Bile Salt Export Pump Expression Research Articles

Ingestion of whey protein and β-conglycinin exerts opposite effects on intestinal FGF15 and serotonin secretion in mice.

Farnesoid X receptor (FXR) and Takeda G protein-coupled Receptor 5 (TGR5), the intestinal bile acid (BA) receptors, regulate the gut-derived hormones including fibroblast growth factor 15/19 (FGF15/19) and serotonin (5-hydrooxytryptamine, 5-HT). Here we show that ingestion of whey protein isolate, a milk protein, significantly decreased expression of heteromeric organic solute transporter Ostα and Ostβ, which is the basolateral BA transporter in the enterocyte, and increased the expression of FXR and FGF15 in C57BL6J mouse ileum and plasma FGF15 levels. In addition, the ingestion of whey protein isolate significantly suppressed expression of hepatic cholesterol-7α hydroxylase (CYP7A1), which induces the primary BA synthesis, bile salt export pump (BSEP) and sodium-taurocholate cotransporting polypeptide (NTCP), which are the key transporters for the BA excretion and uptake in the liver, and genes involved in gluconeogenesis, and decreased the primary BAs including cholic acid, taurocholic acid, glycocholic acid, and taurochenodeoxycholic acid in the liver compared with controls. Moreover, ingestion of whey protein isolate significantly decreased the expression of TGR5, glucagon-like peptide 1 (GLP-1), and tryptophan hydroxylase1 (Tph1) in the small intestine, leading to decreases in plasma 5-HT and insulin levels. On the other hand, ingestion of the soy protein β-conglycinin significantly increased the expression of Ostα and Ostβ, and decreased the expression of FGF15 in the ileum and plasma FGF15 levels, leading to the increases in expression of hepatic CYP7A1, BSEP, NTCP, and genes involved in gluconeogenesis, and the primary BAs in the liver. Moreover, ingestion of β-conglycinin significantly increased the expression of intestinal TGR5, GLP-1, and Tph1, leading to increases in plasma 5-HT and insulin levels. These findings suggest that whey protein and β-conglycinin have opposite effects on intestinal FGF15 and 5-HT secretion in mice.

The Pivotal Mediating Role of Adenosine Monophosphate-Activated Protein Kinase (AMPK) in Liver Tight Junctions and Liver Regeneration of a Partial-Hepatectomy Mouse Model

This study aims to explore the pivotal mediating role of adenosine monophosphate-activated protein kinase (AMPK) in liver tight junctions and liver regeneration of a partial hepatectomy (PH) mouse model. A 70% PH mouse model was used. Firstly, mice were randomly divided into sham, 70% PH, AMPK-activated, and AMPK-inhibited groups. Then serum levels of alanine aminotransferase, aspartate transaminase, total bilirubin, direct bilirubin, albumin, and prealbumin were tested on postoperative days 1, 2 and 3. Furthermore, the expression of tight junction proteins like occludin, claudin-3, and ZO-1, together with bile salt export pump (BSEP), which reflects liver function, and AMPK were measured by Western blot and quantitative real-time polymerase chain reaction. Moreover, the expression of tight junction proteins, BSEP, and Ki-67 were examined by immunohistochemistry. After 70% PH, without intervention, the changes in expression of hepatic tight junction proteins (occludin, claudin-3, and ZO-1) were consistent with that of BSEP, which could reflect liver function. After treatment with AMPK activator, the high expression status of tight junction proteins occurred in advance and was maintained stably and for a longer time. It was beneficial to liver function and liver regeneration was promoted at early periods and enhanced continuously after PH. Activation of AMPK could effectively enhance the expression of hepatic tight junction proteins after PH. Therefore, it could speed up the recovery of liver function and promote liver regeneration especially early after PH.

Silymarin protects the liver from α-naphthylisothiocyanate-induced cholestasis by modulating the expression of genes involved in bile acid homeostasis

Cholestasis is characterized by impaired bile flow which results in inflammation, cirrhosis, and ultimately liver failure. The current study is aimed to evaluate the anti-cholestatic effect of silymarin against α-naphthylisothiocyanate (ANIT) induced cholestasis. Mice were gavaged with various doses of silymarin or ursodeoxycholic acid (UDCA) for 19 days. Then they were challenged with α-naphthylisothiocyanate (ANIT) and after 48 hours the animals were sacrificed to obtain blood and liver sections. Serum levels of bilirubin, aspartate transaminase (AST), alanine transaminase (ALP), and liver histology were analyzed. mRNA expression of selected transporters (Bile salt export pump (BSEP) and sodium taurocholate cotransporting polypeptide (NTCP)) and proteins (farnesoid x receptor (FXR) and Cytochrome P450 Family 7 Subfamily A Member 1 (Cyp7a1)) involved in bile acids biosynthesis, excretion and uptake were also evaluated by quantitative PCR. The results indicated that the serum levels of bilirubin, AST, and ALP were significantly higher in a cholestatic model group as compared to an untreated control group. However, in silymarin groups, the serum level of these parameters is significantly lower than in a cholestatic model group. Liver histology also showed that silymarin prevents ANIT-induced hepatic injury. mRNA expression of FXR, BSEP, and NTCP was downregulated and expression of Cyp7a1 was upregulated in a cholestatic model group as compared to an untreated control group. However, in silymarin treatment groups, the expression of FXR, BSEP and NTCP was upregulated and the expression of Cyp7a1 was downregulated as compared to the cholestatic model group. In conclusion, silymarin could alleviate hepatic injury by modulating the expression of genes involved in bile acid homeostasis.

Tanshinone IIA alleviate rifampicin-induced cholestasis by regulating the expression and function of bile salt export pump.

Objective:Rifampicin (RFP) induces cholestasis due to long-term tubercular therapy. Impairment of the canalicular bile acids efflux via the bile salt export pump (BSEP) is a well-recognized cause of cholestasis. Tanshinone IIA (TAN IIA) has a protective effect on the liver. However, there are limited studies on the effects of RFP and TAN IIA on BSEP. In present study, we aimed to elucidate the effects of RFP and TAN IIA on BSEP and provide evidence to support the treatment of RFP-induced cholestasis with TAN IIA.Methods:Firstly, liver histopathological examination and serum biochemical tests were evaluated in rats. Secondly, we evaluated BSEP expression by qRT-PCR and western blotting to explore whether RFP and TAN IIA influence liver function through BSEP. Thirdly, the accumulation of BSEP substrate taurocholic acid (TCA) in bile ducts was determined to investigate the effects of RFP and TAN IIA on BSEP function.Results:Apparent histopathological alterations and significantly increased serum biomarkers were observed in the RFP group (200 mg/kg), while these changes were attenuated in the combination groups. The mRNA and protein levels of BSEP were decreased by RFP. Whereas TAN IIA reversed the downward regulation of BSEP caused by RFP. And RFP primarily inhibited TCA excretion but co-administration of TAN IIA markedly induced TCA excretion mediated by BSEP.Conclusion:Our findings collectively demonstrated that RFP-induced cholestasis could be related to the inhibition of BSEP, and TAN IIA had the potential to prevent RFP-induced cholestasis by regulating BSEP.

Nifuroxazide mitigates cholestatic liver injury by synergistic inhibition of Il-6/Β-catenin signaling and enhancement of BSEP and MDRP2 expression

Cholestasis is a complex hepatic disorder underlined with retention of the highly toxic bile components within the hepatocytes. Nifuroxazide (NIF); a nitrofuran derivative, is widely used drug for treatment of acute and chronic diarrhea. The current study was performed to investigate the curative effect of NIF (25 and 50 mg/kg) on lithocholic acid (LCA)-induced cholestasis and compare the observed impact to that of ursodeoxycholic acid (UDCA). Intriguingly, NIF significantly attenuated LCA-induced cholestatic injury. NIF successfully reversed cholestatic injury to a similar extent compared to the mainstay drug, UDCA. NIF administration remarkably attenuated liver/body index and restored liver functions. Moreover, it restored the disrupted balance in oxidative homeostasis. On the other hand, NIF induced a marked improvement in histopathological and immuno-histochemical analysis of liver specimens. Ultimately, NIF mitigated inflammatory response and proliferative ability of hepatocytes with significant reduction in hepatic expression of proliferatingcellnuclearantigen(PCNA), cluster of differentiation 68 (CD68), interlukin-6 (Il-6) and β-catenin. Interestingly, NIF successfully increased bile transformation with increased the hepatic expression of bile salt export pump (BSEP) and multidrug resistance-associated protein 2 (MDRP2). Nevertheless, molecular docking of NIF with β-catenin and BSEP showed a better alignment inside the pocket with strong interaction for both protein binding sites. In conclusion, NIF attenuated experimentally-induced cholestatic dysfunction with an underlined synergistic inhibition of Il-6/Β-catenin pathways and direct enhancement of bile acids transporters gene expression.

ABCB11 deficiency presenting as transient neonatal cholestasis: Correlation with genotypes and BSEP expression.

ABCB11 deficiency presenting in infancy is believed generally to manifest as persistent/progressive cholestasis. We describe a group of patients with biallelic ABCB11 variants whose disorder manifested as transient neonatal cholestasis (TNC). Neonatal intrahepatic cholestasis in 68 children (31 males) with biallelic predictedly pathogenic variants (PPV) in ABCB11 was classified as transient (TNC group, n=23, 11 males), intermittent (benign recurrent intrahepatic cholestasis [BRIC] group, n=3, 1 male) or persistent/ progressive (progressive familial intrahepatic cholestasis [PFIC] group, n=42, 19 males). Clinical, genetic and bile salt export pump (BSEP) expression information was correlated with outcomes. The median onset age of jaundice was 3days (birth to 2months) for the TNC group and 10.5days (birth to 3months) for the PFIC group (P=.034). The median length of follow-up of TNC patients was 44months (12months-168months). At presentation, hepatobiliary-injury biomarker values were similar between the groups (P>.05). TNC patients (17/23) more often than PFIC patients (20/42, P=.041) harboured biallelic non-null variants (predicted not to terminate translation prematurely). TNC patient livers (7/7) more often than PFIC patient livers (5/16, P=.005) expressed immunohistochemically detectable BSEP. Kaplan-Meier analysis showed better prognosis for patients with BSEP expression (P=.009). Too few BRIC patients were available for statistical study. Neonatal cholestasis associated with biallelic PPV in ABCB11 can resolve temporarily or persistently in one third of cases. Resolution is more likely in patients with biallelic non-null PPV or with liver BSEP expression.

Picroside II alleviates liver injury induced by alpha-naphthylisothiocyanate through AMPK-FXR pathway

Alpha-naphthylisothiocyanate (ANIT) is a typical hepatotoxicant that causes cholestasis, which causes toxic bile acid accumulation in the liver and leads to liver injury. Picroside II (PIC), one of the dominant effective components extracted from Picrorhiza scrophulariiflora Pennell, exhibits many pharmacological effects. However, the role of AMP-activated protein kinase (AMPK)-Farnesoid X receptor (FXR) pathway in the hepatoprotective effect of PIC against ANIT-induced cholestasis remains largely unknown. This study aimed to investigate the mechanisms of PIC on ANIT-induced cholestasis in vivo and in vitro. Our results showed that PIC protected against ANIT-induced liver injury in primary mouse hepatocytes, and decreased serum biochemical markers and lessened histological injuries in mice. ANIT inhibited FXR and its target genes of bile acid synthesis enzymes sterol-12α-hydroxylase (CYP8B1), and increase bile acid uptake transporter Na + −dependent taurocholate transporter (NTCP), efflux transporter bile salt export pump (BSEP) and bile acid metabolizing enzymes UDP-glucuronosyltransferase 1a1 (UGT1A1) expressions. PIC prevented its downregulation of FXR, NTCP, BSEP and UGT1A1, and further reduced CYP8B1 by ANIT. Furthermore, ANIT activated AMPK via ERK1/2-LKB1 pathway. PIC inhibited ERK1/2, LKB1 and AMPK phosphorylation in ANIT-induced cholestasis in vivo and in vitro. AICAR, an AMPK agonist, blocked PIC-mediated changes in FXR, CYP8B1 and BSEP expression in vitro. Meanwhile, U0126, an ERK1/2 inhibitor, further repressed ERK1/2-LKB1-AMPK pathway phosphorylation. In conclusion, PIC regulated bile acid-related transporters and enzymes to protect against ANIT-induced liver injury, which related to ERK1/2-LKB1-AMPK pathway. Thus, this study extends the understanding of the anti-cholestasis effect of PIC and provides new therapeutic targets for cholestasis treatment.

Mutations in Myosin 5B in Children With Early-onset Cholestasis.

Mutations in Myosin 5B (MYO5B) are known to be associated with microvillous inclusion disease (MVID) a genetic cause of neonatal intractable diarrhoea. More recently, they have been reported in children with cholestasis but without typical gastrointestinal symptoms of MVID. We describe our series of children with cholestasis and mutations in MYO5B. Clinical, laboratory, and histological data were collected from patients with cholestasis and pathogenic mutations in MYO5B, found by next generation sequencing (NGS) but with minimal gastrointestinal disease. Six patients (3 boys) were identified. Median age at presentation was 19 months (range, 3-92). Presenting features were jaundice, pale stools, pruritus, and failure to thrive. Patients 5 and 6 had intractable diarrhoea until the age of 3 and 7 years, respectively, but currently are on full enteral diet with no intestinal symptoms. Median values for serum total bilirubin were 55 μmol/L (2-500), alanine aminotransferase 73I IU/L (32-114), γ-glutamyltransferase 7 IU/L (7-10), and serum bile acids 134 μmol/L (18-274). Three patients underwent 1 or more types of biliary diversion for symptom control. Median follow-up was 5 years (2-22). At most recent follow-up, they all reported pruritus while on antipruritics. Patient 1 had a liver transplant. We identified 6 patients, with mutations in MYO5B, early-onset cholestasis and pruritus, with variable response to biliary diversion without typical MVID.

Metformin Disrupts Bile Acid Efflux by Repressing Bile Salt Export Pump Expression.

The bile salt export pump (BSEP), a key player in hepatic bile acid clearance, has been the center of research on drug-induced cholestasis. However, such studies focus primarily on the direct inhibition of BSEP, often overlooking the potential impact of transcriptional repression. This work aims to explore the disruption of bile acid efflux caused by drug-induced BSEP repression. BSEP activity was analyzed in human primary hepatocytes (HPH) using a traditional biliary-clearance experiment and a modified efflux assay, which includes a 72-h pretreatment prior to efflux measurement. Relative mRNA and protein expressions were examined by RT-PCR and Western blotting, respectively. Metformin concentration-dependently repressed BSEP expression in HPH. Although metformin did not directly inhibit BSEP activity, longer metformin exposure reduced BSEP transport function in HPH by down-regulating BSEP expression. BSEP repression by metformin was found to be AMP-activated protein kinase-independent. Additional screening of 10 reported cholestatic non-BSEP inhibitors revealed that the anti-cancer drug tamoxifen also markedly repressed BSEP expression and reduced BSEP activity in HPH. Repression of BSEP alone is sufficient to disrupt hepatic bile acid efflux. Metformin and tamoxifen appear to be prototypes of a class of BSEP repressors that may cause drug-induced cholestasis through gene repression instead of direct BSEP inhibition.

Protective effect of pyrrolidine dithiocarbamate on isoniazid/rifampicin‑induced liver injury in rats.

Isoniazid (INH) and rifampicin (RIF) continue to be first line anti‑tuberculosis (TB) drugs. However, the use of these drugs is associated with hepatotoxicity. Nuclear factor‑κB (NF‑κB) plays a crucial role in regulating immunity and inflammation. It has been reported that pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF‑κB, exerts a hepatoprotective effect on acute and chronic liver damage. The aim of the present study was to explore the INH/RIF‑induced protective effects and mechanisms of PDTC on liver injury. Rats were intragastrically administered INH (50mg/kg/day) and RIF (50mg/kg/day) daily for 28days. PDTC (50mg/kg/day) was intraperitoneally injected 2h after the co‑administration of INH and RIF to compare liver biochemical indicators in the serum, histopathological damage, NF‑κB activity, oxidative stress, hepatic mRNA expression of tumor necrosis factor (TNF)‑α, bile salt export pump (BSEP), and protein expression of BSEP. It was found that the inhibition of NF‑κB activation by PDTC treatment markedly alleviated liver biochemical and histological injury, decreased oxidative stress and mRNA levels of TNF‑α, and prevented decreases in BSEP mRNA and protein expression induced by the co‑administration of INH and RIF. Collectively, the present data suggested that INH/RIF‑induced liver injury is dependent on the activation of NF‑κB. PDTC exerted a therapeutic effect on INH/RIF‑induced liver injury by increasing BSEP expression, and exhibiting antioxidant and anti‑inflammatory activities.

Development of an in vitro cholestatic drug-induced liver injury evaluation system using HepG2-hNTCP-C4 cells in sandwich configuration

Toxicological approaches in screening drugs that cause drug-induced liver injury (DILI) are urgently needed to reduce the risk of developing DILI and avoid immense costs resulting from late-stage drug withdrawal from clinical trials. Cholestatic DILI is characterized by bile acid (BA) accumulation in hepatocytes, typically caused by drug-induced inhibition of important bile transporters, such as bile salt export pump (BSEP) and multidrug resistance-associated protein 2/3/4 (MRP2/3/4). Therefore, NTCP expression is essential for construction of an in vitro hepatocellular toxicity evaluation system. Here, we investigated whether sandwich-cultured HepG2-hNTCP-C4 (SCHepG2-hNTCP-C4) cells were applicable for evaluation of cholestatic DILI. In SCHepG2-hNTCP-C4 cells, NTCP and MRP2/4 expression levels were comparable to those in human primary hepatocytes; however, BSEP expression was low. In addition, the substrates tauro-nor-THCA-24 DBD and CDF confirmed the functionality of NTCP and MRP2, respectively. When 22 known hepatotoxins were exposed to BAs to evaluate cholestatic DILI, cytotoxicity in SCHepG2-hNTCP-C4 cells was more frequent than that in SCHepG2 cells. Thus, SCHepG2-hNTCP-C4 cells may be useful preclinical screening tools to predict the risk of cholestatic DILI induced by drug candidates. However, further studies are needed to determine why the cholestatic cytotoxicity of some compounds would be still insufficient in SCHepG2-hNTCP-C4 cells.

Emodin Rescues Intrahepatic Cholestasis via Stimulating FXR/BSEP Pathway in Promoting the Canalicular Export of Accumulated Bile.

AimBile salt export pump (BSEP) have been confirmed to play an important role for bile acid canalicular export in the treatment of cholestasis. In this study, we investigated the stimulatory effect of emodin on BSEP signaling pathway in cholestasis.MethodsCell and animal experiments were given different concentrations of emodin. The BSEP upstream molecule farnesoid X receptor was down-regulated by small interfering RNA (siRNA) technology or guggulsterones and up-regulated by lentivirus or GW4064. Real-time PCR and Western blotting was employed to detect the mRNA and protein levels of BSEP in LO2 cell, rat primary hepatocytes and liver tissue. Immunohistochemistry (IHC) was used to examine the expression of BSEP in liver tissues. Rat liver function and pathological changes of liver tissue were performed by biochemical test and hematoxylin and eosin (HE) staining.ResultsEmodin could increase the mRNA and protein expression of BSEP and FXR. When down-regulating farnesoid X receptor expression with the siRNA or inhibitor guggulsterones, and up-regulating farnesoid X receptor expression with the lentivirus or agonist GW4064, emodin could increase the mRNA level of BSEP and FXR and the protein level of BSEP, FXR1, and FXR2. Emodin also had a notable effect on rat primary hepatocytes experiment, rat pathological manifestation, BSEP, FXR1, and FXR2 positive staining in liver tissues and the test of liver function.ConclusionEmodin has a protective effect and a rescue activity on cholestasis via stimulating FXR/BSEP pathways in promoting the canalicular export of accumulated bile.

In vitro bile acid-dependent hepatocyte toxicity assay system using human induced pluripotent stem cell-derived hepatocytes: Current status and disadvantages to overcome

Cholestatic drug-induced liver injury (DILI) is a type of hepatotoxicity. Its underlying mechanisms are dysfunction of bile salt export pump (BSEP) and multidrug resistance-associated protein 2/3/4 (MRP2/3/4), which play major roles in bile acid (BA) excretion into the bile canaliculi and blood, resulting in accumulation of BAs in hepatocytes. The sandwich-cultured hepatocyte (SCH) model can simultaneously analyze hepatic uptake and biliary excretion. Therefore, we investigated whether sandwich-cultured human induced pluripotent stem cell (iPS cell)-derived hepatocytes (SCHiHs) are suitable for evaluating cholestatic DILI. Fluorescent N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)]amino-3α,7α,12α-trihydroxy-27-nor-5β-cholestan-26-oyl)-2′-aminoethanesulfonate (tauro-nor-THCA-24-DBD, a BSEP substrate) was accumulated in bile canaliculi, which supports the presence of a functional bile canaliculi lumen. MRP2 was highly expressed in the Western blot analysis, whereas the mRNA expression of BSEP was hardly detectable. MRP3/4 mRNA levels were maintained. Of the 22 compounds known to cause DILI with BAs, 7 showed significant cytotoxicity. Most high-risk drugs were detected using the developed SCHiH system. However, a shortcoming was the considerably low expression level of BSEP, which prevented the detection of some relevant drugs whose risks should be detected in primary human hepatocytes.

Modeling the Effect of BSEP Inhibitors in Lipid Bilayers by Means of All-Atom Molecular Dynamics Simulation

The human bile salt export pump (BSEP) is a membrane protein expressed on the canalicular plasma membrane domain of hepatocytes, which mediates the active transport of unconjugated and conjugated bile salts from liver cells into bile. Genetically inherited defects in BSEP expression or activity causes cholestatic liver injury, and many drugs that cause cholestatic drug-induced liver injury (DILI) in humans have been shown to inhibit BSEP activity in vitro and in vivo, suggesting that this could be one of the mechanisms that initiates human DILI. The relationship between BSEP inhibition and molecular physicochemical properties has been previously investigated, identifying calculated lipophilicity and molecular weight to be significantly correlated with the BSEP inhibition. Predictive BSEP classification models, constructed through multiple quantitative structure–activity relationship modeling approaches, exhibit significant anomalies with differences in experimental IC50 values of three orders of magnitude...

Functional analysis of the correlation between ABCB11 gene mutation and primary intrahepatic stone.

The adenosine 5′-triphosphate binding cassette subfamily B member (ABCB)11 gene is involved in bile transport, and mutations in this gene are associated with cholestasis and cholelithiasis. Therefore, the aim of the present study was to investigate the association between ABCB11 gene mutation and primary intrahepatic stone (PIS)s and to investigate the mechanism through which ABCB11 gene mutations affect the expression of the corresponding protein. Mutations of the ABCB11 gene in 443 PIS patients and 560 healthy participants were detected by exon sequencing. The expression levels of ABCB11 mRNA and bile salt export pump (BSEP) protein in the liver tissues of patients with PISs were measured by quantitative polymerase chain reaction and western blot analysis. The mutant plasmids constructed by site-directed mutagenesis of the human BSEP gene were transfected into human embryonic kidney 293 (293) cells and Madin-Darby canine kidney (MDCK) cells, and the expression and distribution of rs118109635 of BSEP was measured. There were two significant mutations in the ABCB11 gene of the PIS patients compared with the healthy population; a missense mutation, rs118109635 (P=0.025), and a synonymous mutation, rs497692 (P=0.006). The two mutations were associated with the occurrence of preoperative jaundice (P=0.026, and P=0.011, respectively). The expression levels of BSEP in PIS patients with the missense mutation rs118109635 was decreased, whereas its mRNA expression levels remained unchanged. In PIS patients with the synonymous mutation rs497692, the expression levels of ABCB11 were decreased at both the mRNA and protein level. It was also found that mutation A865V reduced the expression levels of BSEP in 293 cells at the cellular level; its distribution in MDCK cell membranes was decreased, whereas its mRNA levels remained unchanged. The mutated loci at rs118109635 and rs497692 of the ABCB11 gene were correlated with PISs, causing a decreased expression of BSEP and reduced distribution of the protein in the cell membrane. Therefore, mutations at rs118109635 and rs497692 of the ABCB11 gene may be risk factors for PISs.

Therapeutic effects of emodin on acute choiestatic hepatitis and mechanism in rats

Objective To investigate the therapeutic effects of emodin on acute cholestatic hepatitis and mec-hanism thereof. Methods Fifty Sprague-Dawley (SD) rats were randomly divided into 5 groups and were treated with emodin, ursodeoxycholic acid, dexamethasone, or normal saline respectively for 4 days.On the fifth day gastric perfusion of alpha-naphthylisothiocyanate(ANIT) was performed to establish models of choiestatic hepatitis.Four to six hours after the establishment of model the above mentioned agents were given continuously.Forty-eight hours after the model establishment blood samples were collected from abdominal aorta to examine the total bilirubin(TB), direct bilirubin(DB), alanine aminotransferase(ALT), total bile acid(TBA), aspartate aminotransferase(AST), alkaline phosphatase(ALP), gamma glutamine transferase(GGT). Specimen of liver was collected to undergo pathological examination.Real-time PCR was used to detect the mRNA expression of farnesoid X receptor(FXR), small heterodimer partner(SHP), bile salt export pump(BSEP), uridine diphosphate glucuronosyltransferase 2 family polypeptide B4(UGT2B4). Results The serum levels of total bilirubin (TB), direct bilirubin (DB), alanine aminotransferase (ALT), total bile acids (TBA), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) of the model group were respectively (68.1±26.1) μmol/L, (46.3±20.1) μmol/L, (483±228) U/L, (159.1±57.9) μmol/L, (2.0±0.5) U/L, (996±382) U/L, (324±120) U/L.The levels of TB, DB, ALT, TBA, AST, ALP of the emodin group were respectively (15.0±8.7) μmol/L, (10.8±3.9) μmol/L, (147±71) U/L, (60.1±22.7) μmol/L, (295±104) U/L, (222±59) U/L, and were all significantly lower than those of the model group (all P<0.05). The levels of TB, DB, ALT, TBA, AST, GGT, ALP of the emodin group were all significantly lower than those of the ursodeoxycholic acid group (all P<0.05). The levels of TB, DB, ALT, TBA, GGT, AST were all significantly lower than those of the dexamethasone group (all P<0.01). The expression levels of FXR, SHP, BSEP, UGT2B4 mRNA in the emodin group (1.087±0.285, 0.892±0.390, 0.902±0.149, 1.785±0.403) were all significantly higher than those of the model group (0.152±0.088, 0.559±0.194, 0.561±0.123, 0.177±0.039, all P<0.05). Conclusions By decreasing the levels of TB, DB, ALT, TBA, AST, ALP and reducing pathological changes, emodin has a protective effect on cholestatic hepatitis.It has better effects than ursodeoxycholic acid and dexamethasone.These effects may be due to promoting FXR, SHP, BSEP and UGT2B4 expression. Key words: Emodin; Cholestasis; Hepatitis; Farnesoid X receptor; Small heterodimer partner; Uridine diphosphate glucuronosyltransferase 2 family polypeptide B4; Bile salt export pump

Cholestatic Drug Induced Liver Injury: A Function of Bile Salt Export Pump Inhibition and Farnesoid X Receptor Antagonism

Introduction:In vitro bile salt export pump (BSEP) inhibition alone does not accurately predict in vivo drug-induced liver injury in humans, suggesting that other mechanisms may be involved. Inhibition of BSEP has been shown to increase the hepatocellular concentrations of bile acids (BAs), and hepatobiliary disposition of BAs is tightly regulated by the farnesoid X receptor (FXR). Activation of FXR leads to decreased synthesis of BAs and increased expression of BA efflux transporters, BSEP, and organic solute and steroid transporter (OSTα/β). The link between BSEP inhibition and activation of the BA compensatory mechanism has not been clearly demonstrated. Materials and Methods: Therefore, we utilized BSEP inhibitors, cyclosporine A (CsA) and troglitazone (Trog), to explore this “triggering” event using Transporter Certified™ sandwich-cultured human hepatocytes (SCHH). Results: Biliary excretion of glycine-cholic acid (GCA) as a percentage of total GCA accumulation was reduced in SCHH treated with either CsA or Trog. Within 12 hours, CsA treatment concomitantly increased intracellular concentrations (ICCs) of GCA and FGF19 mRNA content, an FXR-target gene. Separately, a synergistic 76.6-fold increase of OSTβ mRNA was observed following CsA and chenodeoxycholic acid co-exposure. In contrast, Trog exposure prevented the synergistic increase of OSTβ, a component of the basolateral BA efflux transporter, under the same conditions. Finally, BA toxicity potency was increased in SCHH exposed to Trog but not CsA. Discussion and Conclusions: Overall, these results suggested that BSEP inhibition activates the BA compensatory mechanism reducing ICCs of BAs. However, Trog possesses both BSEP inhibition and FXR antagonist properties blocking the activation of the compensatory mechanism resulting in BA-induced hepatotoxicity.

Effect of YHHJ on the expression of the hepatocellular bile acid transporters multidrug resistance-associated protein 2 and bile salt export pump in ethinylestradiol-induced cholestasis.

The herbal medicine Yin Huang Mixture (YHHJ; patent no. 200910031240.7) is an aqueous extract composed from various herbs, including Artemisia capillaries Thunb, Hypericum japonicum Thunb, Eucommia ulmoides Oliver, Rheum officinale Baill, Gardenia jasminoides Ellis, Poria cocos Wolf and Dictamnus dasycarpus Turcz. Previous studies have indicated that YHHJ treatment has a beneficial effect on ameliorating itching and reducing serum bile acid levels in patients with intrahepatic cholestasis of pregnancy (ICP). However, the molecular mechanisms of action of YHHJ in ICP have not been fully elucidated. Therefore, the present study investigated an experimental hepatocellular cholestasis model to explore the regulatory role of YHHJ on the expression of the bile acid carriers, multidrug resistance-associated protein 2 (MRP2) and the bile salt export pump (BSEP). Initially, 5 mg/kg/day 17-α ethinylestradiol (EE) was used to induce cholestasis in rats and primary isolated rat hepatocytes. Subsequently, 9 or 36 g/kg/day YHHJ water extract was administrated. Blood samples were collected and serum biochemical parameters of total bile acids (TBA), total bilirubin (TBil), alanine transaminase and aspartate aminotransferase levels were determined. Rat livers and primary isolated rat hepatocytes were obtained and the protein and mRNA expression levels of MRP2 and BSEP were analyzed by western blot analysis and reverse transcription-quantitative polymerase chain reaction, respectively. Results revealed that EE-induced hepatocellular cholestasis was associated with a significant increase in serum TBA and TBil levels, whereas, YHHJ treatment significantly reversed this effect (P<0.01). Further experiments on the molecular mechanism revealed that EE significantly decreased the expression of MRP2 and BSEP compared with the control group, whereas YHHJ treatment significantly upregulated MRP2 and BSEP expression in vivo and in vitro compared with no YHHJ treatment (P<0.01). In addition, to establish whether upregulation of MRP2 and BSEP protein expression levels resulted from increased expression of their respective mRNA, the mRNA expression levels were determined. Results indicated that YHHJ treatment significantly increased MRP2 and BSEP mRNA expression levels in EE-induced hepatocellular cholestasis compared with no YHHJ treatment (P<0.01). In conclusion, the present findings suggest that YHHJ effects EE-induced cholestasis and this process may be mediated through regulating hepatobiliary transporters, MRP2 and BSEP.

Mechanistic insights into geniposide regulation of bile salt export pump (BSEP) expression.

Geniposide (GE) is a major component isolated from Gardenia jasminoides Ellis, which has been used to treat cholestasis liver diseases. Our previous study has shown that GE could notably increase mRNA and protein expressions of BSEP in cholestatic rats. BSEP plays a critical role in maintenance of the enterohepatic circulation of bile acids. BSEP could be regulated by the transactivation pathway of farnesoid X receptor (FXR) and nuclear factor erythroid 2-related factor (Nrf2). Here the mechanisms for BSEP regulation by GE were investigated. GE induced the mRNA levels of BSEP in HepG2 cells and cholestatic mice, and knockdown of FXR and Nrf2 reduced the mRNA expression of BSEP at varying degrees after treatment of GE. FXR acts as the major regulator of BSEP transcription. The involvement of FXR regulated BSEP expression by GE was further investigated. An enhancement was observed in FXR-dependent BSEP promoter activation using luciferase assay. ChIP assay further confirmed the interaction between FXR and BSEP after GE treatment. Using siRNA and ChIP assays, we demonstrated that peroxisome-proliferator-activated receptor γ co-activator-1α (PGC-1α) and co-activator-associated arginine methyltransferase 1 (CARM1) were predominantly recruited to the BSEP promoter upon FXR activation by GE. In conclusion, GE regulated the expression of BSEP through FXR and Nrf2 signaling pathway. The FXR transactivation pathway was enhanced by increasing recruitment of coactivators PGC-1α and CARM1 upon GE treatment, coupled with an increased binding of FXR to the BSEP promoter.

Phytosterols Have Limited Direct Effect on Hepatocyte Transporter Expression but Synergize with Endotoxin to Enhance Kupffer Cell Inflammatory Response

Phytosterols, non‐nutritive components of soybean oil‐based lipid emulsions (SO), have been implicated in the development of parenteral nutrition associated liver disease (PNALD). Phytosterols may drive the pathogenesis of PNALD through a two‐hit mechanism: suppression of hepatic bile clearance through downregulation of the bile salt export pump (BSEP) and increased hepatic inflammation. Inflammation can further suppress BSEP and potentially the ATP‐binding cassette transporters ABCG5/8, the hepatic phytosterol clearance transporter, creating a feed‐forward loop leading to further hepatic injury. Two nuclear hormone receptors, Farnesoid X Receptor (FXR) and Liver X Receptor (LXR) regulate bile acid metabolism and have been shown to suppress the inflammatory response. The aim of our study was to first, determine if the hepatic transport dysfunction and inflammatory response is hepatocyte independent or requires activation of the resident macrophages, Kupffer cells; second, determine if ligand‐mediated activation of FXR and LXR can restore inflammation‐induced suppression of transporter function. Preterm piglets were administered parenteral nutrition for 10 d containing SO lipid emulsion. On day 10, piglets were given an 8‐h infusion of saline or LPS (10 μg*kg−1*h−1). In a separate experiment, primary hepatocytes and Kupffer cells were isolated from neonatal piglets. Cells were treated with media supplemented with 1% SO with or without the addition of a phytosterol mixture containing β‐sitosterol, stigmasterol, and campesterol. After incubation for 24 h, cells were additionally treated with FXR ligand Obeticholic acid (OCA), or LXR ligand GW3694 in the presence or absence of 50 ng/mL LPS or 10 ng/mL IL‐1β for 24 h. In piglets, administration of LPS lead to a significant suppression of BSEP and ABCG5/8 expression compared to saline infused piglets. In cell culture, administration of IL‐1β, but not LPS or phytosterols, led to suppression of both BSEP and ABCG5/8. In hepatocytes co‐treated with IL‐1β and OCA or GW3694, only BSEP expression was rescued. In Kupffer cells, phytosterols alone failed to activate expression of cytokines, however, when treated with both LPS and phytosterols increased cytokine expression significantly more than with LPS alone. From this study, we conclude inflammation can suppress the transporters for bile acid and phytosterol clearance. Our in vitro data shows that Kupffer cells are susceptible to enhanced activation of cytokine expression by phytosterols when cells are primed with LPS. These results suggest that phytosterols may contribute to the progression of liver disease through inflammation.Support or Funding InformationAmerican Society of Parenteral and Enteral Nutrition Rhoads Research Foundation, National Institute of Health Grant DK‐094616