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1、ORIGINAL ARTICLE Cytoprotective properties of rifampicin are related to the regulation of detoxifi cation system and bile acid transporter expression during hepatocellular injury induced by hydrophobic bile acids Rau l Gonza lezAdolfo CruzGustavo Ferr nPedro Lo pez-Cillero Javier Bricen oMiguel A. G
2、o mez Sebastia n Rufi a nJavier Padillo Manuel De la MataJose J. G. MarinJordi Muntane Published online: 28 April 2011 ? Japanese Society of Hepato-Biliary-Pancreatic Surgery and Springer 2011 Abstract Background/PurposeRifampicin has been used for the treatment of patients with jaundice and pruritu
3、s. This study evaluated the effect of rifampicin on the expression of different detoxifi cation systems and bile acid transporters duringin-vivoandin-vitroexperimentalmodelsof cholestasis. MethodsRifampicin was administered to glycochenode- oxycholic acid (GCDCA)-treated human hepatocytes and bile d
4、uct-obstructed rats. Different parameters related to cell death, and the expression of phase I and II drug metabolizing enzymes (DME) and bile acid transporters were determined. ResultsThe induction of hepatocellular injury induced by cholestasis was associated with a reduction in cytochrome P4503A4
5、 (CYP3A4), CYP7A1, and UDP-glucuronosyl- transferase 2B4 (UGT2B4) expression, as well as an increase in import (Na?-taurocholate co-transporting poly- peptide, NTCP) system expression. The benefi cial proper- ties of rifampicin were associated with an increase in DME and export bile acid systems (mu
6、ltidrug resistance- associated protein 4, MRP4, and bile acid export pump to bile duct, BSEP) expression, as well as a reduction in NTCP expression. ConclusionsThe benefi cialeffectofrifampicinin cholestasis is associated with an increase in DME expres- sion involved in toxic, bile acid and choleste
7、rol metabo- lism, as well as a reduction in the bile acid importing system in hepatocytes. KeywordsCholestasis ? Cytochrome P450 ? Glucuronosyltransferase ? Hepatocytes ? Nuclear receptor Abbreviations ABCATP binding cassette BSEPBile acid export pump to bile duct DHEDihydroethidium CARConstitutive
8、androstane receptor FXRFarnesoid X receptor GCDCAGlycochenodeoxycholic acid GRGlucocorticoid receptor LDHLactate dehydrogenase R. Gonza lez ? G. Ferr n ? J. Muntane ( 58 13.6 years old) submitted to surgical interven- tion for primary or secondary liver tumor resection after written consent of the p
9、atient. The procedure of hepatocyte isolation was based on the two-step collagenase method described by Pichard et al. 26. Hepatocytes (8 9 106 cells; 150000 cells/cm2) were seeded in type I collagen- coated dishes (Iwaki, Gyouda, Japan) and cultured in DEM-Ham-F12 and Williams E mediums (1:1) suppl
10、e- mented with 26 mM NaHCO3, 15 mM HEPES, 0.29 g/l glutamine, 50 mg/l vitamin C, 0.04 mg/l dexamethasone, 2 mg/l insulin, 200 lg/l glucagon, 50 mg/l transferrin and 4 ng/l ethanolamine culture medium containing 5% fetal calf serum for 12 h. Afterwards, the medium was replaced by fresh culture medium
11、 without fetal bovine serum. Twenty-four hours after cell stabilization, the culture medium was replaced again. Rifampicin (10 lM) was administered 24 h before the induction of cell death by GCDCA (0.5 mM) administration in hepatocytes. Samples werecollectedatdifferenttimesaccordingtothe parameter.
12、In-vivo study Male Wistar rats (220240 g) were subjected to controlled conditions of temperature (about 2224?C) and illumina- tion (12-h light:12-h dark cycle) and were provided with food (Purina?, Barcelona, Spain) and water ad libitum. Animals were treated according to institutional guidelines, an
13、d the study was approved by the Research Committee of the Reina Sof a University Hospital. Animals (n = 24, 6 in each group) were allocated to the following groups: sham operated (SO), SO ? rifampicin (RIF), obstructive jaun- dice (OJ), and OJ ? rifampicin (OJ ? RIF). All the sur- gical procedures w
14、ere performed in animals anesthetized with ketamine (60 mg/kg i.p.) and midazolam (4 mg/kg i.p.). Cephazoline (17 mg/kg i.m.) was used as antibiotic prophylaxis. SO animals were submitted to laparatomy and abdominal closure without bile duct intervention. The procedure for OJ began with a midline ve
15、ntral incision to expose the extra-hepatic bile duct which was double liga- tured with silk suture and then sectioned. A two-layer running suture was used for abdominal closure with poly- glycolic acid and silk. Rifampicin (50 mg/kg body weight) (Sigma-Aldrich, St Louis, MO, USA) was diluted in 1% s
16、odiumcarboxymethylcelluloseandintraperitoneally injected daily starting on the day of the bile duct ligature and ending the day preceding the sacrifi ce. The animals were sacrifi ced under anesthesia 7 days after OJ. Blood was collected by heart puncture and serum was frozen at -80?C until the measurement of alanine aminotransferease (ALT), aspartate aminotransferease (AST), lactate dehy- drogenase (LDH), total bilirubin, triglycerides (TAG) and cholesterol. Li
