RESUMO
The chemokine interleukin (IL)-8 is a cytokine involved in neutrophil attraction and activation and elevated levels have been observed in intestinal inflammation. Anti-inflammatory activities have been attributed to green tea or its major constituent (-)-epigallocatechin gallate (EGCG). In this study, we investigated the effects of a defined green tea extract (GTE) or EGCG on basal or IL-1beta-induced IL-8 expression and secretion in the human gastrointestinal epithelial cell line Caco-2. mRNA expression levels were determined by quantitative RT-PCR. GTE significantly induced IL-8 mRNA expression, which was not mediated indirectly via an induction of IL-1beta mRNA expression. EGCG only exerted a weak although significant induction of IL-8 mRNA expression at the highest concentration. Intracellular and extracellular protein levels were analyzed by an enzyme-linked immunosorbent assay. GTE and EGCG significantly decreased secreted IL-8 concentrations. Determination of intracellular and secreted IL-8 concentrations after 24 h, 48 h, and 72 h of incubation suggested that GTE specifically inhibited IL-8 secretion while inducing DE NOVO synthesis of IL-8. The IL-1beta-mediated increase of IL-8 secretion was significantly inhibited by GTE in a dose-dependent manner. At the highest concentration, GTE inhibited IL-1beta-induced IL-8 secretion to a similar extent as found for brefeldin A, an inhibitor of vesicular transport. These results suggest that GTE may exert an anti-inflammatory activity in enterocytes, which may be useful for the treatment of intestinal inflammation.
Assuntos
Antioxidantes/farmacologia , Camellia sinensis , Interleucina-8/biossíntese , Fitoterapia , Extratos Vegetais/farmacologia , Antioxidantes/administração & dosagem , Antioxidantes/uso terapêutico , Células CACO-2/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica , Humanos , Interleucina-8/genética , Extratos Vegetais/administração & dosagem , Extratos Vegetais/uso terapêutico , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In this study the influence of green tea extract (GTE) or its component epigallocatechin gallate (EGCG) on the expression of different cytochrome P450 (CYP) isoenzymes was investigated in the human gastrointestinal epithelial cell lines LS-180 and Caco-2. Additionally, the effect of GTE or EGCG on functional activity of different CYP isoenzymes was investigated in vitro. mRNA expression levels were determined by quantitative RT-PCR and compared with protein levels. In LS-180 cells GTE, but not EGCG, significantly induced CYP1A2 mRNA expression, whereas neither CYP1A1 nor CYP3A4 mRNA expression was modulated by GTE or EGCG. In Caco-2 cells CYP1A1 as well as CYP1A2 mRNA expression was significantly increased in a dose-dependent manner by GTE and EGCG. However, EGCG alone was about 3-5-fold less effective than GTE. mRNA expression of CYP1A1 or CYP1A2 induced by the promutagen benzo[a]pyrene was significantly down-regulated by EGCG but not by GTE. CYP1A protein levels in response to GTE in Caco-2 and LS-180 cells confirmed the mRNA expression results. CYP activity was measured with CYP1A2 or CYP3A4 expressed in insect cell membranes using a luminescent method. GTE or EGCG significantly inhibited CYP1A2 and CYP3A4 function in a dose-dependent manner. Therefore, it appears that green tea moderately modulates the expression of drug-metabolizing enzymes but non-specifically inhibits the function of human CYPs. Since CYP enzymes play an important role in detoxification processes, these results might be of relevance for the prediction of the outcome of future clinical studies.
Assuntos
Colo/citologia , Citocromo P-450 CYP1A1/efeitos dos fármacos , Fitoterapia , Extratos Vegetais/farmacologia , Chá , Células CACO-2/efeitos dos fármacos , Catequina/administração & dosagem , Catequina/análogos & derivados , Catequina/farmacologia , Catequina/uso terapêutico , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica , Humanos , Extratos Vegetais/administração & dosagem , Extratos Vegetais/uso terapêutico , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Green tea extracts (GTE) might modulate ABC transporter gene expression or function. This may be relevant in the treatment of cancer or in influencing intestinal drug permeability. To gain more insight on the influence of a GTE on secretory transport proteins we investigated the influence of GTE and several green tea components on the mRNA expression level of P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) in human gastrointestinal epithelial LS-180 cells. Furthermore, the functional activity of MRP2, using glutathione methylfluorescein (GS-MF) or [3H]methotrexate (MTX) as substrate, was investigated in canine kidney cells stably overexpressing human MRP2 (MDCK-MRP2). GTE, at a concentration of 0.01 mg/mL, did not increase mRNA expression of P-gp or MRP2 in LS-180 cells. Functional assays in MDCK-MRP2 cells using GS-MF did not show any effect of 0.01 mg/mL GTE on MRP2 activity. In the same cell line the cellular accumulation of MTX (a specific substrate of MRP2) was significantly increased with the MRP-specific inhibitor MK-571 or with 1 mg/mL GTE, but not with 0.1 mg/mL. The green tea components (-)-epigallocatechin gallate, (-)-epigallocatechin, theanine, or caffeine, each in corresponding concentrations to the respective concentration of GTE, did not show any effect on MRP2 function. These data demonstrate that the mRNA expression patterns of P-gp and MRP2 in LS-180 cells are not altered by 0.01 mg/mL of GTE. However, MRP2 function was inhibited by 1 mg/mL GTE, whereas none of the green tea components tested were responsible for this effect.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Camellia sinensis , Moduladores de Transporte de Membrana , Proteínas de Membrana Transportadoras/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Fitoterapia , Extratos Vegetais/farmacologia , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/uso terapêutico , Linhagem Celular Tumoral/efeitos dos fármacos , Primers do DNA , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Humanos , Proteínas de Membrana Transportadoras/genética , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Extratos Vegetais/administração & dosagem , Extratos Vegetais/uso terapêutico , Folhas de Planta , RNA Mensageiro/análiseRESUMO
Recent reports on sporadic cases of liver disorders (acute hepatitis, icterus, hepatocellular necrosis) after ingestion of dietary supplements based on hydro-alcoholic extracts from green tea leaves led to restrictions of the marketing of such products in certain countries of the EU. Since green tea is considered to exert a number of beneficial health effects, and, therefore, green tea products are widely used as dietary supplements, we were interested in the possible mechanism of hepatotoxicity of green tea extracts and in the components involved in such effects. Seven hours after seeding on collagen, rat hepatocytes in primary culture were treated with various hydro-alcoholic green tea extracts (two different native 80% ethanolic dry extracts and an 80% ethanolic dry extract cleared from lipophilic compounds). Cells were washed, and reduction of resazurin, used as a viability parameter monitoring intact mitochondrial function, was determined. It was found that all seven green tea extracts examined enhanced resazurin reduction significantly at a concentration range of 100-500 microg/ml medium, while a significant decrease was observed at 1-3mg/ml medium. Decreased levels were concomitant with abundant necrosis as observed by microscopic inspection of the cultures and with increased leakage of lactate dehydrogenase activity from the cells. In a separate series of experiments, the green tea constituents (-)-epicatechin, (-)-epigallocatechin-3-gallate, caffeine and theanine were tested at concentrations reflecting their levels in a typical green tea extract. Synthetic (+)-epigallocatechin (200 microM) was used for comparison. Cytotoxicity was found with (-)-epigallocatechin-3-gallate only. The concomitant addition of 0.25 mM ascorbate/0.05 mM alpha-tocopherol had no influence on cytotoxicity. In conclusion, our results suggest that high concentrations of green tea extract can exert acute toxicity in rat liver cells. (-)-Epigallocatechin-3-gallate seems to be a key constituent responsible for this effect. The relatively low bioavailability of catechins reported after oral exposure to green tea argues, however, against a causal role of these constituents in the reported liver disorders.