Effect of genipin on cholestasis induced by estradiol‐17β‐glucuronide and lithocholate‐3‐O‐glucuornide in rats

Aim: Genipin is reported to stimulate the insertion of multidrug resistance protein 2 (Mrp2) in the bile canalicular membrane, thereby causing choleresis by the increased the biliary excretion of glutathione, which has been considered to be a substrate of Mrp2. In the present study, we examined the effect of genipin on cholestasis induced by estradiol‐17β‐glucuronide and lithocholate‐3‐O‐glucuronide, Mrp2 substrates, in rats. Further, the effect of genipin on the biliary excretion of substrates of P‐glycoprotein (P‐gp), vinblastine and erythromycin, was also studied. Methods: The effect of genipin infusion at the rate of 0.5 µmol/min/100 g on cholestasis induced by estradiol‐17β‐glucuronide (0.075 µmol/min/100 g for 20 min) and lithocholate‐3‐O‐glucuronide (0.15 µmol/min/100 g for 40 min) was studied. The effect of genipin infusion on the biliary excretion of a tracer dose of vinblastine and erythromycin infused at the rate of 0.1 µmol/min/100 g was also studied. Results: Genipin relieved estradiol‐17β‐glucuronide‐induced cholestasis, and cumulative biliary estradiol‐17β‐glucuronide excretion for 120 min was increased from 50 ± 20%–81 ± 20% dose. In contrast, genipin had no effect on lithocholate‐3‐O‐glucuronide‐induced cholestasis. Biliary excretion of a tracer dose of vinblastine and the maximum biliary excretion of erythromycin were significantly decreased by genipin. Conclusions: Genipin protected estradiol‐17β‐glucuronide‐induced cholestasis. The mechanism of the protection of cholestasis by genipin is unknown, but it is speculated to be due to a conformational change of P‐gp by genipin, in addition to the stimulation of Mrp2 insertion into the bile canaliculi.     

Effect of penicillin G on the biliary excretion of cholephilic compounds in rats

Aim

Penicillin G is reported to increase bile flow by increasing biliary glutathione excretion, as well as the biliary excretion of penicillin G itself. In order to study the effect of penicillin G on the hepatic excretory pathway, the effect of colchicine and genipin on the increase of biliary glutathione excretion induced by penicillin G was studied in rats. The effect of penicillin G on the biliary excretion of sulfobromophthalein and erythromycin was also studied, together with the effect of penicillin G on cholestasis induced by estradiol-17??-glucuronide.

Methods

After bile duct cannulation, penicillin G was administered to rats at the rate of 0.5???mol/min/100?g. The effect was examined of colchicine pretreatment (0.2?mg/100?g) and genipin administration (0.5???mol/min/100?g) on biliary glutathione excretion increased by penicillin G infused at the rate of 0.5???mol/min/100?g. The effect of penicillin G on the biliary excretion of sulfobromophthalein and erythromycin (0.2 and 0.1???mol/min/100?g for 90?min, respectively) was studied, together with the effect of penicillin G on cholestasis induced by estradiol-17??-glucuronide (0.075???mol/min/100?g for 20?min).

Results

Penicillin G increased bile flow and biliary glutathione excretion, which were not inhibited by colchicine or genipin. Biliary penicillin G excretion was markedly reduced in Eisai hyperbilirubinemic rats (EHBR) and Mrp2-deficient rats. Biliary sulfobromophthalein and erythromycin excretion was unchanged by penicillin G. Cholestasis induced by estradiol-17??-glucuronide was not relieved by penicillin G.

Conclusions

It was shown that colchicine-sensitive vesicular transport has no role on the penicillin G-induced insertion of Mrp2 into the canalicular membrane, as has been observed with genipin. Although the choleresis of penicillin G is thought to be due to the increased biliary excretion of glutathione and penicillin G itself by Mrp2, the mechanism of Mrp2 insertion by penicillin G is thought to be partly different from that by genipin.     

Effects of colchicine on the maximum biliary excretion of cholephilic compounds in rats

BACKGROUND AND AIM: Colchicine, an inhibitor of intracellular vesicular transport, has been reported to inhibit the biliary excretion of bile acids and organic anions, but the previous findings are controversial. In order to systematically evaluate the effect of colchicine on the biliary excretion of cholephilic compounds, we studied the effect of colchicine on the biliary excretion of substrates of various canalicular transporters, which were administered at or above the excretory maximum in rats. METHODS: Substrates of various canalicular adenosine triphosphate-binding-cassette transporters were infused at or above the rate of maximum excretion into rats, and the effect of colchicine (0.2 mg/100 g), which was intraperitoneally injected 3 h before, on the biliary excretion was studied. Furthermore, the effect of tauroursodeoxycholate (TUDC) co-infusion on the biliary excretion of taurocholate (TC) after colchicine treatment was also studied. RESULTS: The biliary excretion of TC and cholate administered at the rate of 1 micro mol/min/100 g was markedly inhibited by colchicine, whereas that of TUDC was not inhibited even with the infusion rate of 2 micro mol/min/100 g. TUDC co-infusion at the rate of 1 micro mol/min/100 g increased the biliary excretion of TC (1 micro mol/min/100 g), which was decreased by the colchicine pretreatment. The biliary excretory maximum of taurolithocholate-sulfate and sulfobromophthalein, substrates of the multidrug resistance protein 2, of erythromycin, a substrate of the P-glycoprotein, and of indocyanine green were not affected by colchicine. CONCLUSIONS: The different excretory maximums of TC and TUDC and the different effect of colchicine on the excretion of these bile acids are considered to be a result of different regulatory mechanisms of vesicular targeting of the bile salt export pump to the canalicular membrane by these bile acid conjugates. The vesicular targeting of the multidrug resistance protein 2 and the P-glycoprotein to the canalicular membrane is considered to be colchicine insensitive in the absence of bile acid coadministration.     

Biliary excretion of bile acids and organic anions in rats with dichloroethylene-induced bile canalicular injury

Background Hepatocytes in zone 1 of the hepatic lobule play a role in the uptake and biliary excretion of bile acids and organic anions under physiological conditions, and hepatocytes in zone 3 may play a role only when there is a high-dose load. To further elucidate the role of hepatic zonation in the hepatic handling of bile acids and organic anions, the biliary excretion of these compounds was studied in rats with dichloroethylene (DCE)-induced selective zone 3 bile canalicular injury.Methods Biliary excretion of various bile acids and organic anions was studied in rats 1h after oral administration of DCE (5mg/100g). The effect of DCE on the immunostaining of multidrug resistance protein 2 (Mrp2; an important canalicular organic anion transporter) in the liver was also examined.Results The biliary excretory maximum of taurocholate and tauroursodeoxycholate was decreased in DCE-treated rats, whereas the biliary excretion of taurolithocholate-sulfate and phenolphthalein-glucuronide was unchanged in DCE-treated rats, and DCE treatment decreased the biliary excretion of sulfobromophthalein and pravastatin. DCE decreased Mrp2 staining in the canalicular membrane of zone 3 hepatocytes on immunohistochemistry.Conclusions These findings indicate that canalicular transport in zone 3 hepatocytes is important in the biliary excretion of bile acids and organic anions, when they are administered at high doses.     

Mechanisms of biliary excretion of lithocholate-3-sulfate in Eisai hyperbilirubinemic rats (EHBR)

Biliary excretion of lithocholate-3-sulfate is markedly impaired in EHBR. To examine the mechanism of biliary lithocholate-3-sulfate excretion in EHBR, the effects of colchicine treatment, a vesicular transport inhibitor, and infusion of taurocholate and organic anions were studied in EHBR and Sprague-Dawley rats. Colchicine treatment and taurocholate infusion had no effect on biliary lithocholate-3-sulfate excretion in EHBR, suggesting that biliary lithocholate-3-sulfate excretion is not mediated by the vesicular transport or by the bile acid excretory pathway. In control Sprague-Dawley rats, both sulfobromophthalein and dibromosulfophthalein infusion inhibited biliary lithocholate-3-sulfate excretion. In contrast, in EHBR dibromosulfophthalein infusion inhibited biliary lithocholate-3-sulfate excretion but BSP infusion did not. Indocyanine green and pravastatin infusion did not affect biliary lithocholate-3-sulfate excretion but pravastatin infusion had no effect in EHBR. These findings indicate that, whether physiologically important or not, two or more excretory pathways for organic anions exist at the canalicular membrane other than the ATP-dependent one.This study was funded in part by a Grant-in-Aid for General Scientific Research (C) 06670583 from the Japanese Ministry of Education, Science and Culture.     

Effect of cyclosporin A on the biliary excretion of cholephilic compounds in rats.

Cyclosporin A (CsA) is known to cause cholestasis. CsA is reported to competitively inhibit the transport of the substrates of the bile salt export pump (Bsep), multidrug resistance protein 2 (Mrp2) and P-glycoprotein (P-gp) in the canalicular membrane vesicles. However, the inhibitory effect of CsA on various substrates of the canalicular ATP-dependent transporters in vivo is unknown. Therefore, in the present study, the acute effect of CsA on the biliary excretion of the substrates of Bsep, Mrp2 and P-gp was examined under the same condition. Ten minutes after the intravenous administration of CsA (25mg/kg), the biliary excretion of various bile acids and organic anions and cations was studied. CsA decreased the biliary excretion of tracer amounts of taurocholate, leukotriene C(4), estradiol-17beta-glucuronide, pravastatin, vinblastine and erythromycin. In contrast, the biliary excretion of high doses of taurocholate and sulfobromophthalein was only slightly or not inhibited by CsA. In conclusion, CsA may competitively inhibit biliary excretion of substrates of Bsep, Mrp2 and P-gp also in vivo, and CsA is considered to inhibit bile acid-dependent bile flow by the competitive inhibition of the canalicular transport of bile acids by Bsep.     

Changes in biliary excretory mechanisms in bile duct-ligated rat

The effects of bile duct ligation on biliary excretion of bile acids, glutathione, and lipids were studied in the rat. The bile duct of the rat was ligated for three days. The biliary bile acid excretion after bile duct cannulation was higher at first, but after 90 min became lower than that in the control rat. The bile flow in the bile duct-ligated rat was higher after bile duct cannulation and gradually decreased to the same level as in the control rat. Biliary glutathione excretion, which has been suggested to be a driving force for the bile acid-independent canalicular bile flow, was markedly decreased in the bile duct-ligated rat. The mannitol clearance was increased and the bile ductules showed proliferation in the bile duct-ligated rat, suggesting an increase in the ductular bile flow. Biliary excretion of lithocholate glucuronide was more markedly impaired than that of taurocholate. When taurocholate was infused at higher rates, which increases bile flow and biliary excretion of bile acid and lipids in the control rat, biliary bile acid and lipid excretion remained constant in the bile duct-ligated rat. These findings indicate that, in the bile duct-ligated rat, the ductular bile flow was increased and bile acid-independent canalicular bile flow was decreased and that, although the biliary excretion of bile acids was not as impaired as that of organic anions, the capacity of bile acid and lipid excretion was markedly decreased.     

Biliary excretion of taurocholate, organic anions and vinblastine in rats with alpha-naphthylisothiocyanate-induced cholestasis

BACKGROUND AND AIMS: alpha-Naphthylisothiocyanate (ANIT) is known to cause cholestasis due to injury of the bile duct epithelial cells. The aim of the present study was to examine the effect of a single dose of ANIT on the biliary excretion of various cholephilic compounds and on the amount of canalicular transporters. METHODS: Twenty-four hours after the oral administration of ANIT (100 mg/kg), the biliary excretion of taurocholate, leukotriene C(4), pravastatin and vinblastine was studied. The protein levels of the bile salt export pump and multidrug resistance protein 2 and the immunostaining of multidrug resistance protein 2 in the liver were also examined. RESULTS: The ANIT treatment markedly decreased the biliary excretion of tracer amounts of taurocholate, leukotriene C(4), pravastatin and vinblastine. The biliary excretory maximum of taurocholate was also markedly decreased after ANIT treatment. The ANIT treatment had no effect on the protein levels of bile salt export pump and multidrug resistance protein 2 and the immunostaining of multidrug resistance protein 2 in the liver. CONCLUSIONS: These findings support canalicular transporters having little effect on the marked impairment of biliary excretion of cholephilic compounds in ANIT-induced cholestasis.     

Biliary excretion of sulfated bile acids and organic anions in zone 1- and zone 3-injured rats

Background and Aims: There are several reports on the biliary excretion of bile acids and organic anions in zone 1‐ and zone 3‐injured rat liver, but the results are controversial. In order to dissolve the discrepancy between previous works about the role of hepatic zonation on the hepatic handling of the substrates of multidrug resistance protein 2, the biliary excretion of sulfated bile acids, pravastatin and phenolphthalein glucuronide was studied in zone 1‐ and zone 3‐injured rats. Methods: Zone 1 and zone 3 injury were caused by allyl alcohol and bromobenzene, respectively. Bile acid sulfates, pravastatin and phenolphthalein glucuronide were administered i.v. to bile duct‐cannulated rats, and their biliary excretion was studied. Results: The biliary excretion of a tracer dose of taurolithocholate‐3‐sulfate and its excretory maximum were unchanged in zone 1 injury, but were diminished in zone 3 injury, whereas the biliary excretion of taurochenodeoxycholate‐3‐sulfate was unchanged in zone 1 and zone 3 injury. The biliary excretion of pravastatin and phenolphthalein glucuronide was markedly decreased only in zone 3 injury, whereas the excretory maximum of phenolphthalein glucuronide was decreased in both zone 1 and zone 3 injury. Conclusions: These findings indicate that zone 3 is important for the biliary excretion of substrates of multidrug resistance protein 2.     

Effect of bile acids on the biliary excretion of pravastatin in rats

Aim: The biliary excretion of pravastatin, an HMG‐CoA reductase inhibitor, is mediated by the multidrug resistance protein 2, but a recent report suggests that pravastatin is also a substrate of the bile salt export pump, which transports bile acids. We examined the effects of bile acids on biliary pravastatin excretion in rats. Methods: The effect of taurocholate on biliary pravastatin excretion, and that of pravastatin on biliary taurocholate excretion was examined in bile‐drained rats. Results: Taurocholate had no effect on biliary pravastatin excretion, whereas pravastatin with a dose to cause biliary excretory maximum significantly inhibited biliary taurocholate excretion. Conclusion: These data indicate that increased serum bile acids will not affect the pharmacokinetics of pravastatin in patients with hepatobiliary diseases. Although pravastatin inhibited biliary taurocholate excretion, it is unlikely that pravastatin significantly inhibits biliary bile acid excretion by its therapeutic doses.     

Genipin enhances Mrp2 (Abcc2)-mediated bile formation and organic anion transport in rat liver

Inchin-ko-to (ICKT), an herbal medicine, and its ingredients exert potent choleretic effects by a "bile acid-independent" mechanism. The current study was designed to determine whether ICKT or its ingredients potentiate multidrug resistance-associated protein 2 (Mrp2; Abcc2)-mediated choleresis in vivo. Biliary secretion of Mrp2 substrates and the protein mass, subcellular localization, and messenger RNA (mRNA) level of Mrp2 were assessed in rat liver after infusion of genipin, an intestinal bacterial metabolite of geniposide, a major ingredient of ICKT. The function of Mrp2 was also assessed by the adenosine triphosphate (ATP)-dependent uptake of Mrp2-specific substrates using canalicular membrane vesicles (CMVs) from the liver. Infusion of genipin increased bile flow by 230%. It also increased biliary secretion of bilirubin conjugates and reduced glutathione (GSH) by 513% and 336%, respectively, but did not increase bile acid secretion. The ATP-dependent uptake of estradiol 17-beta-D-glucuronide (E(2)17 beta G; by 265%), leukotriene C4 (LTC(4); by 161%), taurolithocholate-3-sulfate (TLC-3S; by 266%), and methotrexate (MTX; by 234%) was significantly stimulated in the CMVs from the liver. These effects were not observed in Mrp2-deficient rats. Under these conditions, genipin treatment increased the protein mass of Mrp2 in the CMVs but not the mRNA level. In immunoelectron microscopic studies, a marked increase in Mrp2 density in the canalicular membrane (CM) and microvilli was observed in the genipin-treated liver tissue sections when compared with the vehicle-treated liver tissue sections. In conclusion, genipin may enhance the bile acid-independent secretory capacity of hepatocytes, mainly by stimulation of exocytosis and insertion of Mrp2 in the bile canaliculi. ICKT may be a potent therapeutic agent for a number of cholestatic liver diseases.     

Characterization of organic anion transporter regulation, glutathione metabolism and bile formation in the obese Zucker rat

BACKGROUND/AIMS: Alterations in hepatobiliary transporters may render fatty livers more vulnerable against various toxic insults. METHODS: We therefore studied expression and function of key organic anion transporters and their transactivators in 8-week-old obese Zucker rats, an established model for non-alcoholic fatty liver disease. RESULTS: Compared to their heterozygous littermates, obese animals showed a significant reduction in canalicular bile salt secretion, which was paralleled by significantly diminished Oatp2 mRNA and protein levels together with reduced nuclear HNF3beta, while expression of bile salt export pump, organic anion transporter (Oatp) 1 and multidrug resistance-associated protein (Mrp) 4 were unchanged. Impaired bile salt-independent bile flow in obese rats was associated with a 50% reduction of biliary secretion of the Mrp 2 model-substrates glutathione disulfide and S-(2,4-dinitrophenyl)glutathione. In line Mrp2 protein expression was reduced by 50% in obese rats. CONCLUSIONS: Oatp2 and Mrp2 expression is decreased in fatty liver and may impair metabolism and biliary secretion of numerous xenobiotics. Reduction of bile salt secretion and absence of biliary GSH excretion may contribute to impaired bile flow and posthepatic disorders associated with biliary GSH depletion.     

Altered localization and activity of canalicular Mrp2 in estradiol-17beta-D-glucuronide-induced cholestasis

Estradiol-17beta-D-glucuronide (E(2)17G), an endogenous metabolite of estradiol, induces a potent dose-dependent and reversible inhibition of bile flow in the rat. We analyzed the effect of a single dose of E(2)17G (15 micromol/kg, intravenously) to female rats on bile flow and the endocytic retrieval and function of the canalicular multidrug resistance-associated protein 2 (Mrp2) and the effect of pretreatment with dibutyryl-cyclic AMP (DBcAMP; 20 micromol/kg) on these measures. Bile flow was maximally inhibited by 85% within 10 minutes of E(2)17G and returned to 50% and 100% of control levels within 75 and 120 minutes, respectively. Western analysis of total homogenates and mixed plasma and intracellular membranes suggested partial internalization of Mrp2 during the acute phase of cholestasis at 20 minutes and during the period of recovery from cholestasis at 75 minutes, which returned to control levels by 180 minutes after E(2)17G. Confocal analysis confirmed Western studies and demonstrated endocytic retrieval of Mrp2 from the canalicular membrane into pericanalicular and intracellular domains. The biliary concentration and excretion of the model Mrp2 substrate, dinitrophenyl-S-glutathione (DNP-SG), was impaired in parallel with the extent of Mrp2 retrieval. Pretreatment with DBcAMP partially protected against maximal E(2)17G cholestasis and the endocytic retrieval and decreased function of Mrp2 at 20 minutes and significantly accelerated the exocytic insertion of Mrp2 into the canalicular membrane and the recovery of bile flow and biliary excretion of DNP-SG. In conclusion, these data indicate that E(2)17G induces endocytic internalization of Mrp2, which occurs in parallel with decreased bile flow and Mrp2 transport activity.     

Effects of colchicine and phenothiazine on biliary excretion of organic anions in rats

Vesicular transport inhibitors have been reported to inhibit biliary excretion of some organic anions, suggesting that vesicular transport has a role in intracellular transport of these compounds. However, these inhibitors are substrates for P-glycoprotein. To examine whether P-glycoprotein has a role in canalicular transport of organic anions in addition to the canalicular multispecific organic anion transporter, we studied the effect of colchicine, a vesicular transport inhibitor, and phenothiazine to increase P-glycoprotein expression on biliary excretion of various organic anions in rats. Colchicine treatment slightly but significantly inhibited biliary excretion of indocyanine green, dinitrophenylglutathione and pravastatin, and had no effect on biliary excretion of sulphobromophthalein and dibromosulphophthalein. Phenothiazine treatment did not affect biliary excretion of indocyanine green and pravastatin, but it increased biliary sulphobromophthalein-glutathione excretion. In conclusion, the present findings suggest that P-glycoprotein plays an additive role on biliary excretion of some organic anions in addition to the canalicular multispecific organic anion transporter.     

Dose-Dependent Conjugation of Sulfobromophthalein and Hepatic Transit Time in Bile Fistula Rats

Sulfobromophthalein (BSP) is selectively taken up by the liver and secreted into the bile as unconjugated and conjugated forms. Our previous study demonstrated that unconjugated BSP, but not conjugated BSP, caused the dissociation of biliary lipid secretion from that of bile acids, suggesting that the hepatic BSP conjugation rate partly regulated biliary lipid secretion. To evaluate the mechanisms through which biliary lipid secretion is regulated by exogenous organic anions, we intravenously administered BSP to male Sprague–Dawley rats at various doses either continuously or as a bolus. Then the relationship of the dose of BSP to its conjugation rate, hepatic transit time, and biliary lipid secretion was determined. BSP decreased biliary secretion of cholesterol and phospholipids in a dose-dependent manner without affecting bile acid secretion. In contrast, the proportion of conjugated BSP in bile was associated with the dose. Although the serum clearance of BSP after bolus infusion was constant regardless of the dose administered (50 or 200 nmol/100 g), BSP secretion was delayed with increasing doses: unconjugated BSP was secreted predominantly in the early phase (0–15 min after bolus injection), and conjugated BSP was the predominant form in the late phase (15–30 min). Pretreatment with colchicine reduced the conjugation rate and hepatic transit time of BSP, suggesting that the microtubule-dependent vesicle pathway plays a role in biliary excretion and conjugation of BSP. We conclude that biliary lipid secretion is influenced by organic anions with an affinity for bile acids such as BSP and that this effect is dependent upon the hepatic metabolic rate, i.e., conjugation rate. The hepatic transit time also plays a key role in this process by influencing metabolism.     

Altered disposition of acetaminophen in mice with a disruption of the Mrp3 gene

MRP3 is an ABC transporter localized in the basolateral membrane of epithelial cells such as hepatocytes and enterocytes. In this study, the role of Mrp3 in drug disposition was investigated. Because Mrp3 preferentially transports glucuronide conjugates, we investigated the in vivo disposition of acetaminophen (APAP) and its metabolites. Mrp3+/+ and Mrp3-/- knockout mice received APAP (150 mg/kg), and bile was collected. Basolateral and canalicular excretion of APAP was also assessed in the isolated perfused liver. In separate studies, mice received 400 mg APAP/kg for assessment of hepatotoxicity. No differences were found in the biliary excretion of APAP, APAP-sulfate, and APAP-glutathione between Mrp3+/+ and Mrp3-/- mice. However, 20-fold higher accumulation of APAP-glucuronide (APAP-GLUC) was found in the liver of Mrp3-/- mice. Concomitantly, plasma APAP-GLUC content in Mrp3-/- mice was less than 10% of that in Mrp3+/+ mice. In addition, APAP-GLUC excretion in bile of Mrp3-/- mice was tenfold higher than in Mrp3+/+ mice. In the isolated perfused liver, we also found a strong decrease of APAP-GLUC secretion into the perfusate of Mrp3-/- livers. Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), and histopathology showed that Mrp3-/- mice are more resistant to APAP hepatotoxicity than Mrp3+/+ mice, which is most likely a result of the faster repletion of hepatic GSH. In conclusion, basolateral excretion of APAP-GLUC in mice is nearly completely dependent on the function of Mrp3. In its absence, sufficient hepatic accumulation occurs to redirect some of the APAP-GLUC to bile. This altered disposition in Mrp3-/- mice is associated with reduced hepatotoxicity.     

Effect of dexamethasone on biliary excretion of bile acids and organic anions in rats

Multidrug resistance protein 2 (Mrp2) is an important transporter for biliary excretion of organic anions, and is reported to be up-regulated by dexamethasone. In the present study, effect of dexamethazone (1 mg/kg) on biliary excretion of bile acids and organic anions was studied in rats. After bile duct cannulation, bile acids and organic anions were intravenously administered, and their biliary excretion was studied. Biliary excretion of tracer doses of taurocholate and taurolithocholate-sulfate was unchanged with dexamethasone. Dexamethasone increased the excretory maximum of taurolithocholate-sulfate and sulfobromophthalein to 1.8 and 1.5 times, respectively, whereas it did not change the excretory maximum of phenolphthalein-glucuronide and taurocholate. Dexamethasone also increased biliary glutathione excretion. These results obtained by in vivo studies can be explained by the up-regulation of Mrp2, which had been reported in in vitro studies.     

Effects of Japanese herbal medicine inchin-ko-to on endotoxin-induced cholestasis in the rat

Background/Objective. Inchin-ko-to (ICKT) is an herbal medicine used in Japan to treat jaundice and liver fibrosis. We investigated the effect of oral ICKT supplementation on endotoxin-induced cholestasis in the rat.Material and methods.Lipopolysaccharide (LPS) injection (1 mg/kg body weight i.p.) was used as a model of sepsis-induced cholestasis. Bile flow, biliary bile salt secretion, biliary glutathione secretion and protein expression of the main hepatobiliary transporters Na(+)-taurocholate-cotransporting peptide (Ntcp), multidrug resistance protein 2 (Mrp2) and bile salt export pump (Bsep) were analyzed by conventional techniques in ICKT treated and non-treated animals.Results.Injection of LPS induced a significant decrease of bile flow (-24%), biliary bile salts (-40%) and glutathione excretion (-70%) as well as a significant decrease in Ntcp (-90%) and Mrp2 (-80%) protein levels. ICKT supplementation partially prevented the effects of LPS determining a less intense reduction in bile flow (-10%), a normalization of glutathione excretion as well as a significant increase in Mrp2 protein levels to 60% of the levels observed in control animals. ICKT administration did not modify the effects of LPS on BS secretion or Ntcp protein levels.Conclusion.Our data show that oral supplementation of ICKT partially prevents LPS-induced cholestasis by increasing Mrp2 protein levels and biliary glutathione excretion thus increasing bile salt-independent flow.