Danning tablets attenuates α-naphthylisothiocyanate-induced cholestasis by modulating the expression of transporters and metabolic enzymes
© Ding et al.; licensee BioMed Central Ltd. 2014
Received: 14 January 2014
Accepted: 9 July 2014
Published: 17 July 2014
The Danning tablets (DNts) is commonly prescribed in China as a cholagogic formula. Our previous studies showed that DNts exerted the protective effect on α-naphthylisothiocyanate (ANIT)-induced liver injury with cholestasis in a dose-dependent mannar. However, the detailed molecular mechanisms of DNts against ANIT-induced cholestasis are still not fully explored.
Danning tablet (3 g/kg body weight/day) was administered orally to experimental rats for seven days before they were treated with ANIT (60 mg/kg daily via gastrogavage) which caused cholestasis. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (T-Bil), direct bilirubin (D-Bil) and total bile acid (TBA) were measured to evaluate the protective effect of Danning tablet at 12, 24 and 48h after ANIT treatment. Meanwhile, total bilirubin or total bile acid in the bile, urine and liver were also measured at 48h after ANIT treatment. Furthermore, the hepatic or renal mRNA and protein levels of metabolic enzymes and transports were investigated to elucidate the protective mechanisms of Danning tablet against ANIT-induced cholestasis.
In this study, we found that DNts significantly attenuated translocation of multidrug resistance-associated protein 2 (Mrp2) from the canalicular membrane into an intracellular and up-regulated the hepatic mRNA and protein expressions of metabolic enzymes including cytochrome P450 2b1(Cyp2b1) and uridine diphosphate-5¢- glucuronosyltransferase (Ugt1a1)) and transporters including bile salt export pump (Bsep) and multidrug resistance protein 2 (Mdr2)) as well as renal organic solute transporter beta (Ostβ), accompanied by further increase in urinary and biliary excretion of bile acid and bilirubin.
DNts might promote bile acid and bilirubin elimination by regulating the expressions of hepatic and renal transporters as well as hepatic metabolic enzymes.
KeywordsDanning tablet α-naphthylisothiocyanate (ANIT) Cholestasis Transporter Metabolic enzyme
Cholestasis, an impairment or cessation in the flow of bile, leads to hepatic and systemic accumulation of potentially toxic biliary compounds such as bile acids and bilirubin, resulting in acute liver toxicity, jaundice and hypercholesterolemia, and then aggravated outcomes just like hepatic fibrosis, cirrhosis and clinical signs of liver failure[1, 2]. It has been reported that cholestasis may develop from viral hepatitis or the administration of certain drugs and hormones or mechanical obstructions in the extrahepatic bile ducts.
The mechanisms of cholestasis might be related to the altered expressions of hepatic metabolic enzymes and transporters[3, 4]. Drug metabolic enzymes (DMEs) play central roles in the metabolism, elimination and/or detoxification of xenobiotics or exogenous compounds introduced into the body. Metabolic enzymes in the liver are classified into phase 1, such as cytochrome P450 (Cyp), and phase 2, such as uridine diphosphate-5¢- glucuronosyltransferase (Ugt), sulfotransferase (Sult). Several transporters which are related to cholestasis have been identified in hepatocytes, such as multidrug resistance protein (Mdr), bile salt export pump (Bsep), multidrug resistance associated protein (Mrp)[8, 9], organic solute transporter alpha/beta (Ostα/β), Na+ taurocholate cotransporting polypeptide (Ntcp), and organic anion transporting polypeptide (Oatp).
ANIT is well konwn as a cholestatic compound and has been widely used as a model component for induction of intrahepatic cholestasis. In rodents, α-naphthylisothiocyanate (ANIT) is a widely used chemical to mimic human intrahepatic cholestasis. It has been reported that the mRNA expressions of metabolic enzymes and transporters changed in ANIT-induced cholestasis model[14, 15].
Danning tablets (DNts), as a composite prescription of traditional Chinese medicine, consisting of seven medicinal materials: Radix et Rhizoma Rhei, Rhizoma et Radix Polygoni Cuspidati, Pericarpium Citri Reticulatae, Pericarpium Citri Reticulatae Viride, Radix Curcumae, Fructus Crataegi and Rhizoma Imperatae, have been proved to be effective for treatment of cholecystitis and prevention the formation of cholesterol gallstone in human. We previously showed that the protective effect of DNts on ANIT-induced liver injury was probably due to its attenuation of oxidative stress disruption in the liver tissues and neutrophil infiltration. However, the underlying mechanisms about protective effect of DNts on ANIT-induced cholestasis are not explored yet.
In this study, in order to clarify the possible mechanisms for protective effect of DNts on ANIT-induced cholestasis, we investigated the mRNA and protein expressions of metabolic enzymes and transports which were relevant to the excretion of bile acids and bilitubin.
Drugs, chemicals and reagents
DNts were friendly provided by Shanghai Hutchison Pharmaceuticals (Shanghai, China) at July 2010. Standard compounds (emodin, aleo-emodin, rhein, chrysophanol, physcion) purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). ANIT were purchased from Sigma-Aldrich (St. Louis, USA). The purity of each compound was determined to be above 98% by HPLC analysis. Ammonium acetate, formic acid, acetonitrile and methanol (HPLC-grade) were purchased from Fisher Scientific (Nepean, Ont., Canada). Ultrapure water was prepared by a Milli-Q50 SP Reagent Water System (Millipore Corporation, MA, USA) for the preparation of samples and buffer solutions. Serum levels of ALT, AST, ALP, T-Bil, D-Bil and TBA were determined using a commercially available test kits (SHINO-TEST Corporation, Japan) with HITACHI 7080 Automatic Analyzer system (Japan). All other chemicals and solvents used were of analytical grade.
In order to make quality control of DNts and improve the repeatability of experiments, the contents of five anthraquinones, which might be the main active components contained in DNts, were determined by HPLC analysis. The determination was performed on the Agilent 1100 chromatographic system, consisting of a solvent degasser, aquaternary gradient pump before column, diodearray detector (DAD) and a data station with analytical software of Chemstation 8.03 (Agilent, Inc., USA). The method to analyze the anthraquinones in DNts used in this study was well established according to the China Pharmacopoeia Committee (2010 edition). All the separation was performed on a Kromasil® C18 analytical column (250 mm × 4.6 mm, 5 μm) and maintained at 25°C. The mobile phase was a mixture of methanol–0.1% phosphoric acid aqueous solution (85:15, v:v) with the flow rate of 1ml/min. The injection volume was 10 μl and the detection wavelength was set at 254 nm. The standard solutions of five AQs (aloe-emodin 15 μg/mL, emodin 8 μg/mL, chrysophanol 40 μg/mL, rhein 2 μg/mL and physcion 10 μg/mL) were prepared with methanol. Samples were prepared according to China Pharmacopoeia Committee, 2010.
Animal experimental design
Male Wistar rats weighing 220 ± 20 g were used in this study. All rats were maintained in 12-h light/dark cycles and given free access to water and standard chow. All animal care and use procedures were in accordance with the guidelines of the Institutional Animal Care and Use Committee and were approved by the Animal Experimentation Ethics Committee at the Shanghai University of Traditional Chinese Medicine under the guidelines of the National Health and Medical Research Council of China. The animals were randomly divided into the following treatment groups (n = 8 rats/group): (1) Control group (Vehicle group), (2) ANIT group, (3) DNts + ANIT group, (4) DNts group. The rats in Control and ANIT groups were pretreated with 0.5% sodium carboxymethylcellulose (CMCNa) solution alone; the other two groups were intragastrically treated daily with the powder of DNts which was suspended in 0.5% CMCNa solution at a dose of 3.0 g/kg B.W. for 9 consecutive days, respectively. At 12 h after the seventh administration of test drugs, groups 2 and 3 were intragastrically received ANIT dissolved in peanut oil at a dose of 60 mg/kg B.W.. The Control and DNts groups were intragastrically given the peanut oil in an equal volume as for groups 2 and 3 (5 ml/kg B.W.). Blood samples were collected from inferior vena cava at 0, 12, 24, and 48 h after ANIT administration and separated, and then stored at −80°C for later analysis of biochemical parameters.
At 48 h post ANIT administration, the rats were anaesthetized with urethane (1 g/kg B.W., i.p.). The bile duct was cannulated with Closed IV Catheter System (inner diameter, 0.36 mm; outer diameter, 0.71 mm; Becton, Dickinson and Co., Suzhou, China). After the operation, Bile specimens were collected into tubes for 1 h, as previously described. Then, the animals were killed by decapitation, the liver and kidney of rats were collected and stored in liquid nitrogen for analysis of enzyme levels and gene or protein expressions.
Serum biochemistry analysis
Serum was collected at 0, 12, 24, 36 and 48 h after ANIT treatment. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin (TBil), direct bilirubin (DBil) and total bile acid (TBA) were determined using commercially available clinical test kits and an automatic biochemical analyzer (HITACHI 7080, Japan).
Bile acid and bilirubin analysis
Bile acids were extracted from liver samples using 75% ethanol, as previously described. Total bile acids in the liver, bile, and urine were measured using commercial assay kits from the Jiancheng Institute of Biotechnology (Nanjing, China). Total bilirubin in the bile and urine were also measured with commercial assay Kits from the Jiancheng Institute of Biotechnology (Nanjing, China) in accordance with the manufacturer’s protocols.
Quantitative real-time PCR analysis
Primer sequences used for quantitative real-time PCR in this study
(GenBank accession no.)
CTACCTCCTCCCT GATGCCCTT (fw)
TCCTCTGTCCCTGAGCCTTATC (fw) TCTCTGCTTTCACCATTTATTACAA (rev)
Preparation of liver membranes and for Western analysis
Plasma membranes or intracellular microsomal membranes were separated by differential centrifugation. Portions of the liver were homogenized with a teflon pestle (20 strokes at 3,000 rpm) in 0.3 mol/L sucrose containing 0.1 mmol/L PMSF, 25 μg/mL leupeptin, 5μg/mL aprotinin, and 5 μg/mL pepstatin A (50 mg liver/mL buffer) according previous report. The homogenate was used for membrane preparation as described previously. Protein concentrations were measured according to the method of BCA using bovine serum albumin as a standard.
Western blot studies
Immunoblotting and subsequent densitometry were performed with mixed plasma membranes, and intracellular membranes were incubated with antibodies against Mrp2 (ab3373, 1:50, abcam) and Na+/K+-ATPase (#3010, 1:1000, Cell Signaling). Moreover, the crude membranes were prepared according to the protocol of protein extraction kit (BioVision, USA). Equal amounts of crude membranes (40 μg/lane) were separated on 6%-15% SDS-PAGE and transferred to nitrocellulose membrane. Membranes were blocked in 5% skim milk and incubated with antibodies against Bsep (sc-25571, 1:200, Santa Cruz), Ntcp (#6522-1, 1:1000, epitomics), Oatp1 (LS-C113034-50, 1:1000, LifeSpan BioSciences), Mrp3(ab3375, 1:50, abcam), Mdr2 (SAB2100008, 1:1000, sigma) and Na+/K+-ATPase (#3010, 1:1000, Cell Signaling), respectively. Blots were incubated with horseradish peroxidase conjugated secondary antibodies (Santa Cruz) and developed by ECL detection regents (Amersham). The protein bands were quantified by the average ratios of integral optic density (IOD) following normalization to Na+/K+-ATPase expression.
Determination of liver Ugt1a1, Cyp2b1 and Sult2a1 levels
Liver segments were homogenized in ice-cold PBS. The homogenates were centrifuged at 3,000 g for 10 min and the supernatants were assayed for the determination of levels of the Ugt1a1, Cyp2b1 (CUSABIO, CN) and Sult2a1 (USCN Life Science,CN) by using ELISA kits according to the manufacturer’s protocols. The results are expressed as folds of control.
The data were analyzed using a SPSS 16.0 statistical package. Multiple comparisons were performed by one-way analysis of variance (ANOVA) followed by LSD t-test. Difference was considered statistically significant when p ≤ 0.05, and very significant when p ≤ 0.01 and p ≤ 0.001. All results are presented as the mean ± SD.
HPLC determination of anthraquinones in DNts
The contents of five anthraquinones in DNts
Effects of DNts on the levels of serum enzymes and components in ANIT-induced cholestasis rats
Effects of DNts on the levels of bile acids and bilirubin in the liver, urine, and bile after ANIT treatment
Effects of DNts on gene expressions of transporter and metabolic enzyme in ANIT-induced cholestasis rats
Secondly, hepatic gene expressions of canalicular efflux transporters were determined (Figure 4B). The mRNA level of Mdr2 was significantly up-regulated by ANIT (Figure 4C; p < 0.01). There were no changes in mRNA levels for Mrp2 and Bsep after ANIT treatment (Figure 4C). DNts pretreatment resulted in substantial elevation of Mdr2 and Bsep mRNA levels in ANIT-intoxicated rats (Figure 4C; p < 0.05).
Thirdly, we examined the hepatic gene expressions of metabolic enzymes such as Cyp2b1, Ugt1a1 and Sult2a1 (Figure 4D). The mRNA levels of Cyp2b1 and Ugt1a1 in ANIT-treated rats were significantly decreased (Figure 4D; p < 0.01). DNts pretreatment significantly reversed ANIT- induced reduction of the Cyp2b1 and Ugt1a1 mRNA levels (Figure 4D; p < 0.05), but did not affect the mRNA expression of Sult2a1 in ANIT-intoxicated rats (Figure 4D). Otherwise, DNts pretreatment alone did not affect mRNA levels of Oatp1, Ntcp, Mrp3, Bsep, Mdr2, Mrp2, Cyp2b1, Ugt1a1 and Sult2a1 (Figure 4D).
Effect of DNts on protein expressions of Ntcp, Oatp1, Mrp3, Mdr2, Bsep and Mrp2 in the liver membranes of ANIT-induced cholestasis rats
Effect of DNts on the protein levels of Cyp2b1, Ugt1a1 and Sult2a1 in the liver of ANIT-induced cholestasis rats
The pretreatment of DNts significantly reversed ANIT-induced elevation of serum ALT, AST, ALP, T-Bil, D-Bil and TBA levels at 48h after ANIT treatment in consistent with previous research. We presented here the evidence that DNts protected rats from the accumulation of bile acids in the liver and subsequent development of liver injury with cholestasis induced by ANIT, in association with an augmentation of bile acid and bilirubin excretion of bile and urine.
In the current study, we further investigated whether the protective effect of DNts against ANIT-induced cholestasis might be related to the expression of metabolic enzymes and transporters. The metabolic enzymes participate in critical processes including the biosynthesis of bile acids and the detoxification of accumulating biliary compounds. The hepatic metabolic enzymes may render hydrophobic substrates less toxic and better soluble for biliary and urinary excretion via phase I hydroxylation and phase II conjugation to counteract cholestatsis. Our findings displayed that DNts pretreatment significantly up-regulated both mRNA and protein expressions of hepatic metabolic enzymes including Cyp2b1 and Ugt1a1 in ANIT-induced cholestasis rats. These results indicated that the up-regulation of mRNA and protein expressions of the phase I hydroxylation enzymes (Cyp2b1) and phase II conjugation enzymes (Ugt1a1) induced by DNts might counteract cholestatic liver damage by detoxification of accumulating biliary compounds.
Basolateral uptake transporters, such as Ntcp and Oatps, transport bile acids and organic anions from sinusoidal blood into hepatocytes[9–11]. In contrast, basolateral export transporters, such as Mrp3, Mrp4 and Ostα/β transport bilirubin glucuronide and bile acids, respectively, from hepatocytes into blood during cholestasis[10, 25]. Bile acids transport at canalicular membrane of the hepatocyte is mediated by ATP-binding cassette transporters, including Bsep, Mrp2, and Mdr2. The export of toxic bile acids by canalicular transporters is probably an alternative escape route during intrahepatic cholestasis. At 48 h after ANIT treatment, we found that spontaneous hepatic anticholestatic defenses which comprised down-regulation of import systems (e.g., Ntcp, Oatp1) and adaptive induction of basolateral alternative export pump (e.g.,Mrp3) as well as the canalicular transporters, such as MDR2 were formed in ANIT- intoxicanted rats. However, intrinsic hepatocellular adaptive induction of transporters in cholestasis is too weak to prevent ongoing liver injury. Indeed, DNts pretreatment induced mRNA and protein expressions of hepatic canalicular export transporters Bsep and Mdr2, beyond what was seen following administration of ANIT alone. In addition, DNts reversed ANIT-induced down-regulation of mRNA and protein levels of Oatp1 and Ntcp for the attenuation of cholestasis has been performed.
Mrp2, which is another key canalicular transporter, belongs to ATP-binding cassette proteins localized to the canalicular domain of the hepatocyte. Recent studies have demonstrated that, Mrp2 undergoes endocytic retrieval from the canalicular membrane into an intracellular compartment in several models of cholestasis[20, 28]. Our results showed that hepatic mRNA expression and protein levels of MRP2 did not changed in ANIT-treated rats. In contrast, Mrp2 in intracellular membranes was significantly increased in ANIT-intoxicated rats. The lack of change of Mrp2 content in mixed plasma membranes in response to ANIT may be tentatively explained by the fact that intracellular accumulation of Mrp2 in enriched microsomal membranes represents a small fraction of the total amount of transporter in the cell according to previous report. It is possible that the protective effects of DNts were through other alternative mechanisms for the elevation of renal and biliary excretion of bile acid and bilirubin with unchanged expression of total Mrp2 in the kidney and liver.
Furthermore, Liver disease is often accompanied by renal dysfunction. After ANIT treatment, renal mRNA expressions of Mrp2, Mrp3, Mrp4 and Ostβ were significantly up-regulated. In contrast, renal mRNA level of Asbt was significantly down-regulated, accompanied by increased concentrations of bile acid and bilirubin in urine. These results indicated that adaptive responses to cholestasis also occur in the kidney in an effort to eliminate excess bile acids and toxic compounds from circulation which is according with previous report. In addition, DNts pretreatment further increased the renal mRNA level of Ostβ in ANIT-intoxicated rats, accompanied by further increase in urinary excretion of bile acid and bilirubin (Figure 7).
In summary, Danning tablets provides protection from ANIT-induced cholestasis with liver injury through adaptive responses in both the kidney and liver that enhance the expressions of hepatic canalicular efflux transporters (Bsep and Mdr2), renal efflux transporter Ostβ and bile acid-detoxifying enzyme (Cyp2b1 and Ugt1a1) and attenuate the hepatic Mrp2 translocation, accompanied by further increase in urinary and biliary excretion of bile acid and bilirubin.
Total bile acid
Uridine diphosphate-5¢-glucuronosyl transferase
Na+ taurocholate cotransporting polypeptide
Organic anion-transporting polypeptide
Organic solute transporter
Multidrug resistance-associated protein
Bile salt export pump
Multidrug resistance protein
Apical sodium dependent bile acid transporter
This work is financially supported by the Natural Science Foundations of China (81073027, 81303186), Shanghai Rising-Star Program (09QA1405500), Shanghai Science and Technology Commission Project (10DZ1970200) and Scientific Innovation Project from Shanghai Municipal Education Commission (09YZ125) and China Postdoctoral Science Foundation (2013M531202).
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