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.

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.

Metabolic profiling of emodin drug-induced liver injury and silybin treatment in rats using ultra-performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry: A metabolomic and mechanistic approach.

Silybin, an active component in the plant Silybum marianum (L.) Gaertn., is commonly used to protect against liver disease. We investigated silybin's protective potential in rat liver against emodin-induced liver injury 4 weeks. It was found that aspartate aminotransferase and direct bilirubin serum biomarkers for liver toxicity significantly increased, and liver histopathology revealed cholestasis and necrosis in rats administered emodin alone, whereas aspartate aminotransferase and total bile acid levels in rats administered emodin and silybin simultaneously were changed compared to rats administered emodin alone. Liver mRNA and protein levels of Cyp7a1-which plays roles in cholesterol metabolism and bile acid synthesis-and Abcb11 (Bsep)-which facilitates bile salt secretion in hepatocyte canaliculi-were significantly altered with emodin, whereas cotreatment with silybin attenuated emodin's adverse effect. Metabolomic analysis using ultra-performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry determined eight potential metabolite biomarkers in serum, urine, and liver tissue. Network analysis was conducted to conceptualize the interplay of genes, metabolites, and metabolic pathways for cholesterol metabolism and bile acid synthesis for liver injury. Overall, rats administered only emodin were shown to be a sound model to investigate fat-associated drug-induced hepatoxicity or liver injury and cotreatment of emodin with silybin prevents fatty liver injury. This metabolomic study revealed that emodin-induced fatty liver injury disrupted bile acid synthesis, vitamin B6 , and glycerophospholipid metabolism pathways and that silybin ameliorates liver injury on these compromised pathways.

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.

Dolomiaea souliei ethyl acetate extract protected against α-naphthylisothiocyanate-induced acute intrahepatic cholestasis through regulation of farnesoid x receptor-mediated bile acid metabolism.

BACKGROUND: Cholestasis is characterized by accumulation of bile components in liver and systemic circulation. Restoration of bile acid homeostasis via activating farnesoid x receptor (FXR) is a promising strategy for the treatment of cholestasis. FXR-SHP (small heterodimer partner) axis plays an important role in maintaining bile acid homeostasis. PURPOSE: To investigate the anti-cholestasis effect of Dolomiaea souliei (Franch.) C.Shih (D. souliei) and clarify its underlying mechanism against α-naphthylisothiocyanate (ANIT) induced acute intrahepatic cholestasis. METHODS: ANIT-induced Sprague-Dawley rats were employed to investigate the anti-cholestasis effect of D. souliei ethyl acetate extract (DSE). Ursodeoxycholic acid (UDCA) was used as positive control. Bile flow and blood biochemical parameters were measured. Liver histopathological examination was conducted via hematoxylin-eosin staining. Western blot analysis was carried out to evaluate the protein levels related to bile acids metabolism and inflammation. The interactions between FXR and costunolide or dehydrocostus lactone, were conducted by molecular docking experiments. The effect of costunolide and dehydrocostus lactone on aspartate aminotransferase (AST), alanine aminotransferase (ALT) levels and FXR expression were also evaluated using guggulsterone-induced L02 cells. RESULTS: DSE could promote bile excretions and protect against ANIT-induced liver damage in cholestasis rats. Protein levels of FXR, SHP, Na+/taurocholate cotransporter (NTCP), bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2) were increased and the expressions of cholesterol 7α-hydroxylase (CYP7A1) and sterol 27-hydroxylase (CYP27A1) were decreased by DSE. Meanwhile, the anti-inflammatory factors, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) were also significantly increased, and the pro-inflammatory factor, interleukin-10 (IL-10), was significantly decreased in rats of DSE groups. Molecular docking revealed that costunolide and dehydrocostus lactone could be well docked into the FXR protein molecule, and hydrophobic interactions played the main function. Costunolide could reverse the increased AST and ALT levels and increase the FXR expression in guggulsterone-induced L02 cells. CONCLUSION: DSE had an anti-cholestasis effect by activating FXR-SHP axis, inhibiting synthesis of bile acid, and increasing bile secretion, together with inflammatory response and improving liver injury. Costunolide may be the main active component. This study provided a potential therapeutic mechanism for D. souliei as an anti-cholestasis medicine in the treatment of cholestasis liver diseases.

Dehydrodiconiferyl alcohol, a lignan from Herpetospermum pedunculosum, alleviates cholestasis by activating pathways associated with the farnesoid X receptor.

BACKGROUND: In our previous study, we demonstrated the hepatoprotective effect of Herpetospermum pedunculosum in cholestatic rats. A bioassay-guided study also led to the identification and isolation of a lignan, dihydrodiconiferyl alcohol (DA) from the seeds of H. pedunculosum. PURPOSE: To investigate whether DA could alleviate cholestasis and determine the mechanisms underlying such action. METHODS: Male Sprague-Dawley (SD) rats were administered with DA (10, 20 or 40 mg/kg) intragastrically once daily for 7 days prior to treatment with α-naphthylisothiocyanate (ANIT) (60 mg/kg). We then evaluated the levels of a range of serum indicators, determined bile flow, and carried out histopathological analyses. Western blotting was then used to investigate the levels of inflammatory mediators and the Farnesoid X Receptor (FXR), proteins involved in the downstream biosynthesis of bile acids, and a range of transport proteins. Molecular docking was used to simulate the interaction between DA and FXR. Cell viability of human hepatocytes (L-02) cells was determined by MTT. Then, we treated guggulsterone-inhibited L-02 cells, Si-FXR L-02 cells, and FXR-overexpression cells with the FXR agonist GW4064 (6 µM) or DA (25, 50 and 100 µM) for 24 h before detecting gene and protein expression by RT-PCR and western blotting, respectively. RESULTS: DA significantly attenuated ANIT-induced cholestasis in SD rats by reducing liver function indicators in the serum, increasing bile flow, improving the recovery of histopathological injuries in the liver, and by alleviating pro-inflammatory cytokines in the liver. DA also increased the expression levels of FXR and altered the levels of downstream proteins in the liver tissues, thus indicating that DA might alleviate cholestasis by regulating the FXR. Molecular docking simulations predicted that DA was as an agonist of FXR. In vitro mechanical studies further showed that DA increased the mRNA and protein expression levels of FXR, Small Heterodimer Partner 1/2, Bile Salt Export Pump, Multidrug Resistance-associated Protein 2, and Na+/taurocholate Co-transporting Polypeptide, in both guggulsterone-inhibited and Si-FXR L-02 cells. Moreover, DA enhanced the mRNA and protein expression of FXR, and its downstream genes and proteins, in L-02 cells containing an FXR-overexpression plasmid. CONCLUSION: DA may represent an effective agonist for FXR has significant therapeutic potential for the treatment of cholestatic liver injury.

Da-Huang-Xiao-Shi decoction protects against3, 5-diethoxycarbonyl-1,4-dihydroxychollidine-induced chronic cholestasis by upregulating bile acid metabolic enzymes and efflux transporters.

ETHNOPHARMACOLOGICAL RELEVANCE: Chronic cholestasis is a usual clinical pathological process in hepatopathy and has few treatment options; it is classified under the category of jaundice in Chinese medicine. Da-Huang-Xiao-Shi decoction (DHXSD) is a classic Chinese prescription which is used to treat jaundice. AIM OF THE STUDY: We aimed to examine the protective effect of DHXSD on liver and its potential mechanism of action against chronic cholestasis. MATERIALS AND METHODS: Chronic cholestasis was induced using 3, 5-diethoxycarbonyl-1,4-dihydroxychollidine (DDC) in mice. Mice were then administered DHXSD intragastrically at doses of 3.68, 7.35, and 14.70 g/kg for four weeks followed by further analyses. Serum biochemical indices and liver pathology were explored. Eighteen individual bile acids (BAs) in mice serum and liver were quantified using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The expression of BA related metabolic enzymes, transporters, along with nuclear receptor farnesoid X receptor (FXR) was detected by real-time qPCR and Western blot. RESULTS: DHXSD treatment reduced the serum biochemical indices, ameliorated pathological injury, and improved the disordered BA homeostasis. Mice treated with DHXSD showed significantly upregulated expression of the metabolic enzymes, cytochrome P450 2b10 (Cyp2b10), Cyp3a11, and UDP-glucuronosyltransferase 1a1 (Ugt1a1); and the bile acid transporters, multidrug resistance protein 2 (Mdr2), bile salt export pump (Bsep), and multidrug resistance-associated protein 3 (Mrp3). DHXSD treatment also significantly upregulated FXR expression in mice with DDC-induced chronic cholestasis. CONCLUSIONS: DHXSD exerted protective effects on chronic cholestasis in DDC-treated mice by alleviating the disordered homeostasis of BAs through increased expression of BA related metabolic enzymes and efflux transporters.

A Two-Tiered In Vitro Approach to De-Risk Drug Candidates for Potential Bile Salt Export Pump Inhibition Liabilities in Drug Discovery.

Hepatocellular accumulation of bile salts by inhibition of bile salt export pump (BSEP/ABCB11) may result in cholestasis and is one proposed mechanism of drug-induced liver injury (DILI). To understand the relationship between BSEP inhibition and DILI, we evaluated 64 DILI-positive and 57 DILI-negative compounds in BSEP, multidrug resistance protein (MRP) 2, MRP3, and MRP4 vesicular inhibition assays. An empirical cutoff (5 µM) for BSEP inhibition was established based on a relationship between BSEP IC50 values and the calculated maximal unbound concentration at the inlet of the human liver (fu*Iin,max, assay specificity = 98%). Including inhibition of MRP2-4 did not increase DILI predictivity. To further understand the potential to inhibit bile salt transport, a selected subset of 30 compounds were tested for inhibition of taurocholate (TCA) transport in a long-term human hepatocyte micropatterned co-culture (MPCC) system. The resulting IC50 for TCA in vitro biliary clearance and biliary excretion index (BEI) in MPCCs were compared with the compound's fu*Iin,max to assess potential risk for bile salt transport perturbation. The data show high specificity (89%). Nine out of 15 compounds showed an IC50 value in the BSEP vesicular assay of <5µM, but the BEI IC50 was more than 10-fold the fu*Iin,max, suggesting that inhibition of BSEP in vivo is unlikely. The data indicate that although BSEP inhibition measured in membrane vesicles correlates with DILI risk, that measurement of this assay activity is insufficient. A two-tiered strategy incorporating MPCCs is presented to reduce BSEP inhibition potential and improve DILI risk. SIGNIFICANCE STATEMENT: This work describes a two-tiered in vitro approach to de-risk compounds for potential bile salt export pump inhibition liabilities in drug discovery utilizing membrane vesicles and a long-term human hepatocyte micropatterned co-culture system. Cutoffs to maximize specificity were established based on in vitro data from a set of 121 DILI-positive and -negative compounds and associated calculated maximal unbound concentration at the inlet of the human liver based on the highest clinical dose.

Picroside II protects against cholestatic liver injury possibly through activation of farnesoid X receptor.

BACKGROUD: Cholestasis, accompanied by the accumulation of bile acids in body, may ultimately cause liver failure and cirrhosis. There have been limited therapies for cholesteric disorders. Therefore, development of appropriate therapeutic drugs for cholestasis is required. Picroside II is a bioactive component isolated from Picrorhiza scrophulariiflora Pennell, its mechanistic contributions to the anti-cholestasis effect have not been fully elucidated, especially the role of picroside II on bile acid homeostasis via nuclear receptors remains unclear. PURPOSE: This study was designed to investigate the hepatoprotective effect of picroside II against alpha-naphthylisothiocyanate (ANIT)-induced cholestatic liver injury and elucidate the mechanisms in vivo and in vitro. METHODS: The ANIT-induced cholestatic mouse model was used with or without picroside II treatment. Serum and bile biochemical indicators, as well as liver histopathological changes were examined. siRNA, Dual-luciferase reporter, quantitative real-time PCR and Western blot assay were used to demonstrate the farnesoid X receptor (FXR) pathway in the anti-cholestasis effects of picroside II in vivo and in vitro. RESULTS: Picroside II exerted hepatoprotective effect against ANIT-induced cholestasis by impaired hepatic function and tissue damage. Picroside II increased bile acid efflux transporter bile salt export pump (Bsep), uptake transporter sodium taurocholate cotransporting polypeptide (Ntcp), and bile acid metabolizing enzymes sulfate transferase 2a1 (Sult2a1) and UDP-glucuronosyltransferase 1a1 (Ugt1a1), whereas decreased the bile acid synthesis enzymes cholesterol 7α-hydroxylase (Cyp7a1) and oxysterol 12α-hydroxylase (Cyp8b1). In addition, expression of FXR and the target gene Bsep was increased, whereas aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor alpha (PPARα) and their corresponding target genes were not significantly influenced by picroside II under cholestatic conditions. Furthermore, regulation of transporters and enzymes involved in bile acid homeostasis by picroside II were abrogated by FXR silencing in mouse primary cultured hepatocytes. Dual-luciferase reporter assay performed in HepG2 cells demonstrated FXR activation by picroside II. CONCLUSION: Our findings demonstrate that picroside II exerts protective effect on ANIT-induced cholestasis possibly through FXR activation that regulates the transporters and enzymes involved in bile acid homeostasis. Picroside II might be an effective approach for the prevention and treatment of cholestatic liver diseases.

Quantitative systems toxicology (QST) reproduces species differences in PF-04895162 liver safety due to combined mitochondrial and bile acid toxicity.

Many compounds that appear promising in preclinical species, fail in human clinical trials due to safety concerns. The FDA has strongly encouraged the application of modeling in drug development to improve product safety. This study illustrates how DILIsym, a computational representation of liver injury, was able to reproduce species differences in liver toxicity due to PF-04895162 (ICA-105665). PF-04895162, a drug in development for the treatment of epilepsy, was terminated after transaminase elevations were observed in healthy volunteers (NCT01691274). Liver safety concerns had not been raised in preclinical safety studies. DILIsym, which integrates in vitro data on mechanisms of hepatotoxicity with predicted in vivo liver exposure, reproduced clinical hepatotoxicity and the absence of hepatotoxicity observed in the rat. Simulated differences were multifactorial. Simulated liver exposure was greater in humans than rats. The simulated human hepatotoxicity was demonstrated to be due to the interaction between mitochondrial toxicity and bile acid transporter inhibition; elimination of either mechanism from the simulations abrogated injury. The bile acid contribution occurred despite the fact that the IC50 for bile salt export pump (BSEP) inhibition by PF-04895162 was higher (311 µmol/L) than that has been generally thought to contribute to hepatotoxicity. Modeling even higher PF-04895162 liver exposures than were measured in the rat safety studies aggravated mitochondrial toxicity but did not result in rat hepatotoxicity due to insufficient accumulation of cytotoxic bile acid species. This investigative study highlights the potential for combined in vitro and computational screening methods to identify latent hepatotoxic risks and paves the way for similar and prospective studies.

Severe and protracted cholestasis in 44 young men taking bodybuilding supplements: assessment of genetic, clinical and chemical risk factors.

BACKGROUND: Bodybuilding supplements can cause a profound cholestatic syndrome. AIM: To describe the drug-Induced liver injury network's experience with liver injury due to bodybuilding supplements. METHODS: Liver injury pattern, severity and outcomes, potential genetic associations, and exposure to anabolic steroids by product analysis were analysed in prospectively enrolled subjects with bodybuilding supplement-induced liver injury with causality scores of probable or higher. RESULTS: Forty-four males (mean age 33 years) developed liver injury with a median latency of 73 days. Forty-one per cent presented with hepatocellular pattern of liver injury as defined by the R > 5 ([Fold elevation of ALT] ÷ [Fold elevation of Alk Phos] (mean, range = 6.4, 0.5-31.4, n = 42) despite all presenting with clinical features of cholestatic liver injury (100% with jaundice and 84% with pruritus). Liver biopsy (59% of subjects) demonstrated a mild hepatitis and profound cholestasis in most without bile duct injury, loss or fibrosis. Seventy-one per cent were hospitalised, and none died or required liver transplantation. In some, chemical analysis revealed anabolic steroid controlled substances not listed on the label. No enrichment of genetic variants associated with cholestatic syndromes was found, although mutations in ABCB11 (present in up to 20%) were significantly different than in ethnically matched controls. CONCLUSIONS: Patients with bodybuilding supplements liver injury uniformly presented with cholestatic injury, which slowly resolved. The ingested products often contained anabolic steroids not identified on the label, and no enrichment in genetic variants was found, indicating a need for additional studies.

Beyond an Obvious Cause of Cholestasis in a Toddler: Compound Heterozygosity for ABCB11 Mutations.

A 27-month-old girl presented with a short history of jaundice initially attributed to drug-induced liver injury. During the preceding 20 days, she had received a 10-day course of cefprozil and 2 doses of a homeopathic preparation of cantharidin for cystitis. Severe conjugated hyperbilirubinemia was present with normal γ-glutamyl transpeptidase activity. Liver biopsy revealed marked canalicular and hepatocellular cholestasis, with moderate hepatocellular disarray, as well as evidence of chronicity, including moderate portal-tract and perisinusoidal fibrosis. Immunohistochemical studies revealed that bile salt export pump expression was preserved, whereas canalicular γ-glutamyl transpeptidase expression was largely absent. An inherited cholestatic disorder was suspected. The entire coding region of ABCB11, encoding bile salt export pump, was analyzed. The patient was found to be a compound heterozygote for the missense mutation c.3148C>T (p.Arg1050Cys) associated with benign recurrent intrahepatic cholestasis type 2 in the homozygous state and for the nonsense mutation c.3904G>T (p.Glu1302Ter) associated with progressive familial intrahepatic cholestasis type 2. Despite initial improvement with ursodeoxycholic acid, over the course of 5 years the patient developed cirrhosis that required liver transplant. Our report emphasizes the need for molecular studies even in patients with putatively "explained" cholestasis to reveal the entire spectrum of inherited cholestatic disorders.

Polygonum multiflorum Thunb suppress bile acid synthesis by activating Fxr-Fgf15 signaling in the intestine.

ETHNOPHARMACOLOGICAL RELEVANCE: Polygonum multiflorum Thunb (Heshouwu, HSW) is commonly used in clinical medicine, while the hepatotoxicities of HSW are reported increasingly in recent years. Currently, researchers have demonstrated an essential role of Bile Acids (BAs) in liver diseases. The occurrence of hepatotoxicity cases linked to HSW are characterized by jaundice and cholestasis, suggesting an interaction that between BAs and HSW AIM OF THE STUDY: This study was designed to investigate the HSW-induced liver functional and histological changes in mice and the role of HSW on bile acid synthesis, metabolism, clearance and intestinal absorption. MATERIALS AND METHODS: The mice were intragastrically (i.g.) given HSW at doses of 1.275 and 3.825 g/kg (Crude extracts /body weight) once a day for seven days. Liver function was evaluated by measuring the serum levels of enzymes and analyzing the liver histology. The LC/MS analysis was performed to quantify BAs from liver, ileum and serum. Moreover, the expression of bile metabolic-related transporters and metabolic enzymes at both protein and mRNA levels were observed to elucidate the underlying mechanisms. RESULTS: Oral administration of HSW for 7 days could not cause liver damage. A significant change was observed for the concentrations of liver and serum BAs in treatment groups compared with normal control. The mRNA expression levels of bile acid excretory transporter (Bsep) and basolateral uptake transporter (Ntcp) were increased with the development of HSW. The concentrations of unconjugated BAs increased in mice intestines after the administration of HSW. Western blot and qRT-PCR analyses showed that HSW upregulated the protein and mRNA expression of Shp and Fgf15 in the ileum of the mice. CONCLUSION: HSW treatment for 7days did not cause liver damage. HSW accelerated bile acid enterohepatic circulation and changed the composition of intestinal BAs, leding to the activation of Fxr-Fgf15 signal in intestines, and further inhibited the expression of Cyp7a1 in the liver.

Pinelliae Rhizoma Praeparatum Involved in the Regulation of Bile Acids Metabolism in Hepatic Injury.

Pinelliae Rhizoma Praeparatum (PRP) as traditional Chinese medicine had been used for hepatic diseases in combinative forms. However, the effect of PRP was not clear when used alone. So to explore the hepatoprotective/hepatotoxin of PRP is necessary. The activities of PRP were investigated in acetaminophen-induced hepatic injury mice. Liver function markers, hepatic oxidative stress markers were evaluated. Bile acids metabolic transports and nuclear factor erythroid 2-related factor 2 (Nrf2) were detected. As a drug for the treatment of liver diseases, PRP slightly restored the parameters towards normal in model mice only in low dosage, and also had no antioxidant activity and regulate Nrf2. Cholestasis was significantly elevated in model mice when pretreatment with routine or high dosage of PRP, but had no effect on normal mice. Bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2) in model mice were markedly increased when pretreatment with low dose PRP, but significantly decreased when pretreatment in routine or high dosage. Mrp3 was significantly induced in model mice after pretreatment of PRP. But the adjustment effect to bile acids transporters by PRP was not significant in normal mice. These results reveal that PRP has the different effects on bile acids transporter in hepatic injury mice, and therefore, the dosage of PRP need to be paid attention to when it is used in clinical hepatic injury.

Perioperative oral supplementation with fish oil promotes liver regeneration following partial hepatectomy in mice via AMPK activation.

The present study aimed to observe the effects of perioperative oral supplementation with fish oil (FO) on liver regeneration in mice and examine the potential mechanism. A total of 120 male ICR mice were randomly divided into 5 groups: Sham, Control, fish oil (FO), Compound C [the AMP­activated protein kinase (AMPK) inhibitor dorsomorphin], and Compound C + FO. Changes in liver function, alterations in hepatocyte proliferation and in the expression of polarization markers, and activation of AMPK signaling were examined following partial hepatectomy (PH). The results demonstrated that restoration of serum alanine aminotransferase (ALT) and total bilirubin (TBIL) levels were significantly faster in FO­treated mice compared with Control mice, and this effect was suppressed by treatment with Compound C. FO­treated mice exhibited increased numbers of Ki­67 positive hepatocytes and their postoperative liver­to­body weight ratio was significantly increased compared with the Control mice, which was also suppressed by co­treatment with the AMPK inhibitor. Furthermore, protein expression of Occludin, Claudin­3, tight junction protein 1 and bile salt export pump was gradually increased in FO­treated mice compared with Control, whereas Compound C treatment reversed this effect. Therefore, the present study revealed that perioperative oral supplementation with FO may promote liver regeneration and improved liver function in mice following PH through AMPK activation.

Measures of BSEP Inhibition In Vitro Are Not Useful Predictors of DILI.

Inhibition of the bile salt export pump (BSEP) by a drug has been implicated as a risk factor for a drug's potential to cause drug-induced liver injury (DILI) and is thought to be an important mechanism leading to DILI. For a wide variety of drugs a correlation has been observed between the potency of in vitro BSEP inhibition and its propensity to cause DILI in humans. These findings were interpreted to suggest that BSEP inhibition could be an important mechanism to help explain how some drugs initiate DILI. Because the Biopharmaceutics Drug Disposition Classification System (BDDCS) can be useful in characterizing and predicting some important transporter effects in terms of drug-drug interactions, we evaluated the information provided by BDDCS in order to understand the inhibition propensity of BSEP. Here we analyze the relationship between a compound's ability to inhibit BSEP function and cause liver injury in humans using a compilation of published DILI datasets that have screened for BSEP inhibitors, other hepatic transporters and other mechanism-based toxicity key events. Our results demonstrate that there is little support for in vitro BSEP inhibition being universally DILI predictive. Rather we show that most potent BSEP inhibitors are BDDCS class 2 drugs, which we have demonstrated previously is the BDDCS class most likely to be DILI related. Since BDDCS class is not related to any proposed DILI mechanistic hypotheses, we maintain that if measures of BSEP inhibition alone or together with inhibition of other transporters cannot be differentiated from class 2 assignment, there is no support for in vitro BSEP inhibition being DILI predictive.

Genetic and Proteomic characterization of Bile Salt Export Pump (BSEP) in Snake Liver.

Snake gallbladder, a traditional Chinese medicine, has been believed in various Asian countries to improve visual acuity and alleviate rheumatism. Bile acids, a major component of the gallbladder, are toxic to the liver and kidney in humans and animals due to its detergent effects, while also exhibiting therapeutic effects due to an increase in the gallbladder contractions of muscle strips in patients with cholesterol gallstones. Secretion of bile acids in human and mammals depends on the bile salt export pump (BSEP), a liver-specific adenosine triphosphate (ATP)-binding cassette transporter encoded by ABCB11. However, the presence of BSEP in snakes has not been thoroughly explored. Here we confirm the existence of BSEP and its coding DNA sequence in snakes on both the proteomic and genetic level. This work provides information on the snake ABCB11 sequence and helps further potential genetic manipulation to affect bile salt metabolism. Our study provides the foundation for research on bile acid production from snakes by using modern genetic and proteomic methodologies.

A preliminary investigation on single nucleotide polymorphism rs2287622 of bile salt export pump gene in patients with chronic hepatitis C virus infection in Hunan, China.

BACKGROUND: European researchers have underscored associations between single nucleotide polymorphism (SNP) rs2287622 of the hepatobiliary bile salt export pump (BSEP) gene and the risk of hepatitis C virus (HCV) infection. The distributions of SNP rs2287622 are racially specific. This study was aimed to preliminarily investigate the distribution of BSEP gene SNP rs2287622 in the Han patients with chronic HCV-infection (CHC) in Hunan, China. METHODS: BSEP gene SNP rs2287622 of 165 CHC patients, 99 patients with chronic hepatitis B virus infection (CHB) and 99 healthy individuals were analyzed by polymerase chain reaction-restriction fragment length polymorphism analysis and nucleotide sequencing. RESULTS: The overall frequencies of the C allele of BESP gene SNP rs2287622 in the CHC patients, CHB patients and healthy individuals were 74.2, 72.7 and 74.2%, respectively (P > 0.05). The overall odds ratios (ORs) aiming at predicting CHC risk by comparing the ratios of the frequency distribution of alleles or genotypes in the CHC group with those in the non-CHC group had no statistical significance (P > 0.05). However, the CHC ORs of CC vs TT, TC vs TT and CC + CT vs TT among the individuals aged over 40 years were 2.680, 3.122 and 2.824 respectively (P < 0.05), and the higher risk did not relate to gender, HCV genotypes and presence of HCV-related liver cirrhosis. CONCLUSIONS: Among the Han individuals aged over 40 years in Hunan, China, genotype CC or CT of BSEP gene SNP rs2287622 may correlate with higher risk of CHC in comparison with genotype TT. Further study with a larger cohort is essential.