RESUMO
BACKGROUND AND OBJECTIVE: It is known that chronic periodontal infection can magnify the cytokine responses in patients with diabetes. Hyperglycemia increases the proinflammatory status, including the levels of advanced glycation end-products (AGEs), in patients with periodontitis. However, whether AGEs have additional effects on the production of those proinflammatory cytokines in diabetic patients with periodontitis is still unknown. To examine in vitro the effect of hyperglycemia and AGEs on the amounts of interleukin (IL)-6 and IL-8 produced in periodontally infected gingiva, human gingival fibroblasts (HGFs) were stimulated with glucose, AGE-modified bovine serum albumin (AGE-BSA) and Porphyromonas gingivalis LPS in the present study. MATERIAL AND METHODS: Primary culture of HGFs was incubated with various concentrations of AGE-BSA (0, 50, 100 and 200 µg/mL) and LPS (0, 10, 100 or 1000 ng/mL) at two different glucose concentrations - normal glucose (5 mm) and high glucose (25 mm). The amounts of IL-6 and IL-8 produced by HGFs were evaluated using ELISA. Expression of the AGE receptor on HGFs was determined by flow cytometry. RESULTS: High glucose stimulated a significant increase in the production of IL-6 and IL-8 by HGFs compared with normal glucose. This enhanced production of IL-6 and IL-8 could also be observed in the presence of LPS and/or AGE-BSA. When both LPS and AGE-BSA were present, especially at high concentrations (≥ 500 µg/mL of LPS and ≥ 25 µg/mL of AGE-BSA), a synergistic effect on IL-8 production was found in the high-glucose condition. CONCLUSIONS: A synergistic effect of the production of IL-8 could be induced in HGFs with the combination of high glucose, LPS and AGEs.
Assuntos
Fibroblastos/metabolismo , Gengiva/metabolismo , Glucose/farmacologia , Produtos Finais de Glicação Avançada/farmacologia , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipopolissacarídeos/farmacologia , Porphyromonas gingivalis/metabolismo , Adulto , Células Cultivadas , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Feminino , Fibroblastos/citologia , Citometria de Fluxo , Gengiva/citologia , Humanos , Masculino , Adulto JovemRESUMO
Cyclosporine-A (CsA) stimulates heme oxygenase-1 (HO-1) expression in the gingiva, but the regulation and the role of HO-1 in gingival overgrowth are not well-understood. HO-1 is regulated by several transcription factors, such as nuclear factor-κB (NF-κB) and nuclear factor erythroid-2-related factor 2 (Nrf-2). The aim of this study was to examine the role of Nrf-2 in the regulation of CsA-stimulated HO-1 expression in human gingival fibroblasts. Nrf-2 siRNA (siNrf-2), NF-κB, kinase inhibitors, and sulforaphane (SFN) were used to examine the nuclear translocation of Nrf-2 and expression of HO-1 and transforming growth factor-ß1 (TGF-ß1) in cells. Treatment with siNrf-2, but not with an NF-κB inhibitor, reduced CsA-stimulated HO-1 mRNA expression. ERK inhibition significantly decreased CsA-stimulated Nrf-2 nuclear translocation and HO-1 mRNA expression. Pre-treatment with SFN showed that HO-1 plays a role in attenuating CsA-mediated TGF-ß1 expressions. These findings suggest that CsA-stimulated HO-1 expression is mediated through the activation of ERK, and that Nrf-2 plays a protective role against CsA-induced gingival fibrosis by modulating collagen turnover-related genes.
Assuntos
Ciclosporina/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Crescimento Excessivo da Gengiva/metabolismo , Heme Oxigenase-1/biossíntese , Fator 2 Relacionado a NF-E2/fisiologia , Transporte Ativo do Núcleo Celular , Análise de Variância , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Gengiva/citologia , Gengiva/metabolismo , Crescimento Excessivo da Gengiva/induzido quimicamente , Heme Oxigenase-1/genética , Humanos , Isotiocianatos , Sistema de Sinalização das MAP Quinases , Fator 2 Relacionado a NF-E2/genética , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/metabolismo , RNA Interferente Pequeno , Estatísticas não Paramétricas , Sulfóxidos , Tiocianatos/farmacologia , Fator de Crescimento Transformador beta1/biossíntese , Fator de Crescimento Transformador beta1/genética , Regulação para CimaRESUMO
BACKGROUND AND OBJECTIVE: This study aimed to evaluate the expression and bioactivities of endothelin-1 (ET-1) in gingiva during cyclosporine A (CsA) treatment. MATERIAL AND METHODS: After establishing edentulous ridges, experimental rats were fed 30 mg/kg/day CsA while control animals received mineral oil for 4 weeks, after which a reverse transcription-polymerase chain reaction (RT-PCR) and/or immunohistochemistry was used to examine the expression of ET-1, its receptors, proliferating cell nuclear antigen (PCNA) and inducible nitric oxide synthase (iNOS) in gingivae. The roles of the endothelin receptors A and B (ET(A) and ET(B)) in CsA-enhanced expression of PCNA and iNOS were examined in cultured human gingival fibroblasts pretreated with receptor antagonists, by immunocytochemistry and RT-PCR, respectively. RESULTS: The mRNA expression of ET-1, ET(A) and ET(B), as well as of PCNA and iNOS, was significantly greater in edentulous gingiva that received CsA compared with control gingiva. Immunohistochemistry revealed more cells positively stained for ET-1 and its receptors in the tissues of CsA-treated rats than in those of control rats. In fibroblast cultures, enhanced mRNA expression of ET-1, ET(A) and ET(B) was observed after CsA treatment at the concentrations of 10 and 100 ng/mL. Cyclosporine A-enhanced PCNA expression was somewhat reduced by blockade of ET(A), but not ET(B), whereas iNOS expression was somewhat reduced by blockade of ET(B). CONCLUSION: Based on the present findings, we suggest that: (1) CsA upregulates the gingival expression of ET-1 and its receptors; and (2) ET(A) and ET(B) have different bioactivities, ET(A) being involved in cell proliferation and ET(B) being associated with iNOS expression.
Assuntos
Ciclosporina/farmacologia , Endotelina-1/análise , Gengiva/efeitos dos fármacos , Imunossupressores/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Antagonistas do Receptor de Endotelina A , Antagonistas do Receptor de Endotelina B , Endotelina-1/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Gengiva/patologia , Humanos , Masculino , Boca Edêntula/patologia , Óxido Nítrico Sintase Tipo II/análise , Antígeno Nuclear de Célula em Proliferação/análise , Ratos , Receptor de Endotelina A/análise , Receptor de Endotelina B/análise , Regulação para CimaRESUMO
BACKGROUND AND OBJECTIVE: Various inflammatory mediators are involved in the development of cyclosporine A-induced gingival overgrowth. In this study, the gingival expression of cyclooxygenase-2 after cyclosporine A therapy was examined in vivo and in vitro. MATERIAL AND METHODS: After edentulous ridges on maxilla were established, 21 Sprague-Dawley rats received cyclosporine A daily for 4 wk, and a further 21 rats received solvent. After the rats were killed, the expression of cyclooxygenase-2 mRNA, interleukin-1beta mRNA, tumor necrosis factor-alpha mRNA, and interleukin-6 mRNA was examined in the edentulous gingiva. The expression of cyclooxygenase-2 protein and the production of prostaglandin E2 were also evaluated. RESULTS: In cultured human gingival fibroblasts and epithelial cells, the expression of cyclooxygenase-2 mRNA was measured after treatment with cyclosporine A. Significantly lower expression of cyclooxygenase-2 and interleukin-1beta mRNA, but higher interleukin-6 expression, were observed in gingiva from cyclosporine A-treated rats than in those from the control rats. Significantly less prostaglandin E2 production was observed in cyclosporine A-treated rats. Immunohistochemistry revealed that fewer gingival stromal cells were positively stained for cyclooxygenase-2 in cyclosporine A-treated rats. In cultured cells, significantly less cyclooxygenase-2 mRNA was detected after treatment with cyclosporine A. CONCLUSION: The expression of cyclooxygenase-2 was lower in the plaque nonretentive gingivae and the in vitro gingival cells upon treatment with cyclosporine A. Thus, we propose that cyclosporine A inhibits the expression of gingival cyclooxygenase-2.