胆石患者术后胆盐转运子BSEP、MRP2、NTCP的水平变化及意义

目的:探讨腹腔镜联合胆道镜微创手术对胆囊结石患者胆盐转运因子胆盐输出泵(BSEP)、多重耐药蛋白2(MRP2)和牛黄胆酸钠转运蛋白(NTCP)水平的影响。方法:选取我院普外科收治的胆囊结石患者20例排除手术和麻醉禁忌症,予腹腔镜联合胆道镜微创手术治疗。治疗结束后,对比治疗前后患者BSEP、MRP2、NTCP水平变化。结果:1治疗后患者肝脏组织中BSEP、MRP2水平明显比治疗前升高,差异有统计学意义(P0.05);2治疗后患者肝组织中NTCP水平与治疗前水平无明显变化,差异无统计学意义(P0.05)。结论:腹腔镜联合胆道镜微创手术治疗胆囊结石能升高患者胆盐转运因子BSEP、MRP2水平,提高患者术后对胆盐的转运和胆汁酸的代谢,降低胆囊结石复发率,对临床具有指导意义,值得临床推广。     

Regulation of MRP2/ABCC2 and BSEP/ABCB11 Expression in Sandwich Cultured Human and Rat Hepatocytes Exposed to Inflammatory Cytokines TNF-α, IL-6, and IL-1β

In the present study MRP2/ABCC2 and BSEP/ABCB11 expression were investigated in sandwich cultured (SC) human and rat hepatocytes exposed to the proinflammatory cytokines. The investigation was also done in lipopolysaccharide (LPS)-treated rats. In SC human hepatocytes, both absolute protein and mRNA levels of MRP2/ABCC2 were significantly down-regulated by TNF-α, IL-6, or IL-1β. In contrast to mRNA decrease, which was observed for BSEP/ABCB11, the protein amount was significantly increased by IL-6 or IL-1β. A discrepancy between the change in BSEP/ABCB11 mRNA and protein levels was encountered in SC human hepatocytes treated with proinflammatory cytokines. In SC rat hepatocytes, Mrp2/Abcc2 mRNA was down-regulated by TNF-α and IL-6, whereas the protein level was decreased by all three cytokines. Down-regulations of both Bsep/Abcb11 mRNA and protein levels were found in SC rat hepatocytes exposed to TNF-α or IL-1β. Administration of LPS triggered the release of the proinflammatory cytokines and caused the decrease of Mrp2/Abcc2 and Bsep/Abcb11 protein in liver at 24 h post-treatment; however, the Mrp2 and Bsep protein levels rebounded at 48 h post-LPS treatment. In total, our results indicate that proinflammatory cytokines regulate the expression of MRP2/Mrp2 and BSEP/Bsep and for the first time demonstrate the differential effects on BSEP/Bsep expression between SC human and rat hepatocytes. Furthermore, the agreement between transporter regulation in vitro in SC rat hepatocytes and in vivo in LPS-treated rats during the acute response phase demonstrates the utility of in vitro SC hepatocyte models for predicting in vivo effects.     

Molecular regulation of sinusoidal liver bile acid transporters during cholestasis.

Impairment of the hepatic transport of bile acids and other organic anions will result in the clinically important syndrome of cholestasis. Cloning of a number of specific hepatic organic anion transporters has enabled studies of their molecular regulation during cholestasis. The best characterized transport system is a 50-51 kDa sodium-dependent taurocholate cotransporting polypeptide (ntcp), which mediates the sodium-dependent uptake of conjugated bile acids at the sinusoidal plasma membrane of hepatocytes. Under physiologic conditions and after depletion of biliary constituents, ntcp remains constitutively expressed throughout the liver acinus. However, both function and expression of ntcp are rapidly down-regulated in rat liver in various models of experimental cholestasis, such as cholestasis induced by common bile duct ligation, estrogen, endotoxin or cytokine treatment. In addition to ntcp, the sinusoidal organic anion transporting polypeptide oatp-1 is also down-regulated at the protein and steady-state mRNA levels in estrogen-cholestasis, but does not affect sodium-independent uptake of taurocholate. The regulation of a recently cloned member of the organic anion transporter family (oatp-2), which is highly expressed in liver, remains to be studied under cholestatic conditions.     

Regulation of organic anion transport in the liver.

In several liver diseases the biliary transport is disturbed, resulting in, for example, jaundice and cholestasis. Many of these symptoms can be attributed to altered regulation of hepatic transporters. Organic anion transport, mediated by the canalicular multispecific organic anion transporter (cmoat), has been extensively studied. The regulation of intracellular vesicular sorting of cmoat by protein kinase C and protein kinase A, and the regulation of cmoat-mediated transport in endotoxemic liver disease, have been examined. The discovery that the multidrug resistance protein (MRP), responsible for multidrug resistance in cancers, transports similar substrates as cmoat led to the cloning of a MRP homologue from rat liver, named mrp2. Mrp2 turned out to be identical to cmoat. At present there is evidence that at least two mrp''s are present in hepatocytes, the original mrp (mrp1) on the lateral membrane, and mrp2 (cmoat) on the canalicular membrane. The expression of mrp1 and mrp2 in hepatocytes appears to be cell-cycle-dependent and regulated in a reciprocal fashion. These findings show that biliary transport of organic anions and possibly other canalicular transport is influenced by the entry of hepatocytes into the cell cycle. The cloning of the gene for cmoat opens up new possibilities to study the regulation of hepatic organic anion transport.     

Molecular cloning and characterization of the murine bile salt export pump

Hepatic bile salt secretion and bile formation are essential functions of the mammalian liver, and the rate-limiting step of hepatocellular secretion of bile salts is canalicular secretion. Recently, the rat sister-of-p-glycoprotein/bile salt export pump (spgp/BSEP) was demonstrated to encode for the rat ATP-dependent canalicular bile salt export protein, and mutations of human BSEP were identified as the cause of PFIC 2. Since mouse models are vital for studies in hepatocellular transport and metabolism, cloning and characterization of the murine gene are essential. In this study, we have cloned a full-length, functional cDNA for the mBsep. The deduced amino acid sequence encodes for a 1321-amino-acid protein and is 94% similar to rat and 89% similar to human bsep. Western immunoblotting using an antibody directed against a carboxy-terminal peptide of mbsep protein reveals a 160kDa protein, which is highly enriched in mouse canalicular membranes. Transfection of mBSEP into Sf-9 insect cells or mammalian Balb-3T3 cells confers functional transport of the bile salt taurocholate. The mBsep mRNA is expressed in murine liver, but not in other tissues. Hepatic mBsep levels appear highly regulated, being markedly diminished in both LPS and estrogen models of cholestasis. These data are important for further murine studies of hepatocellular transport physiology and metabolism.     

Histone H3K4 trimethylation by MLL3 as part of ASCOM complex is critical for NR activation of bile acid transporter genes and is downregulated in cholestasis

The nuclear receptor Farnesoid x receptor (FXR) is a critical regulator of multiple genes involved in bile acid homeostasis. The coactivators attracted to promoters of FXR target genes and epigenetic modifications that occur after ligand binding to FXR have not been completely defined, and it is unknown whether these processes are disrupted during cholestasis. Using a microarray, we identified decreased expression of mixed lineage leukemia 3 (MLL3), a histone H3 lysine 4 (H3K4) lysine methyl transferase at 1 and 3 days of post-common bile duct ligation (CBDL) in mice. Chromatin immunoprecipitation analysis (ChIP) analysis revealed that H3K4me3 of transporter promoters by MLL3 as part of activating signal cointegrator-2 -containing complex (ASCOM) is essential for activation of bile salt export pump (BSEP), multidrug resistance associated protein 2 (MRP2), and sodium taurocholate cotransporting polypeptide (NTCP) genes by FXR and glucocorticoid receptor (GR). Knockdown of nuclear receptor coactivator 6 (NCOA6) or MLL3/MLL4 mRNAs by small interfering RNA treatment led to a decrease in BSEP and NTCP mRNA levels in hepatoma cells. Human BSEP promoter transactivation by FXR/RXR was enhanced in a dose-dependent fashion by NCOA6 cDNA coexpression and decreased by AdsiNCOA6 infection in HepG2 cells. GST-pull down assays showed that domain 3 and 5 of NCOA6 (LXXLL motifs) interacted with FXR and that the interaction with domain 5 was enhanced by chenodeoxycholic acid. In vivo ChIP assays in HepG2 cells revealed ligand-dependent recruitment of ASCOM complex to FXR element in BSEP and GR element in NTCP promoters, respectively. ChIP analysis demonstrated significantly diminished recruitment of ASCOM complex components and H3K4me3 to Bsep and Mrp2 promoter FXR elements in mouse livers after CBDL. Taken together, these data show that the "H3K4me3" epigenetic mark is essential to activation of BSEP, NTCP, and MRP2 genes by nuclear receptors and is downregulated in cholestasis.     

Kupffer cell-mediated down regulation of rat hepatic CMOAT/MRP2 gene expression

Lipopolysaccharides (LPS) induces intrahepatic cholestasis and canalicular multispecific organic anion transporter (CMOAT/MRP2) plays a central role in hepatic bilirubin transport. This study examined the role of Kupffer cell in LPS-induced cholestasis. Rats were injected intravenously with LPS. Kupffer cells were inactivated with gadolinium chloride (Gd). CMOAT/MRP2 mRNA expression was time- and dose-dependently decreased by LPS injection with a decrease in bile flow and an increase in serum bilirubin level. Gd pretreatment inhibited decrease in CMOAT/MRP2 mRNA and bile flow, and increase in serum bilirubin. Kupffer cell-conditioned medium decreased CMOAT/MRP2 expression. Addition of anti-IL-1 or anti-TNFalpha antibody restored CMOAT/MRP2 expression, whereas IL-1 and TNFalpha decreased the expression. MAP kinases were activated by addition of the conditioned medium, and addition of PD98059 or SB203580 restored CMOAT/MRP2 expression. These results suggest that LPS activates Kupffer cells to secrete IL-1 and TNFalpha, which in turn activate MAP kinases and decrease CMOAT/MRP2 expression.     

Oxidative stress is a triggering factor for LPS-induced Mrp2 internalization in the cryopreserved rat and human liver slices

Cholestasis develops during inflammation and is characterized as occurring under oxidative stress. We have described the internalization of multidrug resistance-associated protein 2 (Mrp2), a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid or lipopolysaccharide (LPS)-induced acute oxidative stress in rat liver. However, it remains unclear whether canalicular Mrp2 internalization is observed in human liver under conditions of acute oxidative stress. In this study, we examined the effect of dimerumic acid (DMA), an antioxidant and found in traditional Chinese medicine, on endotoxin-induced Mrp2 internalization in rat and human liver slices. At 1.5 h following LPS treatment (100 μg/mL), canalicular Mrp2 localization was disrupted without changing the expression of Mrp2 protein or the integrity of filamentous actin in the rat and human liver slices. Pretreatment with DMA (10 μM) counteracted LPS-induced subcellular distribution of Mrp2. Our data clearly indicated that LPS-induced short-term rapid retrieval of Mrp2 from the canalicular surface resulted from LPS-induced oxidative stress in rat and human liver slices.     

Lithocholic acid decreases expression of bile salt export pump through farnesoid X receptor antagonist activity

Bile salt export pump (BSEP) is a major bile acid transporter in the liver. Mutations in BSEP result in progressive intrahepatic cholestasis, a severe liver disease that impairs bile flow and causes irreversible liver damage. BSEP is a target for inhibition and down-regulation by drugs and abnormal bile salt metabolites, and such inhibition and down-regulation may result in bile acid retention and intrahepatic cholestasis. In this study, we quantitatively analyzed the regulation of BSEP expression by FXR ligands in primary human hepatocytes and HepG2 cells. We demonstrate that BSEP expression is dramatically regulated by ligands of the nuclear receptor farnesoid X receptor (FXR). Both the endogenous FXR agonist chenodeoxycholate (CDCA) and synthetic FXR ligand GW4064 effectively increased BSEP mRNA in both cell types. This up-regulation was readily detectable at as early as 3 h, and the ligand potency for BSEP regulation correlates with the intrinsic activity on FXR. These results suggest BSEP as a direct target of FXR and support the recent report that the BSEP promoter is transactivated by FXR. In contrast to CDCA and GW4064, lithocholate (LCA), a hydrophobic bile acid and a potent inducer of cholestasis, strongly decreased BSEP expression. Previous studies did not identify LCA as an FXR antagonist ligand in cells, but we show here that LCA is an FXR antagonist with partial agonist activity in cells. In an in vitro co-activator association assay, LCA decreased CDCA- and GW4064-induced FXR activation with an IC(50) of 1 microm. In HepG2 cells, LCA also effectively antagonized GW4064-enhanced FXR transactivation. These data suggest that the toxic and cholestatic effect of LCA in animals may result from its down-regulation of BSEP through FXR. Taken together, these observations indicate that FXR plays an important role in BSEP gene expression and that FXR ligands may be potential therapeutic drugs for intrahepatic cholestasis.     

Vectorial transport of unconjugated and conjugated bile salts by monolayers of LLC-PK1 cells doubly transfected with human NTCP and BSEP or with rat Ntcp and Bsep

Na(+)-taurocholate-cotransporting peptide (NTCP)/SLC10A1 and bile salt export pump (BSEP)/ABCB11 synergistically play an important role in the transport of bile salts by the hepatocyte. In this study, we transfected human NTCP and BSEP or rat Ntcp and Bsep into LLC-PK1 cells, a cell line devoid of bile salts transporters. Transport by these cells was characterized with a focus on substrate specificity between rats and humans. The basal to apical flux of taurocholate across NTCP- and BSEP-expressing LLC-PK1 monolayers was 10 times higher than that in the opposite direction, whereas the flux across the monolayer of control and NTCP or BSEP single-expressing cells did not show any vectorial transport. The basal to apical flux of taurocholate was saturated with a K(m) value of 20 microM. Vectorial transcellular transport was also observed for cholate, chenodeoxycholate, ursodeoxycholate, their taurine and glycine conjugates, and taurodeoxycholate and glycodeoxycholate, whereas no transport of lithocholate was detected. To evaluate the respective functions of NTCP and BSEP and to compare them with those of rat Ntcp and Bsep, we calculated the clearance by each transporter in this system. A good correlation in the clearance of the examined bile salts (cholate, chenodeoxycholate, ursodeoxycholate, and their taurine or glycine conjugates) was observed between transport by human and that of rat transporters in terms of their rank order: for NTCP, taurine conjugates > glycine conjugates > unconjugated bile salts, and for BSEP, unconjugated bile salts and glycine conjugates > taurine conjugates. In conclusion, the substrate specificity of human and rat NTCP and BSEP appear to be very similar at least for monovalent bile salts under physiological conditions.     

Localization of a substrate specificity domain in the multidrug resistance protein.

Multidrug resistance protein (MRP) confers resistance to a number of natural product chemotherapeutic agents. It is also a high affinity transporter of some physiological conjugated organic anions such as cysteinyl leukotriene C(4) and the cholestatic estrogen, 17beta-estradiol 17(beta-D-glucuronide) (E(2)17betaG). We have shown that the murine orthologue of MRP (mrp), unlike the human protein, does not confer resistance to common anthracyclines and is a relatively poor transporter of E(2)17betaG. We have taken advantage of these functional differences to identify region(s) of MRP involved in mediating anthracycline resistance and E(2)17betaG transport by generating mrp/MRP hybrid proteins. All hybrid proteins conferred resistance to the Vinca alkaloid, vincristine, when transfected into human embryonic kidney cells. However, only those in which the COOH-terminal third of mrp had been replaced with the corresponding region of MRP-conferred resistance to the anthracyclines, doxorubicin, and epirubicin. Exchange of smaller segments of the COOH-terminal third of the mouse protein by replacement of either amino acids 959-1187 or 1188-1531 with those of MRP produced proteins capable of conferring some level of resistance to the anthracyclines tested. All hybrid proteins transported cysteinyl leukotriene C(4) with similar efficiencies. In contrast, only those containing the COOH-terminal third of MRP transported E(2)17betaG with an efficiency comparable with that of the intact human protein. The results demonstrate that differences in primary structure of the highly conserved COOH-terminal third of mrp and MRP are important determinants of the inability of the murine protein to confer anthracycline resistance and its relatively poor ability to transport E(2)17betaG.     

介导多药耐药的ABC转运蛋白超家族与MTX耐药性的关系研究

细胞耐药性的产生是导致肿瘤化疗失败的重要因素, 尤其是多药耐药是目前研究的一个重点。ABC转运蛋白超家族成员介导药物的外排, 与多药耐药密切相关。为了解该家族成员与MTX耐药的相关性, 进一步探讨MTX的耐药机制, 应用SuperArray基因芯片对MTX耐药前后编码ABC转运蛋白超家族成员的mdr1、mrp1、mrp2、mrp3、mrp5、mrp6和abcg2 7个基因进行检测, 并对MRP1和MRP5蛋白表达进行了验证。结果显示, 与MTX耐药性相关的ABC转运蛋白超家族成员主要为多药耐药相关蛋白, 其中mrp1和mrp5呈现高表达, 并且, 在MTX抗性细胞中, MRP5在mRNA及蛋白水平的表达均明显增强, 提示其在MTX耐药机制中起重要作用, 可能为潜在的药物作用靶点。     

Enhanced expression of basolateral multidrug resistance protein isoforms Mrp3 and Mrp5 in rat liver by LPS

Lipopolysaccharide (LPS) induces hepatocellular down-regulation and endocytic retrieval of multidrug resistance protein 2 (Mrp2, Abcc2). Basolateral Mrp isoforms may compensate for the intracellular metabolic changes in cholestasis. Therefore, the effect of LPS on the zonal localization of Mrp2 and Mrp3 and the expression of Mrp3, Mrp4, Mrp5, and Mrp6 mRNA were investigated in rat liver. In normal rat liver Mrp3 was found in pericentral hepatocytes also expressing glutamine synthetase. In LPS-treated rat liver the decrease in Mrp2 protein was most pronounced in pericentral hepatocytes, with only minor down-regulation in periportal hepatocytes. Conversely, induction of Mrp3 was found in pericentral hepatocytes with a low expression of Mrp2. Furthermore, we found a strong induction of Mrp5 mRNA. Likewise, Mrp6 mRNA was up-regulated, however Mrp6 protein expression was not significantly altered. It is concluded that Mrp3 is inversely regulated to Mrp2 in a zonal pattern and may compensate for the LPS-induced loss of Mrp2 in the perivenous area. Induction of pericentral Mrp3 and up-regulation of Mrp5 mRNA may play an important role in the hepatocellular clearance of cholephilic substances and cyclic nucleotides accumulating after LPS treatment.     

Taurine supplementation induces multidrug resistance protein 2 and bile salt export pump expression in rats and prevents endotoxin-induced cholestasis

The effect of oral taurine supplementation on endotoxin-induced cholestasis was investigated in rat liver. At 12h following lipopolysaccharide (LPS) injection (4mg/kg body weight i.p.) bile flow and bromosulfophthalein (BSP) and taurocholate (TC) excretion were determined in the perfused liver and the expression of the canalicular transporters multidrug resistance protein 2 (Mrp2) and bile salt export pump (Bsep) was analyzed. Injection of LPS induced a significant decrease of bile flow ( 2.2+/-0.2 microl/g liver wet weight/min vs 3.3+/-0.1 microl/g liver wet weight in controls), biliary BSP excretion (10.8+/-2.2 nmol/g/min vs 21.0+/-3.8 nmol/g/min), and biliary TC excretion (114+/-23 nmol/g/min vs 228+/-8 nmol/g/min). These effects were due to transporter retrieval from the canalicular membrane and downregulation of Mrp2 and Bsep expression. In taurine-supplemented rats bile flow was 30% higher than that in untreated rats and the expression of Mrp2 and Bsep protein was increased two- to threefold. In taurine-supplemented rats there was no significant reduction of bile flow or of BSP and TC excretion at 12h following LPS injection. This protective effect of taurine was due to higher Mrp2 and Bsep protein levels compared to nonsupplemented LPS-treated rats, whereas relative Mrp2 retrieval from the canalicular membrane induced by LPS was not significantly different. LPS-induced tumor necrosis factor alpha and interleukin-1beta release were lower in taurine-fed rats; however, downregulation of Mrp2 and Bsep expression by LPS was delayed but not prevented. The data show that oral supplementation of taurine induces Mrp2 and Bsep expression and may prevent LPS-induced cholestasis.     

Discovery that theonellasterol a marine sponge sterol is a highly selective FXR antagonist that protects against liver injury in cholestasis

Background

The farnesoid-x-receptor (FXR) is a bile acid sensor expressed in the liver and gastrointestinal tract. Despite FXR ligands are under investigation for treatment of cholestasis, a biochemical condition occurring in a number of liver diseases for which available therapies are poorly effective, mice harboring a disrupted FXR are protected against liver injury caused by bile acid overload in rodent models of cholestasis. Theonellasterol is a 4-methylene-24-ethylsteroid isolated from the marine sponge Theonella swinhoei. Here, we have characterized the activity of this theonellasterol on FXR-regulated genes and biological functions.

Principal Findings

Interrogation of HepG2 cells, a human hepatocyte cell line, by microarray analysis and transactivation assay shows that theonellasterol is a selective FXR antagonist, devoid of any agonistic or antagonistic activity on a number of human nuclear receptors including the vitamin D receptor, PPARs, PXR, LXRs, progesterone, estrogen, glucorticoid and thyroid receptors, among others. Exposure of HepG2 cells to theonellasterol antagonizes the effect of natural and synthetic FXR agonists on FXR-regulated genes, including SHP, OSTα, BSEP and MRP4. A proof-of-concept study carried out to investigate whether FXR antagonism rescues mice from liver injury caused by the ligation of the common bile duct, a model of obstructive cholestasis, demonstrated that theonellasterol attenuates injury caused by bile duct ligation as measured by assessing serum alanine aminostrasferase levels and extent of liver necrosis at histopathology. Analysis of genes involved in bile acid uptake and excretion by hepatocytes revealed that theonellasterol increases the liver expression of MRP4, a basolateral transporter that is negatively regulated by FXR. Administering bile duct ligated mice with an FXR agonist failed to rescue from liver injury and downregulated the expression of MRP4.

Conclusions

FXR antagonism in vivo results in a positive modulation of MRP4 expression in the liver and is a feasible strategy to target obstructive cholestasis.