Polypeptide from Chlamys farreri inhibits UVB-induced apoptosis of HaCaT cells via iNOS/NO and HSP90

Polypeptide from Chlamys farreri(PCF) is a novel marine bioactive product that was isolated from the gonochoric Chinese scallop Chlamys farreri,and was found to be an effective antioxidant in our recent studies.In this study,we investigated the effect of PCF on ultraviolet B(UVB)-induced apoptosis of HaCaT cells and the intracellular signaling pathways involved.Pretreatment with the inducible nitric oxide synthase(iNOS) inhibitor S-methylisothiourea sulfate inhibited UVB-induced apoptosis,indicating that iNOS and NO play important roles in apoptosis.On the other hand,the inhibition of UVB-induced apoptosis in the immortalized keratinocyte(HaCaT) cells by PCF was estimated using a DNA ladder.PCF treatment inhibited UVB-induced iNOS activation,as determined by RT-PCR,NO production,as determined by ESR,and up-regulated heat shock protein(HSP) 90 activation,as determined by Western blotting.Our results indicate that iNOS and NO are involved in UVB-induced apoptosis of HaCaT cells and the protective effect of PCF against UVB irradiation is exerted by suppressing the expression of iNOS,followed by inhibition of NO release and enhanced activation of HSP90.

UVB suppresses PTEN expression by upregulating miR-141 in HaCaT cells

MicroRNAs （miRNAs） are 21 to 24 nucleotide, non-coding RNA molecules that post-transcriptionally regulate the expression of target genes. Ultraviolet B （UVB） radiation has been shown to inhibit phosphatase and tensin homolog deleted on chromosome 10 （PTEN） expression in HaCaT cells through an unknown mechanism. In this study, we investigated whether miR-141 can regulate UVB exposure-mediated inhibition of PTEN expression. Real-time RT-PCR, annexin V/fluorescein isothiocyanate staining, Western blotting and anti-miRNA oligonucleotide transfection were employed in this study. We found that upregulation of miR-141 expression after UVB irradiation was inversely correlated with PTEN expression levels in HaCaT cells. Furthermore, miR-141 expression increased apoptosis, while anti-miR-141 partly restored PTEN expression and reversed the pro-apoptosis effect of UVB. UVB suppresses the expression of PTEN by upregulating miR-141 in HaCaT cells. Therefore, miR-141 is a potential gene therapy target for UVB-induced photodamage.

Effects of Oxymatrine on the NF-kappa B expression of HaCaT cells

Objective： To study the effect of oxymatrine on the expression of nuclear factor-kappa B（NF-K B） of Human benign epidermal keratinocytes line（HaCaT cells）. Methods：HaCaT cells were cultured with different concentration of Oxymatrine（10 μ g/ml, 50 μ g/ ml and 100 μg/ml） for 24 h, then 10.5 mol/L Substance P was added to the cells. After 30 min, NF-K B expression in the cells was observed by immunocytochemistry, NF-K B P65 protein expression was evaluated by Western blot, and the mRNA expression of NF-K B P65 was evaluated by reverse transcription polymerase chain reaction（RT-PCR）. The 10.5 mol/L Substance P and culture medium were added to the Substance P group and control group, respectively. Results：In control group, expression rate of positive cells, the expressions of protein and mRNA of NF-K B were all low. In Substance P group, when 10μ mol/L Substance P was added, the expressions were all increased（P 〈 0.05）. But in Oxymatrine groups, the expression rate of positive cells, the expressions of protein and mRNA were all descended in a concentration-dependent manner（P 〈 0.05 or P 〈 0.01）. Conclusion：Oxymatrine can down-regulate the expression of NF-K B of the HaCaT cells and may play an important role in regulating anti-inflammation and immunity.

Effects of different doses of acitretin on FGF10 mRNA transcription and its protein translation in HaCaT cells

Objective： To observe the effects of different doses of acitretin on the transcription of FGF10 mRNA and the translation of FGF10 protein in cultured HaCaT cells. Methods： HaCaT cells were treated with different doses of acitretin for 48 h, then the changes on the transcription of FGF10 mRNA and the translation of FGF10 protein in these cells were detected by immunofluorescence and in situ hybridization assay. Results： Compared with the control group, the transcription of FGF10 mRNA and the translation of FGF10 protein were gradually decreased along with the increasing dose of aeitretin. There were significant differences between different groups （P〈0.01）. Conclusion： Aeitretin could inhibit the transcription of FGF10 mRNA and the translation of FGF10 protein in HaCaT cells. With the dose of aeitretin increased, the stains of both FGF10 mRNA and FGF10 protein in HaCaT cells are reduced.

Effect of Red <i>Panax ginseng</i>on Mitochondrial Dynamics and Bioenergetics in HaCaT Cells Exposed to Urban Pollutants

Background: Urban air pollution contributes to lung and cardiovascular system dysfunction, making it a major concern for human health. Its impact on skin integrity, associated with increased occurrence of atopic dermatitis, is now recognized, but its cellular mechanisms remain poorly understood. Objective: In the present study we aimed at establishing the impact of urban pollutant on mitochondrial dynamics and bioenergetics using the HaCaT cell model. We also sought to establish the protective effect of ECH-5195 (red Panax ginseng extract), standardized in ginsenosides, in reversing pollution-induced mitochondrial defects. Methods: Urban pollution exposure was mimicked by 1 h exposure of HaCaT cells with standardized atmospheric particulate matter containing PAHs, nitro-PAHs, PCB congeners, and chlorinated pesticides with a mean particulate diameter of 5.85 μm (SRM1648). Results: The presence of urban pollutant in the cultures increased the prevalence of hyperfission by 1.41-fold (p = 0.023) and fission by 1.35 fold (p = 0.006) in the reticular mitochondrial network. ECH-5195 reduced both pollution-induced hyperfission by 0.54-fold (p = 0.004) and fission by 0.68-fold (p = 0.0006) normalizing the mitochondrial reticular network. Pollution exposure was associated with a significant reduction of basal OCR and increased lactate production, pushing the cell to rely on glycolysis for ATP production. When ECH-5195 was used, OCR was significantly increased, and the glycolytic contribution to ATP production was reduced while both oxidative phosphorylation and mitochondrial respiration were increased demonstrating mitochondrial re-engagement in ATP production. Conclusions: Pollution exposure was disruptive for both the mitochondrial network dynamics and mitochondrial respiration. Ginsenosides in ECH-5195 efficiently protected both from pollution-induced defects.

Protective Effects of Jin Bai Mei Yan Prescription on Oxidative Damage and Photoaging Induced by Ultraviolet B in HaCaT Cells

Objective Ultraviolet B(UVB)mainly acts on the skin epidermis,causing oxidative damage and apoptosis of keratinocytes.Jin Bai Mei Yan Prescription(JBMYP)comprises a variety of antioxidant traditional Chinese medicines(TCM).In this study,we aimed to evaluate the effects of JBMYP on the oxidative damage induced by UVB in human immortalized epidermal keratinocytes(HaCaT)cells.Methods HaCaT cells were divided into six groups:control group,model(UVB)group,positive(UVB+vitamin E)group,UVB+JBMYP low dose group(160μg/mL),UVB+JBMYP moderate dose group(800μg/mL),and UVB+JBMYP high dose group(1600μg/mL).HaCaT cells were irradiated with UVB and treated with JBMYP for 24 h.Methyl thiazolyl tetrazolium(MTT)assay and real-time unlabeled cell function analyzer were used to assess the cell survival and proliferation rates,respectively.At the same time,the levels of intracellular reactive oxygen species(ROS),lipid peroxide malondialdehyde(MDA),glutathione(GSH),and hydroxyproline(HYP),as well as the activities of antioxidant enzyme superoxide dismutase(SOD)and catalase(CAT)were evaluated using enzyme linked immunosorbent assay(ELISA).Results Compared with the model group,the survival rate of HaCaT cells in each dosage group of JBMYP was significantly improved(P<0.05).Further,JBMYP could promote the proliferation of HaCaT cells,leading to a reduction in the contents of MDA and ROS,and increase in the contents of SOD,CAT,GSH and HYP in HaCaT cells.Conclusions JBMYP has enhanced protective effect on oxidative damage induced by UVB in HaCaT cells.

Effects of Combination of 1,25(OH)_(2)D_(3) and TLR-4 Inhibitor on the Damage to HaCaT Cells Caused by UVB Irradiation

Objective Vitamin D and Toll-like receptor-4(TLR-4)inhibition are involved in the protection of keratinocytes.The effects of combination of 1,25(OH)_(2)D_(3) and TLR-4 inhibitor on the protection of keratinocytes against ultraviolet radiation B(UVB)irradiation remain unclear.This study was undertaken to explore the effects of combination of 1,25(OH)_(2)D_(3) and TAK-242(TLR-4 inhibitor)on the damage to HaCaT cells caused by UVB irradiation.Methods In vitro,HaCaT cells were treated with 1,25(OH)_(2)D_(3) or/and TAK-242 prior to UVB irradiation at the intensity of 20 mJ/cm^(2),then the production of reactive oxygen species(ROS),cell migration,apoptosis of cells,and the expression of oxidative stress,endoplasmic reticulum stress,and apoptosis related proteins were determined.Results Compared with the HaCaT cells treated with 1,25(OH)_(2)D_(3) or TAK-242,the cells treated with both 1,25(OH)_(2)D_(3) and TAK-242 showed,1)significantly lower production of ROS(P<0.05);2)significantly less apoptosis of HaCaT cells(P<0.05);3)significantly lower expression of NF-κB,Caspase-8,Cyto-C,Caspase-3(P<0.05).Conclusion The combination of 1,25(OH)_(2)D_(3) and TAK-242 could produce a better protection for HaCaT cells via inhibiting the oxidative stress,endoplasmic reticulum stress and apoptosis than 1,25(OH)_(2)D_(3) or TAK-242 alone.

Potential Application of a Wine Extract in Skin Care: How to Benefit from the Antibacterial, Antioxidant and Elastase Inhibiting Properties

Since plant polyphenols have many beneficial properties on health, the aim of this study was to evaluate the potential use of a phenolic wine extract, a by-product of wine production, for skin care on HaCaT cells. In these studies, a significant reduction of reactive oxygen species formation in HaCaT cells and severe elastase inhibition was observed. In contrast, the wine extract caused a major increase in lipase activity. The extract showed no influence on cell proliferation, but an immunomodulatory effect on the release of the interleukins IL-6 and IL-8 was found. The phenolic wine extract demonstrated a strong activity against gram-positive and gram-negative pathogens, yeasts, and fungi. Overall, our results show that the investigated phenolic wine extract is a promising ingredient for anti-aging skin care, could contribute to the improvement of skin appearance and health, and may positively affect cellulite.

Effect of mitomycin on normal dermal fibroblast and HaCat cell:an in vitro study

Objective:To evaluate the effects of mitomycin on the growth of human dermal fibroblast and immortalized human keratinocyte line (HaCat cell),particularly the effect of mitomycin on intracellular messenger RNA (mRNA) synthesis of collagen and growth factors of fibroblast.Methods:The normal dermal fibroblast and HaCat cell were cultured in vitro.Cell cultures were exposed to 0.4 and 0.04 mg/ml of mitomycin solution,and serum-free culture medium was used as control.The cellular morphology change,growth characteristics,cell proliferation,and apoptosis were observed at different intervals.For the fibroblasts,the mRNA expression changes of transforming growth factor (TGF)-β1,basic fibroblast growth factor (bFGF),procollagen Ⅰ,and Ⅲ were detected by reverse transcription polymerase chain reaction (RT-PCR).Results:The cultured normal human skin fibroblast and HaCat cell grew exponentially.A 5-min exposure to mitomycin at either 0.4 or 0.04 mg/ml caused marked dose-dependent cell proliferation inhibition on both fibroblasts and HaCat cells.Cell morphology changed,cell density decreased,and the growth curves were without an exponential phase.The fibroblast proliferated on the 5th day after the 5-min exposure of mitomycin at 0.04 mg/ml.Meanwhile,5-min application of mitomycin at either 0.04 or 0.4 mg/ml induced fibroblast apoptosis but not necrosis.The apoptosis rate of the fibroblast increased with a higher concentration of mytomycin (p<0.05).A 5-min exposure to mitomycin at 0.4 mg/ml resulted in a marked decrease in the mRNA production of TGF-β1,procollagen Ⅰ and Ⅲ,and a marked increase in the mRNA production of bFGF.Conclusions:Mitomycin can inhibit fibroblast proliferation,induce fibroblast apoptosis,and regulate intracellular protein expression on mRNA levels.In additon,mitomycin can inhibit HaCat cell proliferation,so epithelial cell needs more protecting to avoid mitomycin's side effect when it is applied clinically.

Regulation of haptoglobin expression in a human keratinocyte cell line HaCaT by inflammatory cytokines and dexamethasone

Background Haptoglobin （Hp） is one of the acute-phase proteins. Recent studies have demonstrated that Hp exerts immunoregulatory and anti-inflammatory actions and may be one of the inhibitory factors of immune reactions in the skin. In this study we investigated the regulation of Hp expression in a human keratinocyte cell line HaCaT by various cytokines and glucocorticoid. Methods HaCaT cells were cultured with IL-6 （50 ng/ml）, TNF-α （20 ng/ml）, IFN-γ （20 ng/ml） or IL-4 （20 ng/ml） with or without 1 pmol/L dexamethasone in 6-well plates for 12, 24 and 48 hours. Both the cells and the supernatants were collected to detect the changes of Hp expression by reverse-transcription PCR, ELISA and immunohistochemistry. Results The results showed that Hp expression were elevated at both the mRNA and protein level by the combination of IL-6, TNF-α or IL-4 with dexamethasone, whereas the three cytokines alone did not upregulate the Hp expression. IFN-γ showed no effect on the Hp expression in HaCaT cells. Conclusions These findings suggest that different inflammatory cytokines as well as glucocorticoid may be involved in the regulation of Hp expression in keratinocytes, and this may be one of the negative feedback mechanisms in inflammatory skin diseases.