratinocytes against hydrogen peroxide and methylglyoxal induced cytotoxicity

Reactive carbonyl species including methylglyoxal (MGO) are oxidation metabolites of glucose and precursors of advanced glycation end products (AGEs). They are important mediators of cellular oxidative stress and exacerbate skin complications. Published data supports that certain phenolic compounds can exert cellular protective effects by their antioxidant activity. A phenolic‐enriched maple syrup extract (MSX) was previously reported to show protective effects against AGEs‐ and MGO‐induced cytotoxicity in human colon cells but its skin protective effects remain unknown. The protective effects of MSX were evaluated against hydrogen peroxide (H₂O₂)‐ and MGO‐induced cytotoxicity in human keratinocytes (HaCaT cells). Cellular viability and antioxidant activity were evaluated by the luminescent cell viability CellTiter‐Glo assay and the reactive oxygen species (ROS) assay, respectively. A single‐cell gel electrophoresis (Comet assay) was used to measure the strand breaks in the DNA of HaCaT cells. MSX (at 50 μg/mL) ameliorated H₂O₂‐ and MGO‐induced cytotoxicity by increasing cell viability by 21.5% and 25.9%, respectively. MSX reduced H₂O₂‐ and MGO‐induced ROS production by 69.4% and 56.6%, respectively. MSX also reduced MGO‐induced DNA damage by 47.5%. MSX showed protective effects against H₂O₂‐ and MGO‐induced cytotoxicity in HaCaT cells supporting its potential for dermatological and/or cosmeceutical applications.     

Differential response of normal human epidermal keratinocytes and HaCaT cells to hydrogen peroxide-induced oxidative stress

Background. Normal human epidermal keratinocytes (NHEKs) and HaCaT cells are the most common models used to study the effects of various factors on skin cells. These cell lines share some common characteristics, but little is known about their differences in handling hydrogen peroxide (H₂O₂)‐induced oxidative stress. Aim. To investigate the differential response of NHEKs and HaCaT cells to H₂O₂‐induced oxidative stress. Methods. We examined differences in NHEKs and HaCaT cells after H₂O₂ treatment, assessing changes in cell viability; levels of intracellular reactive oxygen species (ROS), superoxide dismutase (SOD) and caspase‐3/7; percentage of cells arrested in G1 phase; number of senescence‐associated β‐galactosidase (SA‐β‐Gal)‐positive cells and; expression of senescence‐related protein Klotho. Results. The viability of NHEKs and HaCaT cells decreased in a concentration‐dependent and time‐dependent manner after exposure to H₂O₂. The inhibitory effect of 150 μmol/L H₂O₂ on cell viability was greater in HaCaT cells than in NHEKs (P < 0.05). Levels of ROS and caspase‐3/7, and the percentage of cells arrested in G1 phase, were higher in HaCaT cells than in NHEKs, whereas intracellular SOD was higher in NHEKs than in HaCaT cells after exposure to 150 μmol/L H₂O₂ (P < 0.05). SA‐β‐Gal positive cells increased significantly in NHEKs after treatment with H₂O₂ (P < 0.05). Klotho was significantly downregulated in both NHEKs and HaCaT cells after H₂O₂ treatment, but no SA‐β‐Gal‐positive HaCaT cells were seen, even after treatment with H₂O₂. Conclusions. Normal human epidermal keratinocytes are more resistant than HaCaT cells to H₂O₂‐induced oxidative stress. HaCaT cells have senescence phenotypes, but do not express β‐Gal.     

Ultraviolet-B-induced oxidative DNA base damage in primary normal human epidermal keratinocytes and inhibition by a hydroxyl radical scavenger

To evaluate the effects of ultraviolet-induced environmental trauma on human skin cells, primary normal human epidermal keratinocytes were exposed to ultraviolet-B radiation (290-320 nm). We found that relatively low doses of ultraviolet-B (62.5-500 mJ per cm2) caused dose-dependent increases in 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a biomarker of oxidative DNA damage. Unirradiated normal human epidermal keratinocytes contained 1.49 (+/- 0.11) 8-oxo-dG per 10(6) 2'-deoxyguanosine (dG) residues in cellular DNA, which increased linearly to as high as 6.24 (+/- 0.85) 8-oxo-dG per 10(6) dG after irradiation with 500 mJ per cm2. Further, this oxidative damage was reduced by 60.7% when the cells were pretreated with 1 mM mannitol. As hydrogen peroxide (H2O2) is known to be generated during oxidative stress, its accumulation in ultraviolet-B-irradiated normal human epidermal keratinocytes was also assessed and correlated to 8-oxo-dG formation. An ultraviolet-B-induced increase in H2O2 was observed in normal human epidermal keratinocytes and its production was inhibited by the addition of catalase. Based on the ability of a neutral molecule like H2O2 to permeate membranes, our data indicate that, after ultraviolet-B irradiation, H2O2 migrates from the cytosol to the nucleus where it participates in a Fenton-like reaction that results in the production of hydroxyl radicals (OH*), which may then cause 8-oxo-dG formation in cellular DNA. This conclusion is supported by our data showing that OH* scavengers, such as mannitol, are effective inhibitors of oxidative DNA base damage. Although increased levels of 8-oxo-dG were previously found in immortalized mouse keratinocytes exposed to ultraviolet-B radiation, we now report the induction of 8-oxo-dG in normal human skin keratinocytes at ultraviolet-B doses relevant to human skin exposure.     

Attenuation of UVA-induced damage to human keratinocytes by silymarin

BACKGROUND: UV radiation from sunlight is a potent environmental risk factor in skin cancer pathogenesis. UVA is the major portion of UV light reaching the earth surface ( approximately 95%) and it is reported to lead to benign and malignant tumor formation. UVA-mediated cellular damage occurs primarily through the release of reactive oxygen species (ROS) and it is responsible for inflammation, immunosuppression, photoaging and photocarcinogenesis. OBJECTIVE: The aim of our study was to investigate the potency of silymarin, the polyphenol fraction from the seeds of Silybum marianum, to modulate UVA-induced oxidative damage to human keratinocytes. METHODS: Skin epidermal cell line HaCaT, extensively used for studying the influence of UV radiation, was chosen as an experimental model. Silymarin's effect on UVA-disrupted cell viability, proliferation, mitochondrial function, and intracellular ATP and GSH level was measured. Furthermore, silymarin's potency to reduce UVA-induced ROS generation, membrane lipid peroxidation, caspase-3 activation and DNA damage was monitored. RESULTS: Treatment of irradiated HaCaT (20 J/cm(2)) with silymarin (0.7-34 mg/l; 4h) resulted in concentration-dependent diminution of UVA-caused oxidative stress on all studied parameters. Silymarin application extensively reduced GSH depletion and ROS production as well as lipid peroxidation in irradiated cells. Formation of UVA-induced DNA single strand breaks and caspase-3 activity was also significantly decreased by silymarin. CONCLUSION: The results suggest that silymarin may be beneficial in the treatment of UVA-induced skin oxidative injury and inflammation. However, further studies especially whose using human systems are needed to determine efficacy of silymarin in vivo.     

Role of phagocyte oxidase in UVA-induced oxidative stress and apoptosis in keratinocytes

Chronic exposure to ultraviolet radiation including ultraviolet A (315-400 nm) (UVA) may cause photocarcinogenesis and photoaging. The UVA-induced production of reactive oxygen species (ROS) and the resultant oxidative stress exposure play an important role in these biological processes. Here we have investigated the role of phagocyte oxidase (PHOX, gp91phox) in the production of ROS, redox status change, and apoptosis after UVA exposure by using gp91phox-deficient (gp91phox-/-) primary keratinocytes. UVA radiation resulted in increased ROS production and oxidation of reduced glutathione (GSH) to its oxidized form (GSSG). The presence of diphenylene iodonium (DPI) inhibited ROS production by UVA. In comparison with wild-type cells, gp91phox-/- cells produced slightly less ROS and GSH oxidation. UVA radiation induced apoptosis in wild-type keratinocytes as detected by phosphatidylserine (PS) translocation, caspase activation, and DNA fragmentation. As compared with wild-type cells, UVA induced less PS translocation in gp91phox-deficient cells. No difference, however, was observed in caspase activation and DNA fragmentation after UVA exposure in wild-type and gp91phox-/- cells. These findings suggest that gp91phox plays a limited role in the UVA-induced ROS production, oxidative stress, and therefore the PS translocation, but has no effect on UVA-induced caspase activation and DNA fragmentation during apoptosis.     

Voriconazole enhances UV-induced DNA damage by inhibiting catalase and promoting oxidative stress

Cutaneous squamous cell carcinoma (cSCC) is the second most common form of skin cancer and is associated with cumulative UV exposure. Studies have shown that prolonged voriconazole use promotes cSCC formation; however, the biological mechanisms responsible for the increased incidence remain unclear. Here, we show that voriconazole directly increases oxidative stress in human keratinocytes and promotes UV-induced DNA damage as determined by comet assay, 8-oxoguanine immunofluorescence and mass spectrometry. Voriconazole treatment of human keratinocytes potentiates UV-induced apoptosis and activation of the p38 MAP kinase and 53BP1 UV stress response pathways. The p38 MAP kinase activation promoted by voriconazole exposure can be mitigated by pretreating keratinocytes with N-acetylcysteine. Voriconazole increases oxidative stress in keratinocytes by directly inhibiting catalase leading to lower intracellular NADPH levels and the triazole moieties in voriconazole are critical for inhibiting catalase. Furthermore, voriconazole is shown to promote UV-induced dysplasia in an in vivo model. Together, these data demonstrate that voriconazole potentiates oxidative stress in UV-irradiated keratinocytes through catalase inhibition. Use of antioxidants may mitigate the pro-oncogenic effects of voriconazole.     

Impact of reactive oxygen species on keratinocyte signaling pathways

Human epidermal keratinocytes are located on the body surface, which is a specialized location for component cells of human skin tissue. Those cells are always exposed to external stimuli, which constantly generate reactive oxygen species (ROS) in the cells. Regulation of the redox state is a key for maintaining intracellular homeostasis. Originally, each cell type has defensive systems against oxidative stress, thus keratinocytes may have a unique system for regulating ROS levels. Intracellular signaling appropriately reacts to changes of ROS in cooperation with intra- and extra-cellular antioxidant agents, and is sometimes affected by excessive ROS generated by various stresses. We review in this paper the impact of ROS on keratinocytes based on published data and focus on related signaling pathways involved in inflammation and oncogenesis.     

IgE‐independent atopic dermatitis is associated with a β‐2 adrenergic receptor gene polymorphism

IgE levels are not elevated in about 20% of patients with atopic dermatitis (AD). In this intrinsic AD (IAD), allergic mechanisms are not very important and pathogeny could be mainly neurogenic. β2-adrenergic receptors are localized on cells involved in AD: Langerhans' cells, keratinocytes and lymphocytes. We wondered whether IAD could be associated with gene polymorphisms 16 and 27 of this receptor. We studied 98 healthy subjects and 83 subjects suffering from DA (UKWP criteria). IgE levels were normal in 12 of them and elevated in 71 (EAD). After DNA extraction, the genotyping was done by PCR and Direct Sequencing of candidate gene. Statistical analysis was performed with EPI-INFO 6.04 for χ² test. We found a significant association of Gln27Glu polymorphism with IAD ( P  = 0.00071 and χ² = 14.51). There was no difference between healthy subjects and EAD patients. Adrenergic receptor agonists are known to attenuate the proliferative response of human lymphocytes after activation, through the inhibition of interleukin-2 release. It is known that catecholamines inhibit the antigen-presenting capability of epidermal Langerhans' cells. Long-term agonist-promoted downregulation of receptor number is absent when glu is at position 27. We suggest that the suppression of inhibiting effects of catecholamines could be involved in IAD pathogeny. Dichotomic nature of AD (EAD and IAD) is also associated with polymorphisms (SNP) of the interleukin-4/interleukin-13 receptor gene and the differences of cutaneous variables (transepidermal water loss, capacitance and pH). Altogether, these findings indicate that IAD patients exhibit phenotypic and also genotypic features which differ from those patients with EAD. Otherwise, the presence of this polymorphism could provide an explication of rarity of hypertension with AD, because Glu27Gln has been identified as a susceptibility polymorphism for HTA.     

Astragaloside IV attenuates high glucose-induced human keratinocytes injury via TGF-β/Smad signaling pathway

ObjectivesIn this study, we have investigated the effect of Astragaloside IV on keratinocytes’ proliferation, migration, oxidative stress, apoptosis, inflammation, and relevant signaling pathway, using human keratinocytes exposed to high glucose.BackgroundAstragaloside IV is one of the main active ingredients of Astragalus membranaceus (Fisch.) Bunge. Previous studies have found that Astragaloside IV exerts positive effects in various disease models and promotes wound healing.MethodsCell proliferation and migration of keratinocytes, oxidative stress indicators, cell apoptosis rate, inflammatory factors, and key proteins in the TGF-β/Smad signaling pathway were evaluated by molecular biology/biochemical techniques, fluorescence microscope, and flow cytometry.ResultsHigh glucose inhibited the cell proliferation and migration of keratinocytes, upregulated the levels of MDA, ROS, IL-6, IL-8, and Smad7, and decreased the levels of SOD, IL-10, TGF-β1, p-Smad2, and p-Smad3. Astragaloside IV attenuated the dysfunction of keratinocytes, oxidative stress, cell apoptosis, and inflammation, but activated TGF-β/Smad signaling pathway. Meanwhile, the addition of SB431542 (the inhibitor of TGF-β/Smad signaling pathway) eliminated the impact of Astragaloside IV on high glucose-induced keratinocytes.ConclusionsThese results strongly suggest that Astragaloside IV may be a potential drug candidate for accelerating diabetic wound healing, by protecting keratinocytes against damages induced by high glucose and TGF-β/Smad pathway is involved in this process at the cellular level.     

Carboxyfullerenes protect human keratinocytes from ultraviolet-B-induced apoptosis

Carboxyfullerene, a water-soluble carboxylic acid derivative of a fullerene, which acts as a free-radical scavenger, was investigated as a protective agent against ultraviolet-light-induced damage in human keratinocytes. First, we demonstrate that carboxyfullerene is not cytotoxic for these cells. In addition, this compound significantly reduces the ultraviolet-B-induced inhibition of keratinocyte proliferation and protects keratinocytes from apoptosis caused by ultraviolet B irradiation in a time- and dose-dependent fashion. Furthermore, the percentage of cells with depolarized mitochondria is significantly lower in ultraviolet-B-irradiated keratinocytes pretreated with carboxyfullerene than in cells provided with diluent alone. Carboxyfullerene also protects human keratinocytes from apoptosis induced by exposure to deoxy-D-ribose, a sugar that causes cell death through a pathway involving oxidative stress. On the other hand, ultraviolet B downregulates bcl-2 levels in human keratinocytes, and carboxyfullerene fails to prevent this effect. These results suggest that carboxy- fullerene protects human keratinocytes from ultraviolet B damage possibly via a mechanism interfering with the generation of reactive oxygen species from depolarized mitochondria without the involvement of bcl-2.     

Human keratinocyte radiosensitivity is linked to redox modulation

BACKGROUND: Ionising radiation-induced reactive oxygen species (ROS) overproduction induces keratinocyte alterations and constitutes one of the most common effects after therapeutic gamma-irradiation. ROS production is controlled by a complex enzymatic system. OBJECTIVE: The aim of our study is to analyse the role of radiation-induced oxidative stress in keratinocytes death by apoptosis. We hypothesized that keratinocyte capacity to hamper radiation-induced ROS generation may control their radiosensitivity. METHODS: For this purpose, an original human skin explant model was developed and two types of human epidermal cells were used: primary keratinocytes NHEK and spontaneous non-tumourigenic cell line HaCaT. RESULTS: cDNA-arrays analysis was performed 24h after a 20Gy gamma-radiation and revealed down-regulation of genes involved in oxidative stress control and the apoptosis process. This was confirmed by alterations in catalase, GPx and SOD enzymatic activities. This redox modulation was concomitant to the down-regulation of anti-apoptotic genes and up-regulation of some pro-apoptotic genes (caspase 10, ubiquitin C). Interestingly TUNEL labelling revealed an increase in the number of apoptotic cells. We also demonstrated a differential inducibility of the cell antioxidant network in two keratinocyte lines, which results in a differential cellular level of ROS, explaining their different radiosensitivities. CONCLUSION: Keratinocytes apoptosis is partly dependent on ROS production after exposure to gamma-rays. In addition, the differential radiosensitivity of keratinocytes is linked to different oxidative stress responses.     

N-乙酰半胱氨酸对PM2.5引起HaCaT细胞损伤的保护作用

目的：研究N-乙酰半胱氨酸（N-Acetyl-L-cysteine,NAC）对PM2.5诱导的HaCaT细胞损伤的影响。方法：HaCaT细胞分为NAC干预组、不同浓度PM2.5组(25、50、100、200、400 μg/mL)及空白对照组。 DCFH-DA荧光探针法检测HaCaT细胞内活性氧（ROS）的水平,RT-PCR和ELISA法分别检测IL-1β、IL-33、TSLP mRNA表达及蛋白水平。CCK-8法和Western blot法分别检测细胞存活率和NF-κB信号通路相关蛋白表达水平。结果：不同浓度PM2.5组HaCaT细胞ROS水平均显著高于对照组(P<0.05);200 μg/mL和400 μg/mL PM2.5处理组细胞中IL-1β、TSLP、IL-33 mRNA表达水平显著高于对照组(P<0.05)。与PM2.5组比较,NAC干预组HaCaT细胞存活率增高,ROS水平降低,NF-κB p-p65蛋白表达降低,IL-1β、TSLP、IL-33分泌水平降低,差异有统计学意义(P＜0.05)。结论：NAC对PM2.5诱导的HaCaT细胞损伤具有保护作用,其机制可能与其降低细胞内氧化应激水平有关。     

Oxytocin modulates proliferation and stress responses of human skin cells: implications for atopic dermatitis

The neuropeptide hormone oxytocin (OXT) mediates a wide spectrum of tissue‐specific actions, ranging from cell growth, cell differentiation, sodium excretion to stress responses, reproduction and complex social behaviour. Recently, OXT expression was detected in keratinocytes, but expression of its receptor and function are still unexplored in human skin. Here, we showed that both OXT and its receptor are expressed in primary human dermal fibroblasts and keratinocytes. OXT‐induced dose‐dependent calcium fluxes in both cell types demonstrating that the OXT receptor (OXTR) is functionally expressed. We also showed that OXT decreases proliferation of dermal fibroblasts and keratinocytes in a dose‐dependent manner. In order to further investigate OXT‐mediated functions in skin cells, we performed OXTR knockdown experiments. OXTR knockdown in dermal fibroblasts and keratinocytes led to elevated levels of reactive oxygen species and reduced levels of glutathione (GSH). Moreover, OXTR‐depleted keratinocytes exhibited an increased release of the pro‐inflammatory cytokines IL6, CCL5 and CXCL10. Our data indicate that the OXT system modulates key processes which are dysregulated in atopic dermatitis (AD) such as proliferation, inflammation and oxidative stress responses. Furthermore, we detected a downregulation of the OXT system in peri‐lesional and lesional atopic skin. Taken together, these data suggest that the OXT system is a novel neuroendocrine mediator in human skin homoeostasis and clinically relevant to stressed skin conditions like AD.