RESUMO
This study is to investigate the mechanism underlying the anti-apoptotic effects of freeze-dried grape powder (FDGP) and identify the polyphenolic compounds involved. We examined apoptotic signaling pathways affected by FDGP and by its active components, including epicatechin, cyanidin, quercetin, and resveratrol, in human Huh7 hepatoma cells by assaying cell viability assays, the activities of caspase 3 and caspase 7, and the expression of endoplasmic reticulum stress-associated proteins. FDGP dramatically decreased taurodeoxycholic acid (TDCA)-induced production of reactive oxygen species (ROS). Assessment of individual active components revealed that at concentrations corresponding to 300 µg/mL FDGP, only quercetin demonstrated cytoprotective effects against mitochondrial-mediated apoptosis. In contrast, increased concentrations of other individual polyphenolic compounds were required to produce measurable cytoprotective effect. Only combinations of all four polyphenolic compounds (epicatechin, cyanidin, quercetin, and resveratrol) restored a degree of the anti-apoptotic effects seen with FDGP. The pretreatment of FDGP at 30 µg/mL concentration could reverse the thapsigargin-induced effects on the expression of endoplasmic reticulum stress-associated proteins. In conclusion, FDGP reduced oxidative stress, endoplasmic reticulum stress, and apoptosis. The mechanisms involved in the anti-apoptotic effects of FDGP included reduced generation of ROS, and reduced processing of certain caspases. We demonstrated that quercetin, epicatechin, and cyanidin are active compounds within FDGP that attenuate apoptosis. These findings contribute to our understanding of the molecular mechanisms of anti-apoptotic and anti-oxidant effects of grape and are expected to assist in developing clinical protocols to treat a variety of stress-mediated conditions.
Assuntos
Apoptose/efeitos dos fármacos , Alimentos em Conserva , Frutas/química , Vitis/química , Antocianinas/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Caspase 3/metabolismo , Caspase 7/metabolismo , Catequina/farmacologia , Morte Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Retículo Endoplasmático/metabolismo , Liofilização , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Mitocôndrias Hepáticas/metabolismo , Estresse Oxidativo , Quercetina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Resveratrol , Estilbenos/farmacologia , Ácido Taurodesoxicólico/toxicidadeRESUMO
Mechanisms by which hydrophobic bile salts cause tissue changes below their critical micellar concentration (CMC, 1-2mM) and above (4-8mM) remain poorly understood. In this study, rat colonic mucosa was exposed to different concentrations of taurodeoxycholate (TDC), t-butyl-hydroperoxide (t-BH) or glutathione ester with or without pre-incubation with 2mM TDC. Exposure to 2mM TDC was associated with 10% higher tissue levels of total glutathione (GSH, basal values: 33.7+/-3.3 nmol/mg prot). With TDC 8mM, GSH decreased to 16.4+/-2.3 nmol/mg prot (P<0.05), oxidized glutathione (GSSG) increased by 60% (P<0.05), glutathione peroxidase (GSH-Px) and reductase activities were threefold increased, protein carbonyls fourfold increased, protein sulfhydrils decreased by 78%, lactate dehydrogenase (LDH) and GSSG release in the incubation medium were sixfold higher. In 2mM TDC pre-treated tissues, the subsequent incubation with 8mM TDC induced a lower loss of tissue GSH, and a lower release of LDH and GSSG. Pre-incubation with 2mM TDC partly protected against t-BH toxicity, while glutathione ester protected against 8mM TDC toxicity. In conclusion, TDC exposure causes opposite effects depending on CMC: induction of antioxidant protective systems including glutathione system (pre-conditioning effect) was observed with TDC below CMC, oxidative damages pointing to decreased mucosal detoxification potential with above CMC.
Assuntos
Colagogos e Coleréticos/toxicidade , Enteropatias/induzido quimicamente , Precondicionamento Isquêmico , Estresse Oxidativo/efeitos dos fármacos , Ácido Taurodesoxicólico/toxicidade , Animais , Colagogos e Coleréticos/química , Glutationa/metabolismo , Enteropatias/patologia , Mucosa Intestinal/patologia , L-Lactato Desidrogenase/metabolismo , Masculino , Micelas , Oxirredução , Carbonilação Proteica/efeitos dos fármacos , Ratos , Ratos Wistar , Compostos de Sulfidrila/metabolismo , Ácido Taurodesoxicólico/químicaRESUMO
OBJECTIVE: To evaluate the effect of ethyl pyruvate (EP) in improving the survival and ameliorating distant organ damage and to investigate the role of high-mobility group box (HMGB) 1 in rats with established severe acute pancreatitis (SAP). METHODS: Severe acute pancreatitis was induced by retrograde infusion of sodium taurodeoxycholate (5%, 1 mL/kg) into the biliopancreatic ducts in male Wistar rats. The rats were infused intravenously with EP of 40 mg/kg, 4 mg/kg, and 0.4 mg/kg initiating 12 hours, and EP of 40 mg/kg was administered beginning 2 hours before surgery (-2 hours) and 12, 24, and 36 hours after induction of SAP; then, the mortality was recorded. Serum tumor necrosis factor alpha, interleukin (IL) 6, and IL-1beta were measured using enzyme-linked immunosorbent assay. High-mobility group box 1 levels were measured using Western immunoblotting analysis. RESULTS: Serum HMGB1 levels were increased dramatically after 12 hours, remained at high levels for 72 hours, and were significantly higher in rats with SAP than in those with mild and moderate pancreatitis (P < 0.01). Treatment with EP (40 mg/kg) conferred protection from lethality of SAP (EP survival [63%] vs vehicle survival [6.3%]; P < 0.001). No survival advantage occurred when treatment was initiated 36 hours after surgery, but administration beginning 2 hours before operation (-2 hours) and 12 and 24 hours after induction of SAP significantly increased survival. Ethyl pyruvate treatment significantly decreased serum HMGB1, tumor necrosis factor alpha, IL-1beta, and IL-6 levels and ameliorated extrapancreatic organ dysfunction in rats with SAP. CONCLUSIONS: Ethyl pyruvate improves survival and ameliorates distant organ injury of SAP. These beneficial effects of EP are because of the modulation of HMGB1 and other inflammatory cytokine responses.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Proteínas de Grupo de Alta Mobilidade/sangue , Rim/patologia , Fígado/patologia , Pancreatite/tratamento farmacológico , Piruvatos/uso terapêutico , Proteínas Repressoras/sangue , Doença Aguda , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Biomarcadores , Relação Dose-Resposta a Droga , Esquema de Medicação , Avaliação Pré-Clínica de Medicamentos , Proteína HMGB1 , Interleucina-1beta/sangue , Interleucina-6/sangue , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Masculino , Pancreatite/sangue , Pancreatite/induzido quimicamente , Pancreatite/patologia , Piruvatos/administração & dosagem , Distribuição Aleatória , Ratos , Ratos Wistar , Ácido Taurodesoxicólico/toxicidade , Fator de Necrose Tumoral alfa/análiseRESUMO
This study was performed to compare the effects of two hydrophilic bile acids, taurohyodeoxycholic acid (THDCA) and tauroursodeoxycholic acid (TUDCA), on HepG2 cells. Cytotoxicity was evaluated at different times of exposure by incubating cells with increasing concentrations (50-800 micromol/l) of either bile acid, while their cytoprotective effect was tested in comparison with deoxycholic acid (DCA) (350 micromol/l and 750 micromol/l)-induced cytotoxicity. Culture media, harvested at the end of each incubation period, were analyzed to evaluate aspartate transaminase (AST), alanine transaminase and gamma-glutamyltranspeptidase release. In addition, the hemolytic effect of THDCA and TUDCA on human red blood cells was also determined. At 24 h of incubation neither THDCA nor TUDCA was cytotoxic at concentrations up to 200 and 400 micromol/l. At 800 micromol/l both THDCA and TUDCA induced a slight increase in AST release. At this concentration and with time of exposure prolonged up to 72 h, THDCA and TUDCA induced a progressive increase of AST release significantly (P<0.05) higher than that of controls being AST values for THDCA (2.97+/-0.88 time control value (tcv) at 48 h and 4.50+/-1.13 tcv at 72 h) significantly greater than those of TUDCA (1.50+/-0.20 tcv at 48 h and 1.80+/-0.43 tcv at 72 h) (P<0.01). In cytoprotection experiments, the addition of 50 micromol/l THDCA decreased only slightly (-5%) AST release induced by 350 micromol/l DCA, while the addition of 50 micromol/l TUDCA was significantly effective (-23%; P<0.05). Higher doses of THDCA or TUDCA did not reduce toxicity induced by 350 micromol/l DCA, but were much less toxic than an equimolar dose of DCA alone. At the concentration used in this experimental model neither THDCA nor TUDCA was hemolytic; however at a very high concentration (6 mmol/l) both bile acids induced 5-8% hemolysis. We conclude that bile acid molecules with a similar degree of hydrophilicity may show different cytotoxic and cytoprotective properties.
Assuntos
Ácido Tauroquenodesoxicólico/farmacologia , Ácido Taurodesoxicólico/análogos & derivados , Ácido Taurodesoxicólico/farmacologia , Alanina Transaminase/análise , Aspartato Aminotransferases/análise , Ácido Desoxicólico/antagonistas & inibidores , Relação Dose-Resposta a Droga , Eritrócitos/efeitos dos fármacos , Hemólise , Humanos , Ácido Tauroquenodesoxicólico/toxicidade , Ácido Taurodesoxicólico/toxicidade , Fatores de Tempo , Transglutaminases/análise , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Bile acid-induced inhibition of DNA synthesis by the regenerating rat liver in the absence of other manifestation of impairment in liver cell viability has been reported. Because in experiments carried out on in vivo models bile acids are rapidly taken up and secreted into bile, it is difficult to establish steady concentrations to which the hepatocytes are exposed. Thus, in this work, a dose-response study was carried out to investigate the in vitro cytotoxic effect of major unconjugated and tauro- (T) or glyco- (G) conjugated bile acids and to compare this as regards their ability to inhibit DNA synthesis. Viability of hepatocytes in primary culture was measured by Neutral red uptake and formazan formation after 6 h exposure of cells to bile acids. The rate of DNA synthesis was determined by radiolabeled thymidine incorporation into DNA. Incubation of hepatocytes with different bile acid species - cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), in the range of 10-1000 microM - revealed that toxicity was stronger for the unconjugated forms of CDCA and DCA than for CA and UDCA. Conjugation markedly reduced the effects of bile acids on cell viability. By contrast, the ability to inhibit radiolabeled thymidine incorporation into DNA was only slightly lower for taurodeoxycholic acid (TDCA) and glycodeoxycholic acid (GDCA) than for DCA. When the effect of these bile acids on DNA synthesis and cell viability was compared, a clear dissociation was observed. Radiolabeled thymidine incorporation into DNA was significantly decreased (-50%) at TDCA concentrations at which cell viability was not affected. Lack of a cause-effect relationship between both processes was further supported by the fact that well-known hepatoprotective compounds, such as tauroursodeoxycholic acid (TUDCA) and S-adenosylmethionine (SAMe) failed to prevent the effect of bile acids on DNA synthesis. In summary, our results indicate that bile acid-induced reduction of DNA synthesis does not require previous decreases in hepatocyte viability. This suggests the existence of a high sensitivity to bile acids of cellular mechanisms that may affect the rate of DNA repair and/or proliferation, which is of particular interest regarding the role of bile acids in the etiology of certain types of cancer.
Assuntos
Ácidos e Sais Biliares/farmacologia , Replicação do DNA/efeitos dos fármacos , Inibidores do Crescimento/farmacologia , Fígado/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Animais , Ácidos e Sais Biliares/toxicidade , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ácido Quenodesoxicólico/farmacologia , Ácido Quenodesoxicólico/toxicidade , Ácido Cólico/farmacologia , Ácido Cólico/toxicidade , Corantes , Ácido Desoxicólico/farmacologia , Ácido Desoxicólico/toxicidade , Relação Dose-Resposta a Droga , Formazans , Ácido Glicodesoxicólico/farmacologia , Ácido Glicodesoxicólico/toxicidade , Inibidores do Crescimento/toxicidade , Fígado/citologia , Masculino , Vermelho Neutro , Inibidores da Síntese de Ácido Nucleico/toxicidade , Ratos , Ratos Wistar , Ácido Taurodesoxicólico/farmacologia , Ácido Taurodesoxicólico/toxicidade , Ácido Ursodesoxicólico/farmacologia , Ácido Ursodesoxicólico/toxicidadeRESUMO
BACKGROUND: Pancreatic duct epithelial cells form a barrier against parenchymal injury. The capacity of these cells to respond to injury has not been investigated. We hypothesized that epidermal growth factor (EGF), normally found in pancreatic juice, could protect the duct epithelium from damage. METHODS: An explant system of duct cell culture developed in our lab with the bovine main pancreatic duct was used. Explants were exposed to bile acid (taurodeoxycholic acid [TDCA] 0, 0.05, 0.5, and 1 mmol/L) in the presence or absence of EGF (0, 1, 10, and 100 nmol/L) for 48 hours. Epithelial proliferation, damage, and growth out from the explant edge were assessed histologically. Expression of ductal markers and the extent of cell proliferation were determined by immunohistochemistry using specific antibodies. RESULTS: Explant duct cells proliferated and demonstrated continued expression of key duct antigens in culture. TDCA produced dose-dependent mucosal damage and reduced epithelial density and growth from the edge. EGF increased cellular density in the native epithelium, but did not significantly alter growth from the edge. Mucosal damage created by TDCA exposure was significantly decreased with EGF and both growth from the edge and cell density were preserved. CONCLUSIONS: Explants created from the bovine main pancreatic duct serve as an excellent model for the study of duct epithelial cells in vitro. These cells proliferate in response to EGF and are damaged by TDCA at concentrations below those normally associated with detergent-like activity and below levels observed in bile and duodenal secretions. The ability of EGF to protect from this injury suggests a potential physiologic role in the maintenance of the pancreatic duct mucosal barrier.