RESUMEN
Bile acid-induced hepatocyte apoptosis plays an important role in cholestatic liver disease, and the role of apoptosis may be of therapeutic interest in preventing liver disease. The dried root of Salvia miltiorrhiza Bunge (Labiatae) has been used traditionally to treat liver diseases. We investigated the antiapoptotic effects of a standardized fraction of S. miltiorrhiza (PF2401-SF) and its components, tanshinone I, tanshinone IIA, and cryptotanshinone, in primary cultured rat hepatocytes. PF2401-SF was enriched with tanshinone I (11.5%), tanshinone IIA (41.0%), and cryptotanshinone (19.1%). Glycochenodeoxycholic acid (GCDC)-induced apoptosis, as shown by DNA fragmentation, poly(ADP-ribose) polymerase cleavage, and activation of caspases-8, -9, and -3. PF2401-SF and its components, tanshinone I, tanshinone IIA, and cryptotanshinone showed antiapoptotic activity. Treatment with PF2401-SF or with its components significantly inhibited the generation of intracellular reactive oxygen species. Hydrophobic bile acids activate c-Jun-NH(2)-terminal kinase (JNK), p38 mitogen-activated protein kinases (MAPK), and extracellular signal-regulated kinase 1/2, and PF2401-SF inhibited the phosphorylation of JNK and p38. All three components of PF2401-SF inhibited JNK phosphorylation. Addition of inhibitors of MAPK showed that inhibition of JNK decreased apoptosis. These data indicate that PF2401-SF and its components protect hepatocytes from GCDC-induced apoptosis in vitro by inhibiting JNK.
Asunto(s)
Apoptosis/efectos de los fármacos , Ácidos y Sales Biliares/antagonistas & inhibidores , Ácidos y Sales Biliares/toxicidad , Hepatocitos/efectos de los fármacos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Fenantrenos/farmacología , Salvia/química , Abietanos , Animales , Western Blotting , Caspasas/metabolismo , Células Cultivadas , Colagogos y Coleréticos/antagonistas & inhibidores , Colagogos y Coleréticos/toxicidad , Cromatografía Líquida de Alta Presión , Colorimetría , Fragmentación del ADN/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Ácido Glicoquenodesoxicólico/antagonistas & inhibidores , Ácido Glicoquenodesoxicólico/toxicidad , Estrés Oxidativo/efectos de los fármacos , Fosforilación/efectos de los fármacos , Extractos Vegetales/farmacología , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: Taurolithocholate induced cholestasis is a well established model of drug induced cholestasis with potential clinical relevance. This compound impairs bile salt secretion by an as yet unclear mechanism. AIMS: To evaluate which step/s of the hepatocellular bile salt transport are impaired by taurolithocholate, focusing on changes in localisation of the canalicular bile salt transporter, Bsep, as a potential pathomechanism. METHODS: The steps in bile salt hepatic transport were evaluated in rats in vivo by performing pharmacokinetic analysis of (14)C taurocholate plasma disappearance. Bsep transport activity was determined by assessing secretion of (14)C taurocholate and cholyl-lysylfluorescein in vivo and in isolated rat hepatocyte couplets (IRHC), respectively. Localisation of Bsep and F-actin were assessed both in vivo and in IRHC by specific fluorescent staining. RESULTS: In vivo pharmacokinetic studies revealed that taurolithocholate (3 micro mol/100 g body weight) diminished by 58% canalicular excretion and increased by 96% plasma reflux of (14)C taurocholate. Analysis of confocal images showed that taurolithocholate induced internalisation of Bsep into a cytosolic vesicular compartment, without affecting F-actin cytoskeletal organisation. These effects were reproduced in IRHC exposed to taurolithocholate (2.5 micro M). Preadministration of dibutyryl-cAMP, which counteracts taurolithocholate induced impairment in bile salt secretory function in IRHC, restored Bsep localisation in this model. Furthermore, when preadministered in vivo, dibutyryl-cAMP accelerated recovery of both bile flow and bile salt output, and improved by 106% the cumulative output of (14)C taurocholate. CONCLUSIONS: Taurolithocholate impairs bile salt secretion at the canalicular level. Bsep internalisation may be a causal factor which can be prevented by dibutyryl-cAMP.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Ácidos y Sales Biliares/metabolismo , Colagogos y Coleréticos/antagonistas & inhibidores , Colestasis/inducido químicamente , Ácido Taurolitocólico/efectos adversos , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Actinas/metabolismo , Animales , Transporte Biológico , Colagogos y Coleréticos/farmacocinética , Colestasis/metabolismo , Masculino , Ratas , Ratas Wistar , Ácido Taurolitocólico/farmacocinéticaRESUMEN
BACKGROUND/AIMS: Vasopressin has been reported to reduce bile flow, but its effects on bile acid secretion and bile acid-related hepatotoxicity are still unclear. We therefore investigated the influence of vasopressin on the hepatotoxicity and biliary excretion of taurochenodeoxycholic acid in primary cultured rat hepatocytes and isolated perfused rat liver models. METHODS/RESULTS: 1) Addition of vasopressin to hepatocyte cultures significantly decreased lactate dehydrogenase release as compared to cultures exposed to 1 mM taurochenodeoxycholic acid alone, and also reduced intracellular taurochenodeoxycholic acid content from 19.3 +/- 2.2 to 13.0 +/- 1.6 nmol/mg protein. After 30 min of preincubation with 1 mM taurochenodeoxycholic acid, rinsing and reculture of hepatocytes in bile acid-free medium resulted in gradual decrease in the intracellular level of the bile acid, and addition of vasopressin (10(-9) M) to the reculture medium accelerated this process. 2) Superimposition of vasopressin (330 pmol/l) for 10 min on taurochenodeoxycholic acid infusion (1.0 mumol/min: 25 mumol/l) caused a rapid increase in bile flow and biliary excretion of taurochenodeoxycholic acid (697 +/- 42 vs 584 +/- 27 nmol/10 min per g liver) from perfused rat livers, and significantly reduced lactate dehydrogenase release. 3) Superimposition of the PKC blocker H-7 (5 mumol/l) on taurochenodeoxycholic acid infusion (1.0 mumol/min: 25 mumol/l) caused a gradual increase in bile flow and biliary excretion of taurochenodeoxycholic acid. Furthermore, an additional infusion of vasopressin (100 pmol/l) for 10 min in the presence of H-7 produced a greater increase in bile flow and biliary excretion of taurochenodeoxycholic acid as compared with H-7 alone (754 +/- 71 vs. 657 +/- 26 nmol/g liver). 4) Continuous infusion of vasopressin (330 pmol/l) significantly increased the late peak (10-50 min) of horseradish peroxidase excretion from perfused livers (from 8.48 +/- 1.02 to 21.7 +/- 6.02 ng/g liver). CONCLUSIONS: These findings suggest that vasopressin exerts a protective effect against taurochenodeoxycholic acid-induced hepatotoxicity by stimulating the secretion of this bile acid via intracellular vesicular transport systems.
Asunto(s)
Bilis/metabolismo , Colagogos y Coleréticos/antagonistas & inhibidores , Hepatopatías/prevención & control , Hígado/metabolismo , Ácido Tauroquenodesoxicólico/antagonistas & inhibidores , Vasopresinas/farmacología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Peroxidasa de Rábano Silvestre/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Hígado/citología , Hepatopatías/fisiopatología , Masculino , Perfusión , Ratas , Ratas Sprague-Dawley , Tasa de Secreción/efectos de los fármacosRESUMEN
Increasing amounts of porcine secretion (0.05 to 2.00 clinical units/h/100 g body wt) given to rats during a continuous infusion of bilirubin, increased bile flow and the apparent maximal biliary excretion of bilirubin ('Tm'). This increment was caused by an enhanced biliary output of bilirubin monoconjugates. The effect was dose dependent but maximal at a secretin infusion of 0.80 CU. Somatostatin 0.2 and 0.8 microgram/h/100 g body wt caused a dose related inhibition of the hepatic effects of secretin both on bile flow and on biliary output of bilirubin conjugates. As secretin elicits the release of somatostatin, a feed-back system could be envisaged whereby the somatostatin released stops the effects of secretin.