Establishing Transcription Profile of Psoriasiform Cutaneous In Vitro using HaCaT Cells Stimulated with Combination of Cytokines

10.3791/61537 ◽  
2021 ◽  
Author(s):  
Huaping Zheng ◽  
Linna Gu ◽  
Zhen Wang ◽  
Hong Zhou ◽  
Chen Zhang ◽  
...  
Keyword(s):  
Hacat Cells ◽  
Transcription Profile

scholarly journals Water Extract of Rubus coreanus Prevents Inflammatory Skin Diseases In Vitro Models

Plants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1230
Author(s):  
Sumin Pyeon ◽  
Ok-Kyung Kim ◽  
Ho-Geun Yoon ◽  
Shintae Kim ◽  
Kyung-Chul Choi ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Skin Diseases ◽  
Water Extract ◽  
Total Phenolic ◽  
Hacat Cells ◽  
Rubus Coreanus ◽  
Tnf Α ◽  
Ifn Γ ◽  
Inflammatory Skin

Atopic dermatitis (AD) is a chronic inflammatory skin disease caused by immune hypersensitivity reaction. The cause of AD is unclear, but its symptoms have a negative effect on quality of life; various treatment methods to alleviate these symptoms are underway. In the present study, we aimed to evaluate in vitro antioxidant and anti-inflammatory effects of Rubus coreanus water extract (RCW) on AD. Total phenolic compounds and flavonoid content of RCW were 4242.40 ± 54.84 mg GAE/g RCE and 1010.99 ± 14.75 mg CE/g RCW, respectively. RCW reduced intracellular reactive oxygen species level and increased the action of antioxidant enzymes, such as catalase, superoxide dismutase, and glutathione peroxidase in tumor necrosis factor-α (TNF-α)/interferon-γ (IFN-γ)-stimulated HaCaT cells. Moreover, mRNA expression of the pro-inflammatory cytokines, including TNF-α, interleukin-1β, and interleukin-6, was downregulated by RCW in the TNF-α/IFN-γ-stimulated cells. The levels of inflammatory chemokines (thymus- and activation-regulated chemokine; eotaxin; macrophage-derived chemokine; regulated on activation, normal T-cell expressed and secreted; and granulocyte-macrophage colony-stimulating factor) and intercellular adhesion molecule-1 were decreased in the TNF-α/IFN-γ-stimulated HaCaT cells after RCW treatment. Additionally, the mRNA expression levels of filaggrin and involucrin, proteins that form the skin, were increased by RCW. Furthermore, RCW inhibited the nuclear factor kappa-light-chain-enhancer of the activated B cells pathway in the TNF-α/IFN-γ-stimulated HaCaT cells. Collectively, the present investigation indicates that RCW is a potent substance that inhibits AD.

scholarly journals Cell-free fat extract promotes tissue regeneration in a tissue expansion model

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Mingwu Deng ◽  
Xiangsheng Wang ◽  
Ziyou Yu ◽  
Yizuo Cai ◽  
Wei Liu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Growth Factor ◽  
Collagen Type ◽  
Growth Factor Receptor ◽  
Tissue Expansion ◽  
Nuclear Antigen ◽  
Hacat Cells ◽  
Expansion Model

Abstract Background Tissue expansion techniques play an important role in plastic surgery. How to improve the quality of the expanded skin and shorten the expansion period are still worth investigating. Our previous studies found that a cell-free fat extract (CEFFE) possessed pro-angiogenic and pro-proliferative activities. However, the role of CEFFE on tissue expansion has remained unclear. The purpose of this study was to evaluate the effect of CEFFE on tissue expansion. Methods A rat tissue expansion model was used. Animals were treated with CEFFE by subcutaneous injection. After 4 weeks of tissue expansion, the skin necrosis and retraction rates were evaluated, the thicknesses of the epidermis and dermis were determined by histological analyses, blood vessel density was measured by anti-CD31 staining, cell proliferation was assessed by proliferating cell nuclear antigen staining, and the expression of specific proteins was evaluated by western blot analyses. In addition, the effects of CEFFE on the proliferation and cell cycle of cultured HaCaT cells were evaluated in vitro. Results CEFFE treatment significantly decreased the necrosis rate and retraction of the expanded skin. The thickness of the epidermal and dermal layers was higher in CEFFE-treated compared to untreated skin. The density of blood vessels and cell proliferation in the epidermis of the expanded skin was improved by CEFFE treatment. In addition, CEFFE treatment significantly increased the expression of the vascular endothelial growth factor receptor, epidermal growth factor receptor, collagen type 1, and collagen type 3. CEFFE also increased the proliferation of HaCaT cells in culture. Conclusions CEFFE improves the quality of the expanded skin by promoting angiogenesis and cell proliferation. It could be potentially used clinically for augmenting tissue expansion.

scholarly journals 5-Aminolevulinic Acid-Based Photodynamic Therapy Pretreatment Mitigates Ultraviolet A-Induced Oxidative Photodamage

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Hui Hua ◽  
Jiawei Cheng ◽  
Wenbo Bu ◽  
Juan Liu ◽  
Weiwei Ma ◽  
...  
Keyword(s):  
Aminolevulinic Acid ◽  
Epidermal Keratinocytes ◽  
Control Group ◽  
Hacat Cells ◽  
Ultraviolet A ◽  
Hairless Mice ◽  
Human Epidermal Keratinocytes ◽  
Experimental Group

Aim. To determine whether 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) is effective in combating ultraviolet A- (UVA-) induced oxidative photodamage of hairless mice skin in vivo and human epidermal keratinocytes in vitro. Methods. In in vitro experiments, the human keratinocyte cell line (HaCaT cells) was divided into two groups: the experimental group was treated with ALA-PDT and the control group was left untreated. Then, the experimental group and the control group of cells were exposed to 10 J/m2 of UVA radiation. ROS, O2− species, and MMP were determined by fluorescence microscopy; p53, OGG1, and XPC were determined by Western blot analysis; apoptosis was determined by flow cytometry; and 8-oxo-dG was determined by immunofluorescence. Moreover, HaCaT cells were also treated with ALA-PDT. Then, SOD1 and SOD2 were examined by Western blot analysis. In in vivo experiments, the dorsal skin of hairless mice was treated with ALA-PDT or saline-PDT, and then, they were exposed to 20 J/m2 UVA light. The compound 8-oxo-dG was detected by immunofluorescence. Conclusion. In human epidermal keratinocytes and hairless mice skin, UVA-induced oxidative damage can be prevented effectively with ALA-PDT pretreatment.

scholarly journals Indigo Pulverata Levis (Chung-Dae, Persicaria tinctoria) Alleviates Atopic Dermatitis-like Inflammatory Responses In Vivo and In Vitro

2022 ◽  
Vol 23 (1) ◽  
pp. 553
Author(s):  
Ga-Yul Min ◽  
Ji-Hye Kim ◽  
Tae-In Kim ◽  
Won-Kyung Cho ◽  
Ju-Hye Yang ◽  
...  
Keyword(s):  
Atopic Dermatitis ◽  
Immune Cell ◽  
Cell Infiltration ◽  
Immune Cell Infiltration ◽  
Hacat Cells ◽  
Serum Ige ◽  
Tnf Α ◽  
Inflammatory Chemokines

Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with a type 2 T helper cell (Th2) immune response. The IndigoPulverata Levis extract (CHD) is used in traditional Southeast Asian medicine; however, its beneficial effects on AD remain uninvestigated. Therefore, we investigated the therapeutic effects of CHD in 2,4-dinitrochlorobenzene (DNCB)-induced BALB/c mice and tumor necrosis factor (TNF)-α- and interferon gamma (IFN)-γ-stimulated HaCaT cells. We evaluated immune cell infiltration, skin thickness, and the serum IgE and TNF-α levels in DNCB-induced AD mice. Moreover, we measured the expression levels of pro-inflammatory cytokines, mitogen-activated protein kinase (MAPK), and the nuclear factor-kappa B (NF-κB) in the mice dorsal skin. We also studied the effect of CHD on the translocation of NF-κB p65 and inflammatory chemokines in HaCaT cells. Our in vivo results revealed that CHD reduced the dermis and epidermis thicknesses and inhibited immune cell infiltration. Furthermore, it suppressed the proinflammatory cytokine expression and MAPK and NF-κB phosphorylations in the skin tissue and decreased serum IgE and TNF-α levels. In vitro results indicated that CHD downregulated inflammatory chemokines and blocked NF-κB p65 translocation. Thus, we deduced that CHD is a potential drug candidate for AD treatment.

scholarly journals Impact of p53 modulation on interactions between p53 family members during HaCaT keratinocytes differentiation

2020 ◽  
Author(s):  
AL Rusanov ◽  
PM Kozhin ◽  
DD Romashin ◽  
MN Karagyaur ◽  
NG Luzgina
Keyword(s):  
Cell Line ◽  
P53 Protein ◽  
Tp53 Gene ◽  
Negative Regulator ◽  
Elevated Concentration ◽  
Hacat Cells ◽  
Differentiation Markers ◽  
Substantial Impact ◽  
Skin Physiology

HaCaT cell line is a widely used model for studying normal human keratinocytes. However, mutations of TP53 gene are typical for this cell line, which have a substantial impact on functions of the encoded protein. The features of this regulatory circuit should be considered when using HaСaT cells for assessment of human skin physiology and pathology in vitro. The study was aimed to assess the features of differentiation realization in HaCaT cells with modulated activity of p53 protein. The expression of p53 was reduced by knockdown of TP53 gene by shRNA (by 2.2 times, p < 0.05), and the elevated concentration of the p53 active forms was achieved via exposure of cells to Nutlin-3a, the MDM2 inhibitor and the major negative regulator of p53. It has been found that regulation of at least three differentiation markers, СASP14, IVL (expression increase by 3.9 and 3.7 times respectively in the p53-knockdown cells, p < 0.05) and TGM1 (twofold expression decrease in the p53-knockdown cells, and 1.7-fold expression increase under exposure to Nutlin-3a, p < 0.05) in HaCaT cells is p53-mediated. The positive correlation has been revealed for expression of TGM1 and p53 that might be realized indirectly via ΔNp63 expression alteration. At the same time, modulation of p53 does not result in significant alterations in expression of cytokeratins.

scholarly journals Evaluation of anticancer activity in vitro of a stable copper(I) complex with phosphine-peptide conjugate

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Urszula K. Komarnicka ◽  
Barbara Pucelik ◽  
Daria Wojtala ◽  
Monika K. Lesiów ◽  
Grażyna Stochel ◽  
...  
Keyword(s):  
A549 Cells ◽  
Ros Generation ◽  
Dependent Manner ◽  
Hacat Cells ◽  
Icp Ms ◽  
Intracellular Ros ◽  
M Phase ◽  
Lung Adenocarcinoma A549 ◽  
Induced Apoptosis

Abstract[CuI(2,9-dimethyl-1,10-phenanthroline)P(p-OCH3-Ph)2CH2SarcosineGlycine] (1-MPSG), highly stable in physiological media phosphino copper(I) complex—is proposed herein as a viable alternative to anticancer platinum-based drugs. It is noteworthy that, 1-MPSG significantly and selectively reduced cell viability in a 3D spheroidal model of human lung adenocarcinoma (A549), in comparison with non-cancerous HaCaT cells. Confocal microscopy and an ICP-MS analysis showed that 1-MPSG effectively accumulates inside A549 cells with colocalization in mitochondria and nuclei. A precise cytometric analysis revealed a predominance of apoptosis over the other types of cell death. In the case of HaCaT cells, the overall cytotoxicity was significantly lower, indicating the selective activity of 1-MPSG towards cancer cells. Apoptosis also manifested itself in a decrease in mitochondrial membrane potential along with the activation of caspases-3/9. Moreover, the caspase inhibitor (Z-VAD-FMK) pretreatment led to decreased level of apoptosis (more pronouncedly in A549 cells than in non-cancerous HaCaT cells) and further validated the caspases dependence in 1-MPSG-induced apoptosis. Furthermore, the 1-MPSG complex presumably induces the changes in the cell cycle leading to G2/M phase arrest in a dose-dependent manner. It was also observed that the 1-MPSG mediated intracellular ROS alterations in A549 and HaCaT cells. These results, proved by fluorescence spectroscopy, and flow cytometry, suggest that investigated Cu(I) compound may trigger apoptosis also through ROS generation.

scholarly journals HaCaT Cells as a Reliable In Vitro Differentiation Model to Dissect the Inflammatory/Repair Response of Human Keratinocytes

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Irma Colombo ◽  
Enrico Sangiovanni ◽  
Roberta Maggio ◽  
Carlo Mattozzi ◽  
Stefania Zava ◽  
...  
Keyword(s):  
Cell Density ◽  
Skin Diseases ◽  
Inflammatory Responses ◽  
Human Keratinocytes ◽  
Suitable Model ◽  
Hacat Cells ◽  
Primary Human Keratinocytes ◽  
Proinflammatory Mediators ◽  
Cytokines And Chemokines

Cultured primary human keratinocytes are frequently employed for studies of immunological and inflammatory responses; however, interpretation of experimental data may be complicated by donor to donor variability, the relatively short culture lifetime, and variations between passages. To standardize the in vitro studies on keratinocytes, we investigated the use of HaCaT cells, a long-lived, spontaneously immortalized human keratinocyte line which is able to differentiate in vitro, as a suitable model to follow the release of inflammatory and repair mediators in response to TNFα or IL-1β. Different treatment conditions (presence or absence of serum) and differentiation stimuli (increase in cell density as a function of time in culture and elevation of extracellular calcium) were considered. ELISA and Multiplex measurement technologies were used to monitor the production of cytokines and chemokines. Taken together, the results highlight that Ca2+ concentration in the medium, cell density, and presence of serum influences at different levels the release of proinflammatory mediators by HaCaT cells. Moreover, HaCaT cells maintained in low Ca2+ medium and 80% confluent are similar to normal keratinocytes in terms of cytokine production suggesting that HaCaT cells may be a useful model to investigate anti-inflammatory interventions/therapies on skin diseases.

scholarly journals Optimization and characterization of poly(lactic-co-glycolic acid) nanoparticles loaded with astaxanthin and evaluation of anti-photodamage effect in vitro

2019 ◽  
Vol 6 (10) ◽  
pp. 191184 ◽  
Author(s):  
Fangbin Hu ◽  
Weikang Liu ◽  
Liuliu Yan ◽  
Fanhui Kong ◽  
Kun Wei
Keyword(s):  
Electron Microscopy ◽  
Glycolic Acid ◽  
Water Solubility ◽  
Skin Diseases ◽  
Biological Activities ◽  
Drug Loading ◽  
Synthesis Method ◽  
Hacat Cells ◽  
Scanning Calorimetry

Astaxanthin is a xanthophyll carotenoid with high beneficial biological activities, such as antioxidant function and scavenging oxygen free radicals, but its application is limited because of poor water solubility and low bioavailability. Here, we prepared and optimized poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with astaxanthin using the emulsion solvent evaporation technique and investigated the anti-photodamage effect in HaCaT cells. The four-factor three-stage Box–Behnken design was used to optimize the nanoparticle formulation. The experimental determination of the optimal nanoparticle size was 154.4 ± 0.35 nm, the zeta potential was 22.07 ± 0.93 mV, encapsulation efficiency was 96.42 ± 0.73% and drug loading capacity was 7.19 ± 0.12%. The physico-chemical properties of the optimized nanoparticles were characterized by dynamic light scattering, scanning electron microscopy, transmission electron microscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, differential scanning calorimetry and thermo-gravimetric analyser. In vitro study exhibited the excellent cell viability and cellular uptake of optimized nanoparticles on HaCaT cells. The anti-photodamage studies (cytotoxicity assay, reactive oxygen species content and JC-1 assessment) demonstrated that the optimized nanoparticles were more effective and safer than pure astaxanthin in HaCaT cells. These results suggest that our PLGA-coated astaxanthin nanoparticles synthesis method was highly feasible and can be used in cosmetics or the treatment of skin diseases.

scholarly journals Bioimpedance Analysis of L929 and HaCaT Cells in Low Frequency Range

2018 ◽  
Vol 4 (1) ◽  
pp. 115-118 ◽  
Author(s):  
Viviane S. Teixeira ◽  
Jan-Patrick Kalckhoff ◽  
Wolfgang Krautschneider ◽  
Dietmar Schroeder
Keyword(s):  
Low Frequency ◽  
Steel Corrosion ◽  
Frequency Dispersion ◽  
Human Keratinocytes ◽  
Cell Types ◽  
Hacat Cells ◽  
Frequency Range ◽  
Corrosion Resistant ◽  
L929 Mouse

AbstractIn this work, Bioimpedance Spectroscopy (BIS) is used to study fluids and cell solutions. A n ew fourelectrode- terminal (4T) chamber using 3D printing and stainless steel corrosion resistant V4A was designed to measure the impedance of live cell solutions at the frequency range 0.1Hz- 1MHz. At f < 1kHz the double layer (DL) that builds at electrode’s surface raises the impedance substantially preventing the observation of the real impedance of the cells. The new 4T design circumvents the DL, is more robust and cheap, and allows for the repeatability of the results. Experiments were performed in vitro with two cell lines, L929 (mouse fibroblasts) and HaCaT (human keratinocytes). Results show that it is possible to distinguish between the two cell types by means of its BIS measurements in the new setup. Also, a low-frequency dispersion (α-dispersion) was observed in HaCaT cells solution, but not in L929. Furthermore, a potentiostat circuit model was developed in LTSpice to simulate the hardware setup and two different circuit models were used to fit cell’s data.

scholarly journals Evaluation of the Effectiveness of Crotoxin as an Antiseptic against Candida spp. Biofilms

Toxins ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 532
Author(s):  
Amanda Pissinatti Canelli ◽  
Taís Fernanda dos Santos Rodrigues ◽  
Vivian Fernandes Furletti de Goes ◽  
Guilherme Ferreira Caetano ◽  
Maurício Ventura Mazzi
Keyword(s):  
Biofilm Formation ◽  
Colorimetric Assay ◽  
Oral Candidiasis ◽  
Hacat Cells ◽  
Candida Spp ◽  
Oral Infections ◽  
Minimum Fungicidal Concentration ◽  
Antimicrobial Susceptibility Tests ◽  
Susceptibility Tests

The growing number of oral infections caused by the Candida species are becoming harder to treat as the commonly used antibiotics become less effective. This drawback has led to the search for alternative strategies of treatment, which include the use of antifungal molecules derived from natural products. Herein, crotoxin (CTX), the main toxin of Crotalus durissus terrificus venom, was challenged against Candida tropicalis (CBS94) and Candida dubliniensis (CBS7987) strains by in vitro antimicrobial susceptibility tests. Minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), and inhibition of biofilm formation were evaluated after CTX treatment. In addition, CTX-induced cytotoxicity in HaCaT cells was assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) colorimetric assay. Native CTX showed a higher antimicrobial activity (MIC = 47 μg/mL) when compared to CTX-containing mouthwash (MIC = 750 μg/mL) and nystatin (MIC = 375 μg/mL). Candida spp biofilm formation was more sensitive to both CTX and CTX-containing mouthwash (IC100 = 12 μg/mL) when compared to nystatin (IC100 > 47 μg/mL). Moreover, significant membrane permeabilization at concentrations of 1.5 and 47 µg/mL was observed. Native CTX was less cytotoxic to HaCaT cells than CTX-containing mouthwash or nystatin between 24 and 48 h. These preliminary findings highlight the potential use of CTX in the treatment of oral candidiasis caused by resistant strains.

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