Dan-shen Yin promotes bile acid metabolism and excretion to prevent atherosclerosis via activating FXR/BSEP signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Dan-shen Yin (DSY), a traditional prescription, has been demonstrated to be effective in decreasing hyperlipidemia and preventing atherosclerosis (AS), but its mechanism remains unknown. We hypothesized that DSY activates farnesoid X receptor (FXR) to promote bile acid metabolism and excretion, thereby alleviating AS. AIM OF THE STUDY: This study was designed to explore whether DSY reduces liver lipid accumulation and prevents AS by activating FXR and increasing cholesterol metabolism and bile acid excretion. MATERIALS AND METHODS: The comprehensive chemical characterization of DSY was analyzed by UHPLC-MS/MS. The AS models of ApoE-/- mice and SD rats was established by high-fat diet and high-fat diet combined with intraperitoneal injection of vitamin D3, respectively. The aortic plaque and pathological changes were used to evaluate AS. Lipid levels, H&E staining and oil red O staining were used to evaluate liver lipid accumulation. The cholesterol metabolism and bile acid excretion were evaluated by enzyme-linked immunosorbent assay, UPLC-QQQ/MS. In vitro, the lipid and FXR/bile salt export pump (BSEP) levels were evaluated by oil red O staining, real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting. RESULTS: A total of 36 ingredients in DSY were identified by UPLC-MS/MS analysis. In vivo, high-dose DSY significantly inhibited aortic intimal thickening, improved arrangement disorder, tortuosity, and rupture of elastic fibers, decreased lipid levels, and reduced the number of fat vacuoles and lipid droplets in liver tissue in SD rats and ApoE-/- mice. Further studies found that high-dose DSY significantly reduced liver lipid and total bile acids levels, increased liver ursodeoxycholic acid (UDCA) and other non-conjugated bile acids levels, increased fecal total cholesterol (TC) levels, and augmented FXR, BSEP, cholesterol 7-alpha hydroxylase (CYP7A1), ATP binding cassette subfamily G5/G8 (ABCG5/8) expression levels, while decreasing ASBT expression levels. In vitro studies showed that DSY significantly reduced TC and TG levels, as well as lipid droplets, while also increasing the expression of ABCG5/8, FXR, and BSEP in both HepG2 and Nr1h4 knockdown HepG2 cells. CONCLUSION: This study demonstrated that DSY promotes bile acid metabolism and excretion to prevent AS by activating FXR. For the prevent of AS and drug discovery provided experimental basis.

The noncanonical nucleotide binding site 1 of the bile salt export pump is optimized for proper function of the transporter.

The bile salt export pump (ABCB11/BSEP) is a hepatocyte plasma membrane-resident protein translocating bile salts into bile canaliculi. The sequence alignment of the four full-length transporters of the ABCB subfamily (ABCB1, ABCB4, ABCB5 and ABCB11) indicates that the NBD-NBD contact interface of ABCB11 differs from that of other members in only four residues. Notably, these are all located in the noncanonical nucleotide binding site 1 (NBS1). Substitution of all four deviant residues with canonical ones (quadruple mutant) significantly decreased the transport activity of the protein. In this study, we mutated two deviant residues in the signature sequence to generate a double mutant (R1221G/E1223Q). Furthermore, a triple mutant (E502S/R1221G/E1223Q) was generated, in which the deviant residues of the signature sequence and Q-loop were mutated concurrently to canonical residues. The double and triple mutants showed 80% and 60%, respectively, of the activity of wild-type BSEP. As expected, an increasing number of mutations gradually impair transport as an intricate network of interactions within the ABC proteins ensures proper functioning.

Cav-1 regulates the bile salt export pump on the canalicular membrane of hepatocytes by PKCα-associated signalling under cholesterol stimulation.

BACKGROUND AND AIMS: The secretion of bile salts transported by the bile salt export pump (BSEP) is the primary driving force for the generation of bile flow; thus, it is closely related to the formation of cholesterol stones. Caveolin-1 (Cav-1), an essential player in cell signalling and endocytosis, is known to co-localize with cholesterol-rich membrane domains. This study illustrates the role of Cav-1 and BSEP in cholesterol stone formation. METHODS: Adult male C57BL/6 mice were used as an animal model. HepG2 cells were cultured under different cholesterol concentrations and BSEP, Cav-1, p-PKCα and Hax-1 expression levels were determined via Western blotting. Expression levels of BSEP and Cav-1 mRNA were detected using real-time PCR. Immunofluorescence and immunoprecipitation assays were performed to study BSEP and Hax-1 distribution. Finally, an ATPase activity assay was performed to detect BSEP transport activity under different cholesterol concentrations in cells. RESULTS: Under low-concentration stimulation with cholesterol, Cav-1 and BSEP protein and mRNA expression levels significantly increased, PKCα phosphorylation significantly decreased, BSEP binding capacity to Hax-1 weakened, and BSEP function increased. Under high-concentration stimulation with cholesterol, Cav-1 and BSEP protein and mRNA expression levels decreased, PKCα phosphorylation increased, BSEP binding capacity to Hax-1 rose, and BSEP function decreased. CONCLUSION: Cav-1 regulates the bile salt export pump on the canalicular membrane of hepatocytes via PKCα-associated signalling under cholesterol stimulation.

Ursolic acid attenuates cholestasis through NRF2-mediated regulation of UGT2B7 and BSEP/MRP2.

Ursolic acid (UA), a pentacyclic triterpenoid, exhibits various pharmacological actions, such as anti-inflammation, anti-tumor, anti-diabetes, heart protection, and liver protection. However, the role of nuclear factor E2-related factor 2 (NRF2)-mediated regulation of uridine diphosphate glucuronosyltransferase (UGT2B7) and bile salt export pump (BSEP)/multidrug resistance-associated protein 2 (MRP2) in UA against cholestatic liver injury has not been cleared. The purpose of this study is to explore the effect of UA on cholestatic liver injury and its potential mechanism. The results of the liver pathology sections and blood biochemical indices demonstrated that UA significantly attenuated the cholestatic liver injury induced by alpha-naphthylisothiocyanate (ANIT) in a dose-dependent manner. The mRNA and protein levels of UGT2B7 and BSEP/MRP2 were remarkably increased in the liver of ANIT rats and HepG2 cells pretreated with UA, but this activation was suppressed with NRF2 silenced. In conclusion, our findings demonstrate that UA prevents cholestasis, which may be associated with NRF2-mediated regulation of UGT2B7, BSEP/MRP2.

The choleretic role of tauroursodeoxycholic acid exacerbates alpha-naphthylisothiocyanate induced cholestatic liver injury through the FXR/BSEP pathway.

The aim of this study was to determine the effect of tauroursodeoxycholic acid (TUDCA) on the alpha-naphthylisothiocyanate (ANIT)-induced model of cholestasis in mice. Wild-type and farnesoid X receptor (FXR)-deficient (Fxr-/- ) mice were used to generate cholestasis models by gavage with ANIT. Obeticholic acid (OCA) was used as a positive control. In wild-type mice, treatment with TUDCA for 7 days resulted in a dramatic increase in serum levels of alanine aminotransferase (ALT), with aggravation of bile infarcts and hepatocyte necrosis with ANIT-induction. TUDCA activated FXR to upregulate the expression of bile salt export pump (BSEP), increasing bile acids (BAs)-dependent bile flow, but aggravating cholestatic liver injury when bile ducts were obstructed resulting from ANIT. In contrast, TUDCA improved the liver pathology and decreased serum ALT and alkaline phosphatase (ALP) levels in ANIT-induced Fxr-/- mice. Furthermore, TUDCA inhibited the expression of cleaved caspase-3 and reduced the area of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining in the model mice. TUDCA also upregulated anion exchanger 2 (AE2) protein expression, protecting cholangiocytes against excessive toxic BAs. Our results showed that TUDCA aggravated cholestatic liver injury via the FXR/BSEP pathway when bile ducts were obstructed, although TUDCA inhibited apoptotic activity and protected cholangiocytes against excessive toxic BAs.

Protective Effect of Rhus chinensis Mill. Fruits on 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine-Induced Cholestasis in Mice via Ameliorating Oxidative Stress and Inflammation.

This study focused on the preventive effects of the extracts of Rhus chinensis Mill. (RCM) fruits on cholestasis induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) in mice. The results showed that RCM extracts could significantly ameliorate DDC-induced cholestasis via multiple mechanisms, including (1) alleviating liver damage via enhancing antioxidant capacity, such as increasing the contents of glutathione, superoxide dismutase, and catalase and inhibiting the levels of malondialdehyde; (2) preventing liver inflammation by suppressing NF-κB pathway and reducing proinflammatory cytokines secretion (e.g., tumor necrosis factor-α, interleukin-1ß, and interleukin-6); (3) inhibiting liver fibrosis and collagen deposition by regulating the expression of transforming growth factor-ß and α-smooth muscle actin; (4) modulating abnormal bile acid metabolism through increasing the expression of bile salt export pump and multidrug resistance-associated protein 2. This study was the first to elucidate the potential preventive effect of RCM extracts on DDC-induced cholestasis in mice from multiple pathways, which suggested that RCM fruits could be considered as a potential dietary supplement to prevent cholestasis.

Treprostinil Supplementation Ameliorates Hepatic Ischemia Reperfusion Injury and Regulates Expression of Hepatic Drug Transporters: An Isolated Perfused Rat Liver (IPRL) Study.

PURPOSE: IR injury is an unavoidable consequence in deceased donor liver transplantation. Cold preservation and warm reperfusion may change the expression and function of drug transporters in the liver due to vasoconstriction, infiltration of neutrophils and release of cytokines. We hypothesize that vasodilation, anti-platelet aggregation and proinflammatory downregulation activities of treprostinil will diminish the IR injury and its associated effects. METHODS: Livers obtained from male SD rats (n = 20) were divided into 1) Control, 2) IR, 3) Treprostinil-1 (preservation only), and 4) Treprostinil-2 (preservation and reperfusion) groups. Control livers were procured and immediately reperfused. Livers in the other groups underwent preservation for 24 h and were reperfused. All the livers were perfused using an Isolated Perfused Rat Liver (IPRL) system. Periodic perfusate, cumulative bile samples and liver tissue at the end of perfusion were collected. Liver injury markers, bile flow rates, m-RNA levels for uptake and efflux transporters (qRT-PCR) were measured. RESULTS: Cold preservation and warm reperfusion significantly increased the release of AST and ALT in untreated livers. Treprostinil supplementation substantially reduced liver injury. Bile flow rate was significantly improved in treprostinil-2 group. m-RNA levels of Slc10a1, Slc22a1, and Slc22a7 in liver were increased and m-RNA levels of Mdr1a were decreased by IR. Treprostinil treatment increased Abcb11 and Abcg2 m-RNA levels and maintained Slc22a1m-RNA similar to control livers. CONCLUSIONS: Treprostinil treatment significantly reduced liver injury. IR injury changed expression of both uptake and efflux transporters in rat livers. Treprostinil significantly altered the IR injury mediated changes in m-RNA expression of transporters.

FXR Signaling-Mediated Bile Acid Metabolism Is Critical for Alleviation of Cholesterol Gallstones by Lactobacillus Strains.

Cholesterol gallstone (CGS) disease is characterized by an imbalance in bile acid (BA) metabolism and is closely associated with gut microbiota disorders. However, the role and mechanism by which probiotics targeting the gut microbiota attenuate cholesterol gallstones are still unknown. In this study, Limosilactobacillus reuteri strain CGMCC 17942 and Lactiplantibacillus plantarum strain CGMCC 14407 were individually administered to lithogenic-diet (LD)-fed mice for 8 weeks. Both Lactobacillus strains significantly reduced LD-induced gallstones, hepatic steatosis, and hyperlipidemia. These strains modulated BA profiles in the serum and liver, which may be responsible for the activation of farnesoid X receptor (FXR). At the molecular level, L. reuteri and L. plantarum increased ileal fibroblast growth factor 15 (FGF15) and hepatic fibroblast growth factor receptor 4 (FGFR4) and small heterodimer partner (SHP). Subsequently, hepatic cholesterol 7α-hydroxylase (CYP7A1) and oxysterol 7α-hydroxylase (CYP7B1) were inhibited. Moreover, the two strains enhanced BA transport by increasing the levels of hepatic multidrug resistance-associated protein homologs 3 and 4 (Mrp3/4), hepatic multidrug resistance protein 2 (Mdr2), and the bile salt export pump (BSEP). In addition, both L. reuteri and L. plantarum reduced LD-associated gut microbiota dysbiosis. L. reuteri increased the relative abundance of Muribaculaceae, while L. plantarum increased that of Akkermansia. The changed gut microbiota was significantly negatively correlated with the incidence of cholesterol gallstones and the FXR-antagonistic BAs in the liver and serum and with the FXR signaling pathways. Furthermore, the protective effects of the two strains were abolished by both global and intestine-specific FXR antagonists. These findings suggest that Lactobacillus might relieve CGS through the FXR signaling pathways. IMPORTANCE Cholesterol gallstone (CGS) disease is prevalent worldwide. None of the medical options for prevention and treatment of CGS disease are recommended, and surgical management has a high rate of recurrence. It has been reported that the factors involved in metabolic syndrome are highly connected with CGS formation. While remodeling of dysbiosis of the gut microbiome during improvement of metabolic syndrome has been well studied, less is known about prevention of CGS formation after regulating the gut microbiome. We used the lithogenic diet (LD) to induce an experimental CGS model in C57BL/6J mice to investigate protection against CGS formation by Limosilactobacillus reuteri strain CGMCC 17942 and Lactiplantibacillus plantarum strain CGMCC 14407. We found that these L. reuteri and L. plantarum strains altered the bile acid composition in mice and improved the dysbiosis of the gut microbiome. These two Lactobacillus strains prevented CGS formation by fully activating the hepatic and ileal FXR signaling pathways. They could be a promising therapeutic strategy for treating CGS or preventing its recurrence.

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.

Multiple anti-non-alcoholic steatohepatitis (NASH) efficacies of isopropylidenyl anemosapogenin via farnesoid X receptor activation and TFEB-mediated autophagy.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) can develop into cirrhosis, liver failure, or hepatocellular carcinoma without effective treatment. However, there are currently no drugs for NASH treatment, and the development of new therapeutics has remained a major challenge in NASH research. Advances in traditional Chinese medicine to treat liver disease inspired us to search for new NASH candidates from Chi-Shao, a widely used traditional Chinese medicine. PURPOSE: In this research, we aimed to clarify the anti-NASH effect and the underlying mechanism of isopropylidenyl anemosapogenin (IA, 1), which was a new lead compound isolated from Chi-Shao. STUDY DESIGN AND METHODS: Isopropylidenyl anemosapogenin (IA, 1) was first discovered by collagen type I α 1 promoter luciferase bioassay-guided isolation and then characterized by single crystal X-ray diffraction analysis and enriched by semi-synthesis. Using various molecular biology techniques, the multiple anti-NASH efficacies and mechanisms of IA were clarified based on in vitro LX-2 and Huh7 cell models, along with the in vivo choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD)-induced mouse model and bile duct ligation (BDL)-induced rat model. The UPLC-MS/MS method was used to assess the plasma concentration of IA. RESULTS: A new lead compound IA was isolated from the traditional Chinese medicine Chi-Shao, which showed significant anti-liver fibrosis activity in TGF-ß1-treated LX-2 cells and anti-liver steatosis activity in oleic acid-treated Huh7 cells. Furthermore, IA could significantly ameliorate in vivo CDAHFD-induced liver injury by activating the farnesoid X receptor pathway, including its targets Nr0b2, Abcb11, and Slc10a2. Simultaneously, IA activated the autophagy pathway by activating the TFEB factor, thereby promoting lipid degradation. Its liver-protective and anti-fibrosis activities were verified by the BDL-induced rat model. Finally, with an oral administration of 100 mg/kg, IA achieved the maximum plasma concentration of 1.23 ± 0.18 µg/ml at 2.67 ± 0.58 h. CONCLUSION: IA, an unreported lupine-type triterpenoid isolated from Chi-shao, can significantly alleviate liver injury and fibrosis via farnesoid X receptor activation and TFEB-mediated autophagy, which indicates that IA could serve as a novel therapeutic candidate against NASH.

Clarify the potential cholestatic hepatotoxicity components from Chinese Herb Medicine and metabolism's role via hBSEP vesicles and S9/hBSEP vesicles.

In this study, the inhibitory effect of components from Chinese Herb Medicine (CHMs) with potential hepatotoxicity was assessed by human bile salt export pump (hBSEP) vesicles with and without S9 metabolism. Sixty-three compounds from 22 hepatoxicity CHMs were selected as the test articles. In hBSEP vesicles, eighteen of them were found to have moderate or strong inhibitory effect towards BSEP. Further studies were performed to determine the IC50 values of strong inhibitors. For the compounds belong to CHMs reported to cause cholestasis and strong inhibitors defined in hBSEP vesicles, their relative transport activities of Taurocholic acid (TCA) were evaluated in hBSEP vesicles as well as hBSEP vesicles with S9 system (S9/hBSEP vesicles). The differences of their relative transport activities of TCA between the above two system were compared to reveal the net effect of metabolism on BSEP's activity. It was found that the inhibitory effect of Saikogenin A (SGA), Saikogenin D (SGD), Diosbulbin B (DB) and rhein were significantly increased; while the inhibitory effect of isobavachalcone, saikosaponin d and saikosaponin b2 were significantly decreased after S9 metabolizing. Identification of metabolic pathways suggested that CYP3A4 was responsible for aggravating inhibitory effect of SGA and SGD against BSEP.

The hepatocyte export carrier inhibition assay improves the separation of hepatotoxic from non-hepatotoxic compounds.

An in vitro/in silico method that determines the risk of human drug induced liver injury in relation to oral doses and blood concentrations of drugs was recently introduced. This method utilizes information on the maximal blood concentration (Cmax) for a specific dose of a test compound, which can be estimated using physiologically-based pharmacokinetic modelling, and a cytotoxicity test in cultured human hepatocytes. In the present study, we analyzed if the addition of an assay that measures the inhibition of bile acid export carriers, like BSEP and/or MRP2, to the existing method improves the differentiation of hepatotoxic and non-hepatotoxic compounds. Therefore, an export assay for 5-chloromethylfluorescein diacetate (CMFDA) was established. We tested 36 compounds in a concentration-dependent manner for which the risk of hepatotoxicity for specific oral doses and the capacity to inhibit hepatocyte export carriers are known. Compared to the CTB cytotoxicity test, substantially lower EC10 values were obtained using the CMFDA assay for several known BSEP and/or MRP2 inhibitors. To quantify if the addition of the CMFDA assay to our test system improves the overall separation of hepatotoxic from non-hepatotoxic compounds, the toxicity separation index (TSI) was calculated. We obtained a better TSI using the lower alert concentration from either the CMFDA or the CTB test (TSI: 0.886) compared to considering the CTB test alone (TSI: 0.775). In conclusion, the data show that integration of the CMFDA assay with an in vitro test battery improves the differentiation of hepatotoxic and non-hepatotoxic compounds in a set of compounds that includes bile acid export carrier inhibitors.

Pharmacologic activation of hepatic farnesoid X receptor prevents parenteral nutrition-associated cholestasis in mice.

BACKGROUND AND AIMS: Parenteral nutrition (PN)-associated cholestasis (PNAC) complicates the care of patients with intestinal failure. In PNAC, phytosterol containing PN synergizes with intestinal injury and IL-1ß derived from activated hepatic macrophages to suppress hepatocyte farnesoid X receptor (FXR) signaling and promote PNAC. We hypothesized that pharmacological activation of FXR would prevent PNAC in a mouse model. APPROACH AND RESULTS: To induce PNAC, male C57BL/6 mice were subjected to intestinal injury (2% dextran sulfate sodium [DSS] for 4 days) followed by central venous catheterization and 14-day infusion of PN with or without the FXR agonist GW4064. Following sacrifice, hepatocellular injury, inflammation, and biliary and sterol transporter expression were determined. GW4064 (30 mg/kg/day) added to PN on days 4-14 prevented hepatic injury and cholestasis; reversed the suppressed mRNA expression of nuclear receptor subfamily 1, group H, member 4 (Nr1h4)/FXR, ATP-binding cassette subfamily B member 11 (Abcb11)/bile salt export pump, ATP-binding cassette subfamily C member 2 (Abcc2), ATP binding cassette subfamily B member 4(Abcb4), and ATP-binding cassette subfamily G members 5/8(Abcg5/8); and normalized serum bile acids. Chromatin immunoprecipitation of liver showed that GW4064 increased FXR binding to the Abcb11 promoter. Furthermore, GW4064 prevented DSS-PN-induced hepatic macrophage accumulation, hepatic expression of genes associated with macrophage recruitment and activation (ll-1b, C-C motif chemokine receptor 2, integrin subunit alpha M, lymphocyte antigen 6 complex locus C), and hepatic macrophage cytokine transcription in response to lipopolysaccharide in vitro. In primary mouse hepatocytes, GW4064 activated transcription of FXR canonical targets, irrespective of IL-1ß exposure. Intestinal inflammation and ileal mRNAs (Nr1h4, Fgf15, and organic solute transporter alpha) were not different among groups, supporting a liver-specific effect of GW4064 in this model. CONCLUSIONS: GW4064 prevents PNAC in mice through restoration of hepatic FXR signaling, resulting in increased expression of canalicular bile and of sterol and phospholipid transporters and suppression of macrophage recruitment and activation. These data support augmenting FXR activity as a therapeutic strategy to alleviate or prevent PNAC.

Yellow Yeast Rice Prepared Using Aspergillus terreus DSMK01 Lowers Cholesterol Levels by Stimulating Bile Salt Export Pump in Subjects with Mild-to-Moderate Hypercholesterolemia: A Randomized Controlled Trial.

SCOPE: Aspergillus terreus is an industrial microorganism used in the brewing and sauce industries. It produces monacolin K, a natural statin. The study conducted an 8-week randomized controlled trial with hypercholesterolemic subjects to examine the hypocholesterolemic effects and mechanisms of supplementation with yellow yeast rice (YYR) prepared by growing Aspergillus fungi on steamed rice. METHODS AND RESULTS: YYR supplementation markedly reduced total cholesterol, LDL, and apolipoprotein B100 levels in plasma compared with the placebo. In addition, YYR induced a significantly increased ATP binding cassette subfamily B member 11 (ABCB11) gene expression compared with the placebo, indicating the role of YYR in lowering intrahepatic cholesterol availability by stimulating the bile salt export pump. Upregulation of LDL receptor (LDLR) and 3-methylglutaryl-CoA reductase (HMGCR) gene expressions provided additional evidence to support the role of YYR in reducing hepatic cholesterol availability. Plasma metabolomic profiling revealed the possibility of diminishing bile acid absorption. Finally, Spearman rank analysis showed correlations of plasma cholesterol profiles with HMGCR and LDLR gene expressions (negative) and plasma bile acids (positive). Plasma bile acids also correlated with ABCB11 (negative) and LDLR (positive) gene expressions. CONCLUSION: These findings suggest that daily YYR supplementation exerted hypocholesterolemic effects in mild-to-moderate hypercholesterolemic subjects by reducing intrahepatic cholesterol availability through stimulating bile salt export pumps and inhibiting cholesterol biosynthesis.

Novel Bile Acid-Dependent Mechanisms of Hepatotoxicity Associated with Tyrosine Kinase Inhibitors.

Drug-induced liver injury (DILI) is the leading cause of acute liver failure and a major concern in drug development. Altered bile acid homeostasis via inhibition of the bile salt export pump (BSEP) is one mechanism of DILI. Dasatinib, pazopanib, and sorafenib are tyrosine kinase inhibitors (TKIs) that competitively inhibit BSEP and increase serum biomarkers for hepatotoxicity in ∼25-50% of patients. However, the mechanism(s) of hepatotoxicity beyond competitive inhibition of BSEP are poorly understood. This study examined mechanisms of TKI-mediated hepatotoxicity associated with altered bile acid homeostasis. Dasatinib, pazopanib, and sorafenib showed bile acid-dependent toxicity at clinically relevant concentrations, based on the C-DILI assay using sandwich-cultured human hepatocytes (SCHH). Among several bile acid-relevant genes, cytochrome P450 (CYP) 7A1 mRNA was specifically upregulated by 6.2- to 7.8-fold (dasatinib) and 5.7- to 9.3-fold (pazopanib), compared with control, within 8 hours. This was consistent with increased total bile acid concentrations in culture medium up to 2.3-fold, and in SCHH up to 1.4-fold, compared with control, within 24 hours. Additionally, protein abundance of sodium taurocholate co-transporting polypeptide (NTCP) was increased up to 2.0-fold by these three TKIs. The increase in NTCP protein abundance correlated with increased function; dasatinib and pazopanib increased hepatocyte uptake clearance (CLuptake) of taurocholic acid, a probe bile acid substrate, up to 1.4-fold. In conclusion, upregulation of CYP7A1 and NTCP in SCHH constitute novel mechanisms of TKI-associated hepatotoxicity. SIGNIFICANCE STATEMENT: Understanding the mechanisms of hepatotoxicity associated with tyrosine kinase inhibitors (TKIs) is fundamental to development of effective and safe intervention therapies for various cancers. Data generated in sandwich-cultured human hepatocytes, an in vitro model of drug-induced hepatotoxicity, revealed that TKIs upregulate bile acid synthesis and alter bile acid uptake and excretion. These findings provide novel insights into additional mechanisms of bile acid-mediated drug-induced liver injury, an adverse effect that limits the use and effectiveness of TKI treatment in some cancer patients.

Loss of bile salt export pump aggravates lipopolysaccharide-induced liver injury in mice due to impaired hepatic endotoxin clearance.

BACKGROUND AND AIMS: Lipopolysaccharide (LPS) clearance is delayed in cholestatic liver diseases. While compromised clearance by Kupffer cells (KCs) is involved, the role of LPS uptake into hepatocytes and canalicular excretion remains unclear. APPROACH AND RESULTS: Wild-type (WT) and bile salt export pump (Bsep) knockout (KO) mice were challenged i.p. with LPS. Liver injury was assessed by serum biochemistry, histology, molecular inflammation markers, and immune cell infiltration. LPS concentrations were determined in liver tissue and bile. Subcellular kinetics of fluorescently labeled LPS was visualized by intravital two-photon microscopy, and the findings in Bsep KO mice were compared to common bile duct-ligated (BDL) and multidrug resistance protein 2 (Mdr2) KO mice. Changes in gut microbiota composition were evaluated by 16S ribosomal RNA gene amplicon sequencing analysis. Bsep KO mice developed more pronounced LPS-induced liver injury and inflammatory signaling, with subsequently enhanced production of proinflammatory cytokines and aggravated hepatic immune cell infiltration. After LPS administration, its concentrations were higher in liver but lower in bile of Bsep KO compared to WT mice. Intravital imaging of LPS showed a delayed clearance from sinusoidal blood with a basolateral uptake block into hepatocytes and reduced canalicular secretion. Moreover, LPS uptake into KCs was reduced. Similar findings with respect to hepatic LPS clearance were obtained in BDL and Mdr2 KO mice. Pretreatment with the microtubule inhibitor colchicine inhibited biliary excretion of LPS in WT mice, indicating that LPS clearance is microtubule-dependent. Microbiota analysis showed no change of the gut microbiome between WT and Bsep KO mice at baseline but major changes upon LPS challenge in WT mice. CONCLUSIONS: Absence of Bsep and cholestasis in general impair LPS clearance by a basolateral uptake block into hepatocytes and consequently less secretion into canaliculi. Impaired LPS removal aggravates hepatic inflammation in cholestasis.

Interactions of anti-COVID-19 drug candidates with hepatic transporters may cause liver toxicity and affect pharmacokinetics.

Transporters in the human liver play a major role in the clearance of endo- and xenobiotics. Apical (canalicular) transporters extrude compounds to the bile, while basolateral hepatocyte transporters promote the uptake of, or expel, various compounds from/into the venous blood stream. In the present work we have examined the in vitro interactions of some key repurposed drugs advocated to treat COVID-19 (lopinavir, ritonavir, ivermectin, remdesivir and favipiravir), with the key drug transporters of hepatocytes. These transporters included ABCB11/BSEP, ABCC2/MRP2, and SLC47A1/MATE1 in the canalicular membrane, as well as ABCC3/MRP3, ABCC4/MRP4, SLC22A1/OCT1, SLCO1B1/OATP1B1, SLCO1B3/OATP1B3, and SLC10A1/NTCP, residing in the basolateral membrane. Lopinavir and ritonavir in low micromolar concentrations inhibited BSEP and MATE1 exporters, as well as OATP1B1/1B3 uptake transporters. Ritonavir had a similar inhibitory pattern, also inhibiting OCT1. Remdesivir strongly inhibited MRP4, OATP1B1/1B3, MATE1 and OCT1. Favipiravir had no significant effect on any of these transporters. Since both general drug metabolism and drug-induced liver toxicity are strongly dependent on the functioning of these transporters, the various interactions reported here may have important clinical relevance in the drug treatment of this viral disease and the existing co-morbidities.

The spectrum of Progressive Familial Intrahepatic Cholestasis diseases: Update on pathophysiology and emerging treatments.

The Progressive Familial Intrahepatic Cholestasis (PFIC) disease spectrum encompasses a variety of genetic diseases that affect the bile production and the secretion of bile acids. Typically, the first presentation of these diseases is in early childhood, frequently followed by a severe course necessitating liver transplantation before adulthood. Except for transplantation, treatment modalities have been rather limited and frequently only aim at the symptoms of cholestasis, such as cholestatic pruritus. In recent years, progress has been made in understanding the pathophysiology of these diseases and new treatment modalities have been emerging. Herewith we summarize the latest developments in the field and formulate the current key questions and opportunities for further progress.

Disordered farnesoid X receptor signaling is associated with liver carcinogenesis in Abcb11-deficient mice.

ABCB11 encodes the bile salt export pump (BSEP), a key regulator in maintaining bile acid (BA) homeostasis. Although inherited ABCB11 mutations have previously been linked to primary liver cancer, whether ABCB11 deficiency leads to liver cancer remains unknown. Here, we analyzed ABCB11 mRNA expression levels in liver tumor specimens [29 with hepatocellular carcinoma (HCC), one with intrahepatic cholangiocarcinoma (ICC), and one with mixed HCC/ICC] with adjacent normal specimens and published human datasets. Liver tissues obtained from Abcb11-deficient (Abcb11-/- ) mice and wild-type mice at different ages were compared by histologic, RNA-sequencing, and BA analyses. ABCB11 was significantly downregulated in human liver tumors compared with normal controls. Abcb11-/- mice demonstrated progressive intrahepatic cholestasis and liver fibrosis, and spontaneously developed HCC and ICC over 12 months of age. Abcb11 deficiency increased BAs in the liver and serum in mice, most of which are farnesoid X receptor (FXR) antagonists/non-agonists. Accordingly, the hepatic expression and transcriptional activity of FXR were downregulated in Abcb11-/- mouse livers. Administration of the FXR agonist obeticholic acid reduced liver injury and tumor incidence in Abcb11-/- mice. In conclusion, ABCB11 is aberrantly downregulated and plays a vital role in liver carcinogenesis. The cholestatic liver injury and liver tumors developed in Abcb11-/- mice are associated with increased FXR antagonist BAs and thereby decreased activation of FXR. FXR activation might be a therapeutic strategy in ABCB11 deficiency diseases. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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 ß-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 ß-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.