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1.
Toxicol Lett ; 320: 80-86, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31809884

RESUMO

In vitro cell culture experiments are highly important techniques to accelerate drug discovery, conduct safety testing and reduce the need for animal studies. Therefore, automatization may help to enhance the technical precision, reduce external (including operator's) influence on the data and thus improve reliability. Prior to application in scientific studies, validation of automated systems is absolutely necessary. In this study we present the validation of two combined automated pipetting systems to conduct toxicity studies in HaCaT cells consisting of cell seeding, noxious agent exposure and several assays to assess cell survival, apoptosis and interleukin production. After initial validation of pipetting accuracy, we compared homogeneity after automated seeding to plates seeded by expert laboratory technicians. Moreover, automated dispensing of a potentially unstable noxious agent was analyzed in terms of speed and consistency. We found a 2 % technical imprecision for the cell survival assay and 4.5-6 % for the other assays, bioluminescent and ELISA techniques. Thus, we could demonstrate the excellent technical precision of our assays. In a final step, we found that intraday variations, though acceptable, were much larger than technical variations and had to assume an intraday biological variability between different wells of the same experimental group.


Assuntos
Automação Laboratorial/normas , Substâncias para a Guerra Química/toxicidade , Gás de Mostarda/toxicidade , Técnicas de Cultura de Tecidos/normas , Testes de Toxicidade/normas , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/patologia , Teste de Materiais , Necrose
2.
J Photochem Photobiol B ; 202: 111704, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31743829

RESUMO

Ultraviolet B (UVB) induces inflammation and causes skin aging. The signs of skin aging, such as wrinkles, discolored spots, loss of skin moisture, and disruption of the skin barrier, are mostly caused by inflammatory signaling among various skin layers. The cells on the outermost surface of the skin are keratinocytes; these cells protect the skin against environmental stress and play an important role in immunomodulation by secreting cytokines in response to environmental stress. In the present study, we found that UVB activates STAT1 to mediate inflammatory signaling, yet STAT1 (S272) and STAT (Y702) shows different responses against UVB exposure. Anhua drak tea is a post-fermented dark tea produced in Anhua and Xinhua country in Hunan province of China. Treatment with 2S,3R-6-methoxycarbonylgallocatechin (MCGE), an epigallocatechin gallate derivative isolated from black tea (Anhua dark tea), effectively suppresses STAT1 activation and inflammatory cytokines, and activates Nrf2 pathway to protect cells from reactive oxygen species production in UVB exposed keratinocyte cells (HaCaT). Interestingly, the effects of MCGE were independent on MAPK signaling pathway. Moreover, MCGE regulates inflammatory cytokines in monocyte-keratinocyte (THP-1, HaCaT) co-culture and macrophage differentiation models. These results suggest that MCGE potentially can be used as a photoprotective agent against UVB-induced inflammatory responses.


Assuntos
Catequina/farmacologia , Protetores contra Radiação/farmacologia , Transdução de Sinais/efeitos dos fármacos , Chá/química , Raios Ultravioleta , Sítios de Ligação , Catequina/análogos & derivados , Catequina/química , Catequina/isolamento & purificação , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos da radiação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Técnicas de Cocultura , Citocinas/metabolismo , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/efeitos da radiação , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estrutura Terciária de Proteína , Protetores contra Radiação/química , Protetores contra Radiação/isolamento & purificação , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT1/química , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos da radiação , Chá/metabolismo
3.
Toxicol Lett ; 319: 256-263, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31639410

RESUMO

Transcription factor activator protein (AP)-1 can be activated in nitrogen-mustard-injured mouse skin, and is thought to participate in the inflammatory response. AP-1 consists of homo- or heterodimers of Fos [c-Fos, Fos-B, fos-related antigen (Fra)-1 and Fra-2] and Jun (c-Jun, JunB and JunD) family members, and information about their expression, location and function are still unclear. In nitrogen-mustard-exposed mouse skin, we found p-ERK activation increased Fra-1 and FosB. Unlike the nucleus location of c-Fos and FosB, Fra-1 and Fra-2 were mainly expressed in the cytoplasm. In nitrogen-mustard-exposed cultured immortalized human keratinocytes (HaCaT cells), Fra-1 in the nucleus functioned as an inhibitor of inflammatory cytokine interleukin (IL)-8. Co-immunoprecipitation showed that Fra-1 formed dimers with IL-8 transcription factors c-Jun, JunB and JunD. Fra-1 depletion increased c-Fos and FosB in the nucleus, accompanied by increased heterodimers of c-Fos/c-Jun, c-Fos/JunB, c-Fos/JunD, and FosB/JunB. In conclusion, Fra-1 trapped in the cytoplasm after nitrogen mustard exposure might be a driving force for IL-8 over-expression in injured skin.


Assuntos
Substâncias para a Guerra Química/toxicidade , Epiderme/lesões , Epiderme/metabolismo , Interleucina-8/biossíntese , Mecloretamina/toxicidade , Proteínas Proto-Oncogênicas c-fos/metabolismo , Animais , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Humanos , Queratinócitos/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Pelados , RNA Interferente Pequeno/farmacologia
4.
Life Sci ; 241: 117148, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31830478

RESUMO

Mitochondria are fascinating structures of the cellular compartments that generate energy to run the cells. However, inherent disorders of mitochondria due to diabetes can cause major disruption of metabolism that produces huge amount of reactive oxygen species (ROS). Here we study the elevated level of ROS provoked by high glucose (HG) environment triggered mitochondrial dysfunction, inflammatory response and apoptosis via stress signalling pathway in keratinocytes. Our results demonstrated that elevated glucose level in keratinoctes, increase the accumulations of ROS and decrease in cellular antioxidant capacities. Moreover, excess production of ROS was associated with mitochondrial dysfunction, characterized by loss of mitochondrial membrane potential (ΔΨm), increase in mitochondrial mass, alteration of mitochondrial respiratory complexes, cytochrome c (Cyt c) release, decrease in mitochondrial transcription factor A (TFAM) and increase in mitochondrial DNA (mtDNA) fragmentation. Damaged mtDNA escaped into the cytosol, where it engaged the activation of ERK1/2, PI3K/Akt, tuberin and mTOR via cGAS-STING leading to IRF3 activation. Pre-treatment of pharmacological inhibitors, ERK1/2 or PI3K/Akt suppressed the IRF3 activation. Furthermore, our results demonstrated that activation of IRF3 in HG environment coinciding with increased expression of inflammatory mediators. Excess production of ROS interfered with decreased in cell viability, increased lysosomal content and expression of FoxOs, leading to cell cycle deregulation and apoptosis. Pre-treatment of N-acetyl-l-cysteine (NAC) significantly reduced the HG-induced cell cycle deregulation and apoptosis in keratinocytes. In conclusion, increased oxidative stress underlies the decrease in antioxidant capacities and mitochondrial dysfunction in HG environment correlate with inflammation response and apoptosis via ERK1/2-PI3K/Akt-IRF3 pathway in keratinoctes.


Assuntos
Glucose/farmacologia , Queratinócitos/patologia , Mitocôndrias/patologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Pelados , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Edulcorantes/farmacologia
5.
J Photochem Photobiol B ; 202: 111676, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31837583

RESUMO

Wounds origins serious complications of lives of human beings which may leads to death. The important issue for the problem is infection during wound care management which delays wound healing process. These kinds of infections may be caused by the overuse or misuse of antibiotics, antidotes, usage of new drugs, not properly sterilized surgical instruments, not appropriate for pH level and imperfect wound dressing etc. during or after surgery. Hence in this report, antimicrobial action of pH responsive TA/KA composited hydrogel crosslinked with GO-QDs (TA/KA-GOQDs) using citric acid as cross-linker has been reported by demonstrating in-vitro and in-vivo studies for wound care management. The prepared samples of GOQDs, TA/KA hydrogel and TA/KA-GOQDs were characterized using FT-IR, XRD, SEM and TEM techniques. pH responsive hydrogel property of TA/KA was evaluated by swelling studies. In-vitro antibacterial studies was carried out by direct contact test method. Further, the prepared samples were tested in a wound healing model of rate with the wound of size 1.5 cm2 for in-vivo studies. After 16 days of treatment, the prepared samples for wound healing causes 100% wound areas closure. Histological observations were made by MT and HE staining process which proves keratinocytes proliferation by biocompatible and biocomposited TA/KA-GOQDs. The pH responsive TA/KA-GOQDs proved as efficient wound healing agent by faster keratinocytes proliferation within a compact period.


Assuntos
Materiais Biocompatíveis/farmacologia , Grafite/química , Hidrogéis/química , Queratinas/química , Pontos Quânticos/química , Cicatrização/efeitos dos fármacos , Animais , Materiais Biocompatíveis/química , Proliferação de Células/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Queratinócitos/citologia , Queratinócitos/metabolismo , Ratos , Pele/patologia , Staphylococcus aureus/efeitos dos fármacos
6.
Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi ; 54(11): 870-874, 2019 Nov 07.
Artigo em Chinês | MEDLINE | ID: mdl-31795552

RESUMO

Keratin (K) is the main component of the epithelial cell mesenchymal cytoskeleton, which protects the integrity of epithelial cells and maintains the function of normal epithelial cells. The expression of keratin affects epidermal proliferation and differentiation, and so as to be used as a marker for proliferation, differentiation and migration of keratinocytes. Middle ear cholesteatoma is one of the common ear diseases. In the middle ear cholesteatoma, keratinocytes over-proliferate and keratin debris accumulates. In this paper, we reviewed the recent studies on middle ear cholesteatoma and explained the possible mechanisms of keratin in the pathogenesis of middle ear cholesteatoma from the aspects of "proliferation" and " bone resorption ". At the same time, the existing problems as well as the prospect of the future research were discussed.


Assuntos
Colesteatoma da Orelha Média/metabolismo , Citoesqueleto/metabolismo , Orelha Média/metabolismo , Células Epiteliais/metabolismo , Queratinócitos/metabolismo , Queratinas/biossíntese , Reabsorção Óssea , Diferenciação Celular , Movimento Celular , Proliferação de Células , Colesteatoma da Orelha Média/etiologia , Humanos , Mesoderma/metabolismo , Mesoderma/patologia
7.
J Photochem Photobiol B ; 201: 111653, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31710929

RESUMO

Autophagy is an important process for maintaining intracellular homeostasis. Our previous study demonstrated that autophagy was down-regulated in ultraviolet B (UVB)-irradiated keratinocytes. Raffinose is a natural oligosaccharide that serves as a novel activator of autophagy and as a balancing agent to regulate the diversity of environmental stress. However, whether raffinose balances ultraviolet stress through the autophagy activation pathway has yet to be established. In this study, we found that raffinose treatment inhibited the LDH release and trypan blue staining in UVB-challenged human keratinocytes cell line HaCaT but did not affect the cleavage of apoptotic markers Caspase-3 and PARP, as well as translocation into nucleus of other cell death markers Endonuclease G and AIF. Moreover, we confirmed that raffinose treatment enhanced autophagy flux in an MTOR-independent manner in HaCaT cells. Importantly, decrease of LC3-II turnover in UVB-irradiated keratinocytes could be rescued by raffinose treatment, indicating that raffinose treatment increased autophagy in UVB-irradiated HaCaT cells. Furthermore, the effect on cell death by raffinose was inhibited when autophagy was suppressed with either a small interfering RNA targeting ATG5 (siATG5) or autophagic inhibitor wortmannin. In conclusion, we demonstrated that raffinose increases MTOR-independent autophagy and reduces cell death in UVB-irradiated keratinocytes. Our study indicated that the natural agent raffinose presents the potential value in opposing photodamage.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Rafinose/farmacologia , Raios Ultravioleta , Apoptose/efeitos da radiação , Autofagia/efeitos da radiação , Proteína 5 Relacionada à Autofagia/antagonistas & inibidores , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Caspase 3/metabolismo , Linhagem Celular , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo
8.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 50(5): 654-659, 2019 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-31762233

RESUMO

OBJECTIVE: To investigate the expression of ß-catenin in the skin lesions of patients with systemic scleroderma (SSc) and its effect on epithelial-mesenchymal transition (EMT) of human epidermal keratinocytes. METHODS: The expression of ß-catenin, Snail1 and E-cadherin in the skin lesions sample of 45 SSc patients and normal skin sample from 20 healthy adults was detected with SP immunohistochemistry. HaCaT, the human epidermal keratinocytes, were treated with different concentrations of Wnt10b (0 ng/mL (control), 2 ng/mL and 4 ng/mL) for 48 h. then detected the localization of ß-catenin in HaCaT cells by immunofluorescence assay, determined the mRNA levels of Snail1 and Snail2 in HaCaT cells by real-time fluorescent quantitative PCR, detected the proteins expression of ß-catenin, Vimentin, N-cadherin and E-cadherin in HaCaT cells by Western blot. RESULTS: The positive rates of ß-catenin, Snail1 and E-cadherin in skin lesions of SSc patients were 100%, 88.89% and 2.22% respectively, while in healthy adult skin, the corresponding positive rates were 0%, 10.00%, and 95.00%. The difference between the two groups was significant. Compared with control group, treatment with different concentrations of Wnt10b (2 ng/mL and 4 ng/mL) induced up-regulation of ß-catenin expression and promoted translocation of ß-catenin from cytoplasm to nucleus, increased the mRNA levels of Snail1 and Snail2 (P < 0.05), and up-regulated the proteins expression of Vimentin, N-cadherin, down-regulated the E-cadherin protein expression in HaCaT cells (P < 0.05). CONCLUSIONS: Abnormally activated Wnt/ß-catenin signaling pathway and abnormally expressed EMT-related proteins are observed in SSc lesions. Activation of Wnt/ß-catenin signaling pathway may promote EMT in HaCaT cells.


Assuntos
Transição Epitelial-Mesenquimal , Queratinócitos/metabolismo , Escleroderma Sistêmico/metabolismo , Pele/metabolismo , beta Catenina/metabolismo , Adulto , Antígenos CD/metabolismo , Caderinas/metabolismo , Humanos , Queratinócitos/citologia , Escleroderma Sistêmico/patologia , Pele/patologia , Fatores de Transcrição da Família Snail/metabolismo , Vimentina/metabolismo , Via de Sinalização Wnt
9.
Int J Nanomedicine ; 14: 7123-7139, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31564869

RESUMO

Background: Poly(amidoamine) (PAMAM) dendrimers are of considerable interest when used as a carrier for topical drugs for the skin, although little is known about their possible side effects. Therefore, our study was about the impact of 2nd and 3rd generation PAMAM dendrimers on human keratinocytes and fibroblasts cells. Methods: The effect of the tested compounds on collagen biosynthesis was determined using 5[3H]-proline incorporation bioassay. Morphological changes accompanying cell growth inhibition were observed using a confocal microscope. To evaluate the percentage of apoptotic/necrotic cells and the cell growth dynamic of apoptotic features, we performed Annexin V/PI double staining assay, assessed caspase activity, and performed cell cycle analysis by flow cytometry. The flow cytometry method was also used to determine the effect of dendrimers on pro-inflammatory cytokines (IL-6, IL-8 IL-1ß). Results: The obtained results showed that as the concentration and the generation of dendrimers increased, collagen biosynthesis decreased. We also observed abnormalities in cell differentiation, which may have caused disturbed secretion of pro-inflammatory cytokines. We found that dendrimers cause chronic inflammation which may cause adverse changes in the skin, ultimately- leading to apoptosis in the case of dendrimers in lower concentrations or necrosis at higher concentrations (especially 3rd generation dendrimers). In addition, the inflammatory path induced by the tested compounds was caused by damage in the mitochondria, which we observed as a significant decrease in the mitochondrial membrane potential. Conclusion: The results of our study showed that PAMAM dendrimers can cause disorders of cell proliferation and differentiation and may be the cause of cell cycle deregulation and chronic adverse inflammation.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Citocinas/metabolismo , Dendrímeros/farmacologia , Fibroblastos/metabolismo , Mediadores da Inflamação/metabolismo , Queratinócitos/metabolismo , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 8/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Colágeno/biossíntese , Dendrímeros/química , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fluorescência , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Necrose
10.
Nat Commun ; 10(1): 4676, 2019 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-31611556

RESUMO

Resident adult epithelial stem cells maintain tissue homeostasis by balancing self-renewal and differentiation. The stem cell potential of human epidermal keratinocytes is retained in vitro but lost over time suggesting extrinsic and intrinsic regulation. Transcription factor-controlled regulatory circuitries govern cell identity, are sufficient to induce pluripotency and transdifferentiate cells. We investigate whether transcriptional circuitry also governs phenotypic changes within a given cell type by comparing human primary keratinocytes with intrinsically high versus low stem cell potential. Using integrated chromatin and transcriptional profiling, we implicate IRF2 as antagonistic to stemness and show that it binds and regulates active cis-regulatory elements at interferon response and antigen presentation genes. CRISPR-KD of IRF2 in keratinocytes with low stem cell potential increases self-renewal, migration and epidermis formation. These data demonstrate that transcription factor regulatory circuitries, in addition to maintaining cell identity, control plasticity within cell types and offer potential for therapeutic modulation of cell function.


Assuntos
Fator Regulador 2 de Interferon/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Humanos , Fator Regulador 2 de Interferon/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/genética , Ativação Transcricional/fisiologia
11.
Int J Mol Sci ; 20(17)2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31480310

RESUMO

Particulate matter (PM), a widespread air pollutant, consists of a complex mixture of solid and liquid particles suspended in air. Many diseases have been linked to PM exposure, which induces an imbalance in reactive oxygen species (ROS) generated in cells, and might result in skin diseases (such as aging and atopic dermatitis). New techniques involving nanomedicine and nano-delivery systems are being rapidly developed in the medicinal field. Fullerene, a kind of nanomaterial, acts as a super radical scavenger. Lower water solubility levels limit the bio-applications of fullerene. Hence, to improve the water solubility of fullerene, while retaining its radical scavenger functions, a fullerene derivative, fullerenol C60(OH)36, was synthesized, to examine its biofunctions in PM-exposed human keratinocyte (HaCaT) cells. The PM-induced increase in ROS levels and expression of phosphorylated mitogen-activated protein kinase and Akt could be inhibited via fullerenol pre-treatment. Furthermore, the expression of inflammation-related proteins, cyclooxygenase-2, heme oxygenase-1, and prostaglandin E2 was also suppressed. Fullerenol could preserve the impaired state of skin barrier proteins (filaggrin, involucrin, repetin, and loricrin), which was attributable to PM exposure. These results suggest that fullerenol could act against PM-induced cytotoxicity via ROS scavenging and anti-inflammatory mechanisms, and the maintenance of expression of barrier proteins, and is a potential candidate compound for the treatment of skin diseases.


Assuntos
Poluição do Ar/prevenção & controle , Fulerenos/análise , Material Particulado/toxicidade , Água/química , Apoptose/efeitos dos fármacos , Linhagem Celular , Cidades , Fulerenos/química , Humanos , Inflamação/patologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/ultraestrutura , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Solubilidade
12.
Biol Res ; 52(1): 49, 2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492195

RESUMO

BACKGROUND: Psoriasis is a common and intractable skin disease affecting the physical and mental health of patients. The accumulation of ROS is involved in the pathogenesis of psoriasis and antioxidants are believed to be therapeutic. This study aimed to investigate the therapeutic efficacy of astilbin on ROS accumulation in psoriasis. RESULTS: The study showed that 50 µg/ml astilbin could inhibit the growth and reduce the accumulation of ROS in HaCaT cells stimulated by IL-17 and TNF-α. Astilbin could elevate the Nrf2 accumulation in the nuclei, eventually leading to the transcriptional activation of various antioxidant proteins and reducing the expression of VEGF. CONCLUSIONS: Our results collectively suggest that astilbin could induce Nrf2 nucleus translocation, which is contribute to reduce the ROS accumulation and VEGF expression, and inhibit the proliferation of HaCaT cells.


Assuntos
Flavonóis/administração & dosagem , Queratinócitos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/efeitos dos fármacos , Psoríase/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismo , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Interleucina-17/metabolismo , Queratinócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fator 2 Relacionado a NF-E2/metabolismo , Psoríase/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
13.
Tissue Cell ; 59: 33-38, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31383286

RESUMO

Hair-follicle-associated pluripotent (HAP) stem cells reside in the upper part of the bulge area of the the hair follicle. HAP stem cells are nestin-positive and keratin 15-negative and have the capacity to differentiate into various types of cells in vitro. HAP stem cells are also involved in nerve and spinal cord regeneration in mouse models. Recently, it was shown that the DNA-damage response in non-HAP hair follicle stem cells induces proteolysis of type-XVII collagen (COL17A1/BP180), which is involved in hair-follicle stem-cell maintenance. COL17A1 proteolysis stimulated hair-follicle stem-cell aging, characterized by the loss of stemness signatures and hair-follicle miniaturization associated with androgenic alopecia. In the present study, we demonstrate that HAP stem cells co-express nestin and COL17A1 in vitro and in vivo. The expression of HAP stem cell markers (nestin and SSEA1) increased after HAP stem-cell colonies were formed, then decreased after differentiation to epidermal keratinocytes. In contrast COL17A1 increased after differentiation to epidermal keratinocytes. These results suggest that COL17A1 is important in differentiation of HAP stem cells.


Assuntos
Autoantígenos/biossíntese , Diferenciação Celular , Regulação da Expressão Gênica , Folículo Piloso/metabolismo , Queratinócitos/metabolismo , Colágenos não Fibrilares/biossíntese , Células-Tronco Pluripotentes/metabolismo , Animais , Antígenos de Diferenciação/biossíntese , Folículo Piloso/citologia , Queratinócitos/citologia , Camundongos , Nestina/biossíntese , Células-Tronco Pluripotentes/citologia
14.
Int J Mol Sci ; 20(16)2019 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-31398789

RESUMO

The nuclear factor-erythroid 2-related factor 2 (NRF2) transcription factor plays a central role in mediating the cellular stress response. Due to their antioxidant properties, compounds activating NRF2 have received much attention as potential medications for disease prevention, or even for therapy. Accumulating evidence suggests that activation of the NRF2 pathway also has a major impact on wound healing and may be beneficial in the treatment of chronic wounds, which remain a considerable health and economic burden. While NRF2 activation indeed shows promise, important considerations need to be made in light of corresponding evidence that also points towards pro-tumorigenic effects of NRF2. In this review, we discuss the evidence to date, highlighting recent advances using gain- and loss-of-function animal models and how these data fit with observations in humans.


Assuntos
Regulação da Expressão Gênica , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Cicatrização , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Citoproteção , Fibroblastos , Humanos , Queratinócitos/metabolismo , Mutação com Perda de Função , Estresse Oxidativo , Fenótipo
15.
Int J Mol Sci ; 20(16)2019 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-31398894

RESUMO

Claudin-1 (CLDN1) is expressed in the tight junction (TJ) of the skin granular layer and acts as a physiological barrier for the paracellular transport of ions and nonionic molecules. Ultraviolet (UV) and oxidative stress may disrupt the TJ barrier, but the mechanism of and protective agents against this effect have not been clarified. We found that UVB and hydrogen peroxide (H2O2) caused the internalization of CLDN1 and increased the paracellular permeability of lucifer yellow, a fluorescent marker, in human keratinocyte-derived HaCaT cells. Therefore, the mechanism of mislocalization of CLDN1 and the protective effect of an ethanol extract of Brazilian green propolis (EBGP) were investigated. The UVB- and H2O2-induced decreases in CLDN1 localization were rescued by EBGP. H2O2 decreased the phosphorylation level of CLDN1, which was also rescued by EBGP. Wild-type CLDN1 was distributed in the cytosol after treatment with H2O2, whereas T191E, its H2O2-insensitive phosphorylation-mimicking mutant, was localized at the TJ. Both protein kinase C activator and protein phosphatase 2A inhibitor rescued the H2O2-induced decrease in CLDN1 localization. The tight junctional localization of CLDN1 and paracellular permeability showed a negative correlation. Our results indicate that UVB and H2O2 could induce the elevation of paracellular permeability mediated by the dephosphorylation and mislocalization of CLDN1 in HaCaT cells, which was rescued by EBGP. EBGP and its components may be useful in preventing the destruction of the TJ barrier through UV and oxidative stress.


Assuntos
Claudina-1/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Estresse Oxidativo , Própole/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Claudina-1/genética , Endocitose/efeitos dos fármacos , Expressão Gênica , Humanos , Peróxido de Hidrogênio/metabolismo , Mutação , Fosforilação , Transporte Proteico , Espécies Reativas de Oxigênio/metabolismo , Junções Íntimas/metabolismo
16.
Biosens Bioelectron ; 142: 111555, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31408825

RESUMO

Up to today, in vivo studies are the gold standard for testing of new therapeutics for cutaneous wound healing. Alternative in vitro studies are mostly limited to two-dimensional cell cultures and thus only poorly reflect the complex physiological wound situation. Here we present a new three-dimensional wound model based on a reconstructed human epidermis (RHE). We introduce impedance spectroscopy as a time-resolved test method to determine the efficacy of wound healing non-destructively by focusing on the barrier function of the RHE as a main feature of intact skin. We assessed the skin barrier quantitatively and qualitatively by calculating the transepithelial electrical resistance (TEER), by fitting an equivalent circuit and by analyzing the single characteristic frequency. Upon wounding using a 2 mm biopsy punch, the impedance dropped significantly to 3.5% of the initial value. Impedance spectroscopy thereby proved to be a sensitive tool to distinguish between wounds of different sizes. The glucose and lactate concentration in the medium revealed an acute stress reaction of the wounded RHE (wRHE) in the first days after wounding. During monitoring of reepithelialization over fourteen days, the barrier fully recovered. Microscopy and histology images correlate well with these findings, revealing an active wound closure mostly completed by day seven after wounding. These wounded epidermal models can now be applied in therapeutic screenings and with the help of rapid screening by impedance spectroscopy, expensive and time-consuming imaging and histological methods as well as the use of animal models can be reduced.


Assuntos
Técnicas Biossensoriais/instrumentação , Espectroscopia Dielétrica/instrumentação , Epiderme/patologia , Cicatrização , Células Cultivadas , Epiderme/metabolismo , Desenho de Equipamento , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Microscopia Confocal
17.
Int J Mol Sci ; 20(16)2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31426336

RESUMO

Reactive oxygen species (ROS) are generated from diverse cellular processes or external sources such as chemicals, pollutants, or ultraviolet (UV) irradiation. Accumulation of radicals causes cell damage that can result in degenerative diseases. Antioxidants remove radicals by eliminating unpaired electrons from other molecules. In skin health, antioxidants are essential to protect cells from the environment and prevent skin aging. (-)-Epigallocatechin-3-(3″-O-methyl) gallate (3″Me-EGCG) has been found in limited oolong teas or green teas with distinctive methylated form, but its precise activities have not been fully elucidated. In this study, we examined the antioxidant roles of 3″Me-EGCG in keratinocytes (HaCaT cells). 3″Me-EGCG showed scavenging effects in cell and cell-free systems. Under H2O2 exposure, 3″Me-EGCG recovered cell viability and increased the expression of heme oxygenase 1 (HO-1). Under ultraviolet B (UVB) and sodium nitroprusside (SNP) exposure, 3″Me-EGCG protected keratinocytes and regulated the survival protein AKT1. By regulating the AKT1/NF-κB pathway, 3″Me-EGCG augmented cell survival and proliferation in HaCaT cells. These results indicate that 3″Me-EGCG exhibits antioxidant properties, resulting in cytoprotection against various external stimuli. In conclusion, our findings suggest that 3″Me-EGCG can be used as an ingredient of cosmetic products or health supplements.


Assuntos
Antioxidantes/farmacologia , Catequina/análogos & derivados , Citoproteção/efeitos dos fármacos , Ácido Gálico/análogos & derivados , Queratinócitos/efeitos dos fármacos , Antioxidantes/química , Catequina/química , Catequina/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Citoproteção/efeitos da radiação , Ácido Gálico/química , Ácido Gálico/farmacologia , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Protetores contra Radiação/química , Protetores contra Radiação/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Raios Ultravioleta/efeitos adversos
18.
Int J Mol Sci ; 20(17)2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31438472

RESUMO

Atopic dermatitis (AD) is the most common chronic and relapsing inflammatory skin disease. AD is typically characterized by skewed T helper (Th) 2 inflammation, yet other inflammatory profiles (Th1, Th17, Th22) have been observed in human patients. How cytokines from these different Th subsets impact barrier function in this disease is not well understood. As such, we investigated the impact of the canonical Th17 cytokine, IL-17A, on barrier function and protein composition in primary human keratinocytes and human skin explants. These studies demonstrated that IL-17A enhanced tight junction formation and function in both systems, with a dependence on STAT3 signaling. Importantly, the Th2 cytokine, IL-4 inhibited the barrier-enhancing effect of IL-17A treatment. These observations propose that IL-17A helps to restore skin barrier function, but this action is antagonized by Th2 cytokines. This suggests that restoration of IL-17/IL-4 ratio in the skin of AD patients may improve barrier function and in so doing improve disease severity.


Assuntos
Epiderme/efeitos dos fármacos , Epiderme/metabolismo , Interleucina-17/farmacologia , Interleucina-4/farmacologia , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Células Cultivadas , Claudina-4/metabolismo , Dermatite Atópica/metabolismo , Humanos , Técnicas In Vitro , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Fator de Transcrição STAT3/metabolismo , Pele/efeitos dos fármacos , Pele/metabolismo
19.
Life Sci ; 233: 116714, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31376370

RESUMO

Increased levels of particulate matter (PM) air pollutants in East Asia have resulted in detrimental health impacts increasing morbidity and mortality. Epidemiological studies suggest a possible relation between the cutaneous exposure of PM and increased oxidative stress and inflammation which lead to skin lesions. The present study utilizes an integrated cell culture model of keratinocytes and fibroblasts to mimic viable skin layers and investigate the possible effects of PM exposure after penetration through corneocytes. The skin perfection is upheld by homeostatic functionality of epidermal cells and the integrity of connective tissues. Exposure to xenobiotics could alter the skin cell homeostasis aggravating premature skin aging. Stimulation of HaCaT keratinocytes by PM collected from Beijing, China (CPM) increased the intracellular ROS levels triggering a cascade of events aggravating inflammatory responses and connective tissue degradation. In HDF fibroblasts, treatment with preconditioned keratinocyte culture media augmented inflammatory responses, cellular differentiation, and connective tissue degradation. Above events were marked by the increased intracellular ROS, inflammatory mediators, pro-inflammatory cytokines, matrix metalloproteinases (MMP)-1 and -2 levels, collagenase, and elastase activity. Fucosterol treatment of keratinocytes dose-dependently attenuated the detrimental effects both in keratinocytes and fibroblasts restoring the conditions near to physiological levels. Further evaluations could be advanced on developing fucosterol, in forms such as rejuvenating cosmeceuticals which could attenuate detrimental responses of CPM exposure.


Assuntos
Fibroblastos/efeitos dos fármacos , Inflamação/tratamento farmacológico , Queratinócitos/efeitos dos fármacos , Material Particulado/efeitos adversos , Dermatopatias/tratamento farmacológico , Pele/efeitos dos fármacos , Estigmasterol/análogos & derivados , Poluentes Atmosféricos/efeitos adversos , Células Cultivadas , Técnicas de Cocultura , Citocinas/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Queratinócitos/metabolismo , Queratinócitos/patologia , NF-kappa B/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Pele/patologia , Dermatopatias/etiologia , Dermatopatias/metabolismo , Dermatopatias/patologia , Estigmasterol/farmacologia
20.
Environ Toxicol ; 34(12): 1354-1362, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31436008

RESUMO

Ultraviolet-B light (UV-B) is a major cause of skin photoaging, inducing cell death and extracellular matrix collapse by generating reactive oxygen species (ROS). Belamcandae Rhizoma (BR), the rhizome of Belamcanda chinensis Leman, exhibits antioxidant properties, but it remains unknown whether BR extract ameliorates UV-B-induced skin damage. In this study, we evaluated the effects of a standardized BR extract on UV-B-induced apoptosis and collagen degradation in HaCaT cells. BR was extracted using four different methods. We used radical-scavenging assays to compare the antioxidative activities of the four extracts. Cells were irradiated with UV-B and treated with BR boiled in 70% (vol/vol) ethanol (BBE). We measured cell viability, intracellular ROS levels, the expression levels of antioxidative enzymes, and apoptosis-related and collagen degradation-related proteins. The irisflorentin and tectorigenin levels were measured via high-performance liquid chromatography. BBE exhibited the best radical-scavenging and cell protective effects of the four BR extracts. BBE inhibited intracellular ROS generation and induced the synthesis of antioxidative enzymes such as catalase and glutathione. BBE attenuated apoptosis by reducing the level of caspase-3 and increasing the Bcl-2/Bax ratio. BBE reduced the level of matrix metalloproteinase-1 and increased that of type I collagen. The irisflorentin and tectorigenin contents were 0.23% and 0.015%, respectively. From these results, BBE ameliorated UV-B-induced apoptosis and collagen degradation by enhancing the expression of antioxidative enzymes. It may be a useful treatment for UV-B-induced skin damage.


Assuntos
Apoptose/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Iris/química , Extratos Vegetais/farmacologia , Substâncias Protetoras/farmacologia , Raios Ultravioleta , Antioxidantes/metabolismo , Apoptose/efeitos da radiação , Linhagem Celular , Glutationa/metabolismo , Humanos , Iris/metabolismo , Isoflavonas/análise , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Extratos Vegetais/química , Substâncias Protetoras/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rizoma/química , Rizoma/metabolismo
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