Main Bile Acids Research Articles

Cholangiocyte organoids to study drug-induced injury.

Drug induced bile duct injury is a frequently observed clinical problem leading to a wide range of pathological features. During the past decades, several agents have been identified with various postulated mechanisms of bile duct damage, however, mostly still poorly understood. Here, we investigated the mechanisms of chlorpromazine (CPZ) induced bile duct injury using advanced in vitro cholangiocyte cultures. Intrahepatic cholangiocyte organoids (ICOs) were driven into mature cholangiocyte like cells (CLCs), which were exposed to CPZ under cholestatic or non-cholestatic conditions through the addition of a bile acid cocktail. CPZ caused loss of monolayer integrity by reducing expression levels of tight junction protein 1 (TJP1), E-cadherin 1 (CDH1) and lysyl oxidase homolog 2 (LOXL2). Loss of zonula occuludens-1 (ZO-1) and E-cadherin was confirmed by immunostaining after exposure to CPZ and rhodamine-123 leakage further confirmed disruption of the cholangiocyte barrier function. Furthermore, oxidative stress seemed to play a major role in the early damage response by CPZ. The drug also decreased expression of three main basolateral bile acid transporters, ABCC3 (ATP binding cassette subfamily C member 3), SLC51A/B (solute carrier family 51 subunit alpha/beta) and multidrug resistance transporter ABCB1 (ATP binding cassette subfamily B member 1), thereby contributing to bile acid accumulation. CPZ did not induce an inflammatory response by itself, but addition of TNFα revealed a synergistic effect. These results show that ICOs present a model to identify toxic drugs affecting the bile ducts while providing mechanistic insights into hepatotoxicity.

Bile acid receptors and renal regulation of water homeostasis.

The kidney is the key organ responsible for maintaining the body's water and electrolyte homeostasis. About 99% of the primary urine filtered from the Bowman's capsule is reabsorbed along various renal tubules every day, with only 1-2L of urine excreted. Aquaporins (AQPs) play a vital role in water reabsorption in the kidney. Currently, a variety of molecules are found to be involved in the process of urine concentration by regulating the expression or activity of AQPs, such as antidiuretic hormone, renin-angiotensin-aldosterone system (RAAS), prostaglandin, and several nuclear receptors. As the main bile acid receptors, farnesoid X receptor (FXR) and membrane G protein-coupled bile acid receptor 1 (TGR5) play important roles in bile acid, glucose, lipid, and energy metabolism. In the kidney, FXR and TGR5 exhibit broad expression across all segments of renal tubules, and their activation holds significant therapeutic potential for numerous acute and chronic kidney diseases through alleviating renal lipid accumulation, inflammation, oxidative stress, and fibrosis. Emerging evidence has demonstrated that the genetic deletion of FXR or TGR5 exhibits increased basal urine output, suggesting that bile acid receptors play a critical role in urine concentration. Here, we briefly summarize the function of bile acid receptors in renal water reabsorption and urine concentration.

Gut microbiota and fecal metabolic signatures in rat models of disuse-induced osteoporosis.

Assessing the correlation between gut microbiota (GM) and bone homeostasis has increasingly attracted research interest. Meanwhile, GM dysbiosis has been found to be associated with abnormal bone metabolism. However, the function of GM in disuse-induced osteoporosis (DIO) remains poorly understood. In our research, we evaluated the characteristics of GM and fecal metabolomics to explore their potential correlations with DIO pathogenesis. DIO rat models and controls (CON) underwent micro-CT, histological analyses, and three-point bending tests; subsequently, bone microstructures and strength were observed. ELISAs were applied for the measurement of the biochemical markers of bone turnover while GM abundance was observed using 16S rDNA sequencing. Metabolomic analyses were used to analyze alterations fecal metabolites. The potential correlations between GM, metabolites, and bone loss were then assessed. In the DIO group, the abundance of GM was significantly altered compared to that in the CON group. Moreover, DIO significantly altered fecal metabolites. More specifically, an abnormally active pathway associated with bile acid metabolism, as well as differential bacterial genera related to bone/tissue volume (BV/TV), were identified. Lithocholic acid, which is the main secondary bile acid produced by intestinal bacteria, was then found to have a relationship with multiple differential bacterial genera. Alterations in the intestinal flora and metabolites in feces, therefore, may be responsible for DIO-induced bone loss. The results indicated that changes in the abundance of GM abundance and fecal metabolites were correlated with DIO-induced bone loss, which might provide new insights into the DIO pathogenesis. The detailed regulatory role of GM and metabolites in DIO-induced bone loss needs to be explored further.

Microbial Metabolites Target Histone Deacetylases to Trigger Vascular Inflammation in Sickle Cell Disease

The gut microbiota is a fundamental regulator of host immune responses. Our group previously demonstrated that the depletion of the gut microbiota by broad-spectrum antibiotics (ABX) prevented neutrophil aging (a type of circadian priming) and vaso-occlusion and prolonged survival in a mouse model for sickle cell disease (SCD) (Zhang, D et al. Nature 2015, 570(7570):528-532); the mechanisms involved, however, are as yet unknown. Diverse metabolites are generated by the microbiota from the fermentation of exogenous undigested dietary components and endogenous substrates secreted by the host. Complex carbohydrates are metabolized into short-chain fatty acids (SCFAs), while cholesterol derivatives from the liver are converted into secondary bile acids by intestinal bacteria. The major SCFA producers are Bacteroidetes (gram-negative) and Firmicutes (gram-positive) divisions, and dysbiosis of these bacterial divisions is found in both SCD humans and mice, (Lim, S et al. Am J Hematol 2018, 93(4):E91-E93). Herein, we investigate whether and how microbiota-derived metabolites modulate the dynamics of neutrophils in vaso-occlusive processes in SCD mice. First, to C57BL/6 (wild-type, WT) mice we administered either: 1) one of three main SCFAs, namely sodium butyrate (Na-B), sodium propionate (Na-P), or sodium acetate (Na-A); or 2) a combination of two main secondary bile acids, lithocholic and deoxycholic acids. The number of circulating neutrophils with an aged phenotype (i.e., Ly6Ghi CD11bhi CD62Llo CXCR4pos, Casanova-Acebes, M et al. Cell 2013, 153(5):1025-1035) was assessed by flow cytometry over time. Numbers started to increase at 2h after Na-B administration and we observed a peak of accumulation of total and aged-like neutrophils at 4-5h, with a return to basal after 9h. Consistent with this finding, administration of Na-B to SCD mice led to augmented counts of aged-like neutrophils and impaired survival of mice subjected to inflammation. Interestingly, when SCD mice receiving prolonged ABX treatment (40-45 days) were also given daily Na-B for the last days of ABX treatment, the percentage of aged-like neutrophils in the circulation was higher than that found in the blood of SCD mice under ABX, but without Na-B (55% vs 24%), showing that Na-B by itself is able to reverse the reduction in neutrophil aging promoted by ABX. To address the impact of Na-B in SCD vaso-occlusion, intravital microscopy of the cremaster muscle of SCD mice, after inflammatory stimulus with TNFa, revealed that Na-B abrogated the beneficial effects of ABX and triggered vaso-occlusive processes. We found a 3-fold rise in the adhesion of leukocytes to the endothelium and a 2-fold slower blood flow rate in the microvasculature of mice who received Na-B (see Figure). These results support our hypothesis that the gut microbiota modulates inflammation and vaso-occlusion in SCD through the generation of SCFAs. Because Na-B is known to be a potent inhibitor of histone deacetylase (HDAC) enzymes, we induced HDAC inhibition in WT mice. Vorinostat, a potent synthetic HDAC inhibitor, led to a 3-fold greater expansion of total and aged-like neutrophils in the blood, suggesting a role for HDACs in neutrophil biology. Consistently, in vitro treatment of WT mouse neutrophils with vorinostat or Na-B accelerated the progressive acquisition of an aged-like phenotype, characterized by loss of L-selectin (CD62L). Proteome of neutrophils from WT mice after induction of circadian aging by anti-P and anti-E-selectin antibodies showed that aged neutrophils are similar to neutrophils from vorinostat-treated mice, with down-regulation of genes involved in mRNA processing, protein synthesis, and proteasome complex formation, suggesting that HDAC inhibition is the underlying mechanism driving neutrophil aging. Importantly, the down- and up-regulated pathways found in these neutrophils were the same as those found in SCD mouse neutrophils, and an opposite proteomic profile was observed in neutrophils from ABX-treated SCD mice. Our findings indicate that the intestinal microbiota drives vaso-occlusion and neutrophil aging in SCD mice through the production of metabolites that inhibit HDAC. They also define the microbiota as instigator and innate immunity as effector of vascular disease, such that targeting their crosstalk could be effective at alleviating the devastating symptoms in SCD patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Deoxycholic acid induces gastric intestinal metaplasia by activating STAT3 signaling and disturbing gastric bile acids metabolism and microbiota

ABSTRACT Intestinal metaplasia (IM) is the inevitable precancerous stage to develop intestinal-type gastric cancer (GC). Deoxycholic acid (DCA) is the main bile acid (BA) component of duodenogastric reflux and has shown an increased concentration during the transition from chronic gastritis to IM associated with continued STAT3 activation. However, the mechanisms underlying how DCA facilitates IM in the gastric epithelium need exploration. We evaluated IM and bile reflux in corpus tissues from 161 subjects undergoing GC screening. Cell survival and proliferation, proinflammatory cytokine expression and TGR5/STAT3/KLF5 axis activity were measured in normal human gastric cells, cancer cells, and organoid lines derived from C57BL/6, FVB/N and insulin-gastrin (INS-GAS) mice treated with DCA. The effects of DCA on IM development were determined in INS-GAS mice with long-term DCA supplementation, after which the gastric bacterial and BA metabolic profiles were measured by 16S rRNA gene sequencing and LC-MS. We revealed a BA-triggered TGR5/STAT3/KLF5 pathway in human gastric IM tissues. In gastric epithelial cells, DCA promoted proliferation and apoptotic resistance, upregulated proinflammatory cytokines and IM markers, and facilitated STAT3 phosphorylation, nuclear accumulation and DNA binding to the KLF5 promoter. DCA triggered STAT3 signaling and the downstream IM marker KLF5 in mouse gastric organoids in vitro and in vivo. In INS-GAS mice, DCA promoted the accumulation of serum total BAs and accelerated the stepwise development of gastric IM and dysplasia. DCA induced gastric environmental alterations involving abnormal BA metabolism and microbial dysbiosis, in which the Gemmobacter and Lactobacillus genera were specifically enriched. Lactobacillus genus enrichment was positively correlated with increased levels of GCA, CA, T-α-MCA, TCA and β-MCA in DCA-administrated INS-GAS mice. DCA promotes nuclear STAT3 phosphorylation, which mediates KLF5 upregulation associated with gastric inflammation and IM development. DCA disturbs the gastric microbiome and BA metabolism homeostasis during IM induction.

A critical review of bile acids and their receptors in hepatic encephalopathy

Hepatic encephalopathy describes an array of neurological complications that arise due to liver insufficiency. The pathogenesis of hepatic encephalopathy shares a longstanding association with hyperammonemia and inflammation, and recently, aberrant bile acid signaling has been implicated in the development of key features of hepatic encephalopathy. These key features include neuronal dysfunction, neuroinflammation and blood–brain barrier permeability. This review summarizes the findings of recent studies demonstrating a role for bile acids in the pathogenesis of hepatic encephalopathy via one of three main bile acid receptors and speculates on the possible downstream consequences of aberrant bile acid signaling.

Supplementing a Clay Mineral-Based Feed Additive Modulated Fecal Microbiota Composition, Liver Health, and Lipid Serum Metabolome in Dairy Cows Fed Starch-Rich Diets.

Starch-rich diets are a commonly adopted strategy in order to sustain high milk yields in dairy cows. However, these diets are known to increase the risk of gut dysbiosis and related systemic health disorders. This study aimed to evaluate the effects of supplementing a clay mineral-based feed additive (CM; Mycofix® Plus, BIOMIN) on fecal microbiota structure, fecal short-chain fatty acid (SCFA) fermentation, serum metabolome, and liver health in primiparous (PP, n = 8) and multiparous (MP, n = 16) early-lactation Simmental cows (737 ± 90 kg of live body weight). Cows were randomly assigned to either a control or CM group (55 g per cow and day) and transitioned from a diet moderate in starch (26.3 ± 1.0%) to a high starch diet (32.0 ± 0.8%). Supplementation of CM reversed the decrease in bacterial diversity, richness, and evenness (p < 0.05) during high-starch diet, demonstrating that CM supplementation efficiently eased hindgut dysbiosis. The CM treatment reduced levels of Lactobacillus in PP cows during starch-rich feeding and elevated fecal pH, indicating a healthier hindgut milieu compared with that in control. Butyrate and propionate levels were modulated by CM supplementation, with butyrate being lower in CM-treated MP cows, whereas propionate was lower in MP but higher in PP cows. Supplementing CM during high-starch feeding increased the concentrations of the main primary bile salts and secondary bile acids in the serum and improved liver function in cows as indicated by reduced levels of glutamate dehydrogenase and γ-glutamyl-transferase, as well as higher serum albumin and triglyceride concentrations. These changes and those related to lipid serum metabolome were more pronounced in PP cows as also corroborated by relevance network analysis.

Effect of bile acids on the expression of MRP3 and MRP4: An In vitro study in HepG2 cell line

Introduction and objectivesFree and conjugated bile acids (BA's) cannot cross cell membranes; therefore, a particular transport system is required by the cell. Members of the family of ABC (ATP-binding proteins) transporters transfer bile acids in and out of the cell, preventing their accumulation. High intracellular concentrations of bile acids, such as those observed in cholestasis, have been related to oxidative stress and apoptosis, which in many cases are the leading causes of hepatocyte damage. MRP3 and MRP4 (multidrug resistance-associated protein 3 and 4) proteins belong to the ABC subfamily C, and are transporters of the hepatocyte's basolateral membrane with a compensatory role. Both transporters’ increased expression constitutes an essential role in the protective and adaptive responses of bile acid overload, such as cholestasis. This work aimed to analyze both transporters’ mRNA and protein expression in an in vitro model of cholestasis using HepG2 cell line treated with main bile acids. MethodsThe expression of transporters was investigated through confocal microscopy immunofluorescence, Western Blot, and RT-qPCR after the main bile acids in HepG2 line cells. ResultsThe results showed the relation between confluence and expression of both transporters in the plasma membrane. MRP3 showed atypical and heterogeneous distribution in this cell line. CDCA (chenodeoxycholic acid) at low concentrations induced the expression of mRNA of both transporters. In contrast, protein expression was induced by CA (cholic acid) at high concentrations. ConclusionPrimary bile acids (CDCA and CA) induce overexpression of the MRP4 and MRP3 transporters in the HepG2 cell line.

Ursodeoxycholic acid in intrahepatic cholestasis of pregnancy: a secondary analysis of the PITCHES trial.

ObjectiveTo evaluate whether a particular group of women with intrahepatic cholestasis of pregnancy (ICP), based on their presenting characteristics, would benefit from treatment with ursodeoxycholic acid (UDCA).DesignSecondary analysis of the PITCHES trial (ISRCTN91918806).SettingUnited Kingdom.Population or Sample527 women with ICP.MethodsSubgroup analyses were performed to determine whether baseline bile acid concentrations or baseline itch scores moderated a woman’s response to treatment with UDCA.Main outcome measuresBile acid concentration and itch score.ResultsIn women with baseline bile acid concentrations less than 40 μmol/l, treatment with UDCA resulted in increased post‐randomisation bile acid concentrations (geometric mean ratio 1.19, 95% CI 1.00–1.41, P = 0.048). A test of interaction showed no significance (P = 0.647). A small, clinically insignificant difference was seen in itch response in women with a high baseline itch score (–6.0 mm, 95% CI −11.80 to −0.21, P = 0.042), with a test of interaction not showing significance (P = 0.640). Further subgroup analyses showed no significance. Across all women there was a weak relationship between bile acid concentrations and itch severity.ConclusionsThere was no subgroup of women with ICP in whom a beneficial effect of treatment with UDCA on bile acid concentration or itch score could be identified. This confirms that its routine use in women with this condition for improvement of bile acid concentration or itch score should be reconsidered.Tweetable abstractPITCHES: No group of women with ICP has been found in whom UDCA reduces bile acid concentrations or pruritus.

Taurocholic acid lowers the inflammatory response of gingival fibroblasts, epithelial cells, and macrophages.

Taurocholic acid (TCA), a conjugation of cholic acid with taurine, is one of the main bile acids that is elevated in liver disease. Considering the epidemiologic linkage of periodontal disease to liver disease, the question arises about the possible effect of elevated TCA levels on periodontal cells. To answer this question, gingival fibroblasts and human oral squamous cell carcinoma cell line (HSC-2) were pretreated with interleukine1β (IL1β) and tumor necrosis factorα (TNFα) in the presence and absence of TCA. Also, mouse macrophages (RAW 264.7) were incubated with sterile-filtered human saliva with and without TCA. Inflammatory cytokines were measured by real time polynucleotide chain reaction (RT-PCR) and an immunoassay. The nuclear translocation of the p65 subunit was visualized by immunostaining. In pretreated gingival fibroblasts and HSC-2 cells, TCA considerably reduced the expression of IL1β, IL6, and IL8. In support of these observations, TCA lowered the saliva-induced expression of IL1α, IL1β and IL6 in RAW 264.7 cells. An immunoassay confirmed the capacity of TCA to diminish inflammation-induced expression of IL6 in gingival fibroblasts, HSC-2 and RAW 264.7 cells. Consistently, TCA blocked the nuclear translocation of p65 in fibroblasts. These findings suggest that TCA has anti-inflammatory activity in gingival fibroblasts, human oral squamous cell carcinoma cells and macrophages in vitro.

Transcriptome analysis of golden pompano (Trachinotus ovatus) liver indicates a potential regulatory target involved in HUFA uptake and deposition

Promoting highly unsaturated fatty acid (HUFA) uptake and deposition can improve nutritional value of farmed fish and reduce dietary fish oil addition. Previously, we found that the golden pompano Trachinotus ovatus liver HUFA content increased with the increasing of dietary HUFA. Therefore, we examined the common genes and pathways responsible for HUFA uptake and deposition in T. ovatus liver using transcriptome sequencing technology after feeding with either 1.0% or 2.1% HUFA for 8 weeks. Results showed that a total of 140 and 147 genes were significantly upregulated and downregulated, respectively. Five bile acid synthesis-related genes (CYP7A1, CYP8B1, AKR1D1, SCP2 and ACOT8), which are related to dietary fat emulsification were downregulated in 2.1% HUFA group, implying that the cholate synthesized through the classical pathway might be the main bile acid form in fat emulsification. Moreover, fatty acid transport protein (FATP)-6, fatty acid binding protein (FABP)-1, -4, and -6 increased with HUFA deposition, especially FATP6 and FABP4, suggesting that the two genes may be important mediators involved in HUFA uptake and deposition. KEGG analysis showed that most of the differential genes described above were involved in peroxisome proliferator activator receptor (PPAR) signaling pathway, and PPARγ increased with HUFA deposition, indicating that PPARγ might be a key regulator of HUFA uptake and deposition by regulating the genes involved in fatty acid emulsification and transport. This study focused on the liver, which is the center of intermediary metabolism, providing a comprehensive understanding of the molecular regulation of HUFA uptake and deposition in T. ovatus, which should be further investigated to develop potential measures to improve HUFA content.

Defective FXR-FGF15 signaling and bile acid homeostasis in cystic fibrosis mice can be restored by the laxative polyethylene glycol.

The gastrointestinal phenotype of cystic fibrosis (CF) features intestinal bile acid (BA) malabsorption, impaired intestinal farnesoid X receptor (FXR) activation, and consequently reduced fibroblast growth factor 19 (FGF19, FGF15 in mice) production. The osmotic laxative polyethylene glycol (PEG) has been shown to decrease intestinal mucus accumulation in CF mice and could, by doing so, improve BA reabsorption. Here we determined the effect of PEG on BA excretion and FXR-FGF15 signaling in CF mice. Male Cftr-/-tm1Unc (CF) and wild-type (WT) littermates were administered PEG 4000 in drinking water and fed either chow or a semisynthetic diet. PEG was withdrawn for 3 days before termination. Fecal BA excretion was measured at PEG dosages of 37 g/l (100%) and 0 g/l (0%). Ileal FXR activation was assessed by gene expression of its downstream targets Fgf15 and small heterodimer partner ( Shp). In CF mice, PEG withdrawal increased fecal BA excretion on either diet compared with full PEG dosage (chow, 2-fold, P = 0.06; semisynthetic, 4.4-fold, P = 0.007). PEG withdrawal did not affect fecal BA excretion in WT mice on either diet. After PEG withdrawal, gene expression levels of intestinal FXR target genes Fgf15 and Shp were decreased in CF mice but unaffected in WT littermates. PEG did not affect the gene expression of the main intestinal BA transporter apical sodium-dependent bile acid transporter (ASBT). PEG treatment ameliorates intestinal BA malabsorption in CF mice and restores intestinal FXR-FGF15 signaling, independent from Asbt gene expression. These findings highlight the potential of PEG in the prevention and treatment of the gastrointestinal phenotype of CF. NEW & NOTEWORTHY A gastrointestinal feature of cystic fibrosis is bile acid malabsorption and consequent impairment of farnesoid X receptor (FXR)-fibroblast growth factor 15 (FGF15) signaling. FXR-FGF15 signaling regulates various metabolic processes and could be implicated in metabolic and gastrointestinal complications of cystic fibrosis, such as diabetes and liver disease. In cystic fibrosis mice, treatment with the osmotic laxative polyethylene glycol is associated with decreased fecal bile acid loss and restoration of FXR-FGF15 signaling.

Differential View on the Bile Acid Stress Response of Clostridioides difficile.

Clostridioides difficile is an intestinal human pathogen that uses the opportunity of a depleted microbiota to cause an infection. It is known, that the composition of the intestinal bile acid cocktail has a great impact on the susceptibility toward a C. difficile infection. However, the specific response of growing C. difficile cells to diverse bile acids on the molecular level has not been described yet. In this study, we recorded proteome signatures of shock and long-term (LT) stress with the four main bile acids cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), and lithocholic acid (LCA). A general overlapping response to all tested bile acids could be determined particularly in shock experiments which appears plausible in the light of their common steroid structure. However, during LT stress several proteins showed an altered abundance in the presence of only a single or a few of the bile acids indicating the existence of specific adaptation mechanisms. Our results point at a differential induction of the groEL and dnaKJgrpE chaperone systems, both belonging to the class I heat shock genes. Additionally, central metabolic pathways involving butyrate fermentation and the reductive Stickland fermentation of leucine were effected, although CA caused a proteome signature different from the other three bile acids. Furthermore, quantitative proteomics revealed a loss of flagellar proteins in LT stress with LCA. The absence of flagella could be substantiated by electron microscopy which also indicated less flagellated cells in the presence of DCA and CDCA and no influence on flagella formation by CA. Our data break down the bile acid stress response of C. difficile into a general and a specific adaptation. The latter cannot simply be divided into a response to primary and secondary bile acids, but rather reflects a complex and variable adaptation process enabling C. difficile to survive and to cause an infection in the intestinal tract.

Chenodeoxycholic Acid from Bile Inhibits Influenza A Virus Replication via Blocking Nuclear Export of Viral Ribonucleoprotein Complexes.

Influenza A virus (IAV) infection is still a major global threat for humans, especially for the risk groups: young children and the elderly. The currently licensed antiviral drugs target viral factors and are prone to viral resistance. In recent years, a few endogenous small molecules from host, such as estradiol and omega-3 polyunsaturated fatty acid (PUFA)-derived lipid mediator protection D1 (PD1), were demonstrated to be capable of inhibiting IAV infection. Chenodeoxycholic acid (CDCA), one of the main primary bile acids, is synthesized from cholesterol in the liver and classically functions in emulsification and absorption of dietary fats. Clinically, CDCA has been used in the treatment of patients with cholesterol gallstones for more than five decades. In this study, we showed that CDCA attenuated the replication of three subtypes of influenza A virus, including a highly pathogenic H5N1 strain, in A549 and MDCK cell cultures with IC50 ranging from 5.5 to 11.5 μM. Mechanistically, CDCA effectively restrained the nuclear export of viral ribonucleoprotein (vRNP) complexes. In conclusion, as an endogenous physiological small molecule, CDCA can inhibit IAV replication in vitro, at least in part, by blocking vRNP nuclear export, and affords further studies for development as a potential antiviral agent against IAV infections.

Effect of fermentation on components of bile acids in Arisaema Cum Bile and determination of three kinds of free bile acids in Arisaema Cum Bile

The aim of this study is to analyze the compositions of main bile acids in fermented and mixed processing products of arisame cum bile from pig bile, and to establish a method for content determination of bile acids in fermented Arisaema Cum Bile. Fermented and mixed processing products were prepared from arisaematis rhizome and arisaematis rhizoma preparatum with pig bile respectively. Then the differences in bile acids compositions between such two kinds of products were compared by high performance liquid chromatography and evaporative light-scattering detector (HPLC-ELSD). With three kinds of free bile acid compositions as the indicators, HPLC-ELSD method was adopted to determine the content of bile acid compositions in fermented product,on Agilent Eclipse XDB C₁₈(4.6 mm×250 mm, 5 μm) chromatographic column, with acetonitrile and 0.1% glacial acetic acid solution (55:45) as mobile phase, at a flow rate of 1 mL·min⁻¹, column temperature of 30 °C, drift tube temperature of 90 °C, and a nitrogen flow rate of 2.2 mL·min⁻¹. The results showed that the bile acids in fermented bile Arisaema were mainly in a free form, while in mixed processing product, the compositions were mainly in a conjugated form. Three kinds of free bile acids, namely porcine cholic acid (HCA), porcine deoxycholic acid (HDCA) and chenodeoxycholic acid (CDCA) in fermented product, showed a good linear relationship in the range of quantification. The average recovery rate was 95.99%-104.3%, complying with the requirements. The results showed that the conjugated bile acids could be transformed into free bile acids during the fermentation of arisaema cum bile. This established method can effectively control the content of bile acids compositions in fermenting arisaema cum bile.

Lithocholic Acid-Based Peptide Delivery System for an Enhanced Pharmacological and Pharmacokinetic Profile of Xenopus GLP-1 Analogs.

GLP-1 analogs suffer from the main disadvantage of a short in vivo half-life. Lithocholic acid (LCA), one of the four main bile acids in the human body, possesses a high albumin binding rate. We therefore envisioned that a LCA-based peptide delivery system could extend the half-life of GLP-1 analogs by facilitating the noncovalent binding of peptides to human serum albumin. On the basis of our previously identified Xenopus GLP-1 analogs (1-3), a series of LCA-modified Xenopus GLP-1 conjugates were designed (4a-4r), and the bioactivity studies of these conjugates were performed to identify compounds with balanced in vitro receptor activation potency and plasma stability. 4c, 4i, and 4r were selected, and their LCA side chains were optimized to further increase their stability, affording 5a-5c. Compound 5b showed a more increased albumin affinity and prolonged in vitro stability than that of 4i and liraglutide. In db/ db mice, 5b exhibited comparable hypoglycemic and insulinotropic activity to liraglutide and semaglutide. Importantly, the enhanced albumin affinity of 5b resulted in a prolonged in vivo antidiabetic duration. Finally, chronic treatment investigations of 5b demonstrated the therapeutic effects of 5b on HbA1c, body weight, blood glucose, and pancreatic endocrine deficiencies on db/ db mice. Our studies revealed 5b as a promising antidiabetic candidate. Furthermore, our study suggests the derivatization of Xenopus GLP-1 analogs with LCA represents an effective strategy to develop potent long-acting GLP-1 receptor agonists for the treatment of type 2 diabetes.

Quantitative profiling of 19 bile acids in rat plasma, liver, bile and different intestinal section contents to investigate bile acid homeostasis and the application of temporal variation of endogenous bile acids

Bile acid homeostasis is maintained by liver synthesis, bile duct secretion, microbial metabolism and intestinal reabsorption into the blood. When drug insults result in liver damage, the variances of bile acids (BAs) are related to the physiological status of the liver. Here, we established a method to simultaneously quantify 19 BAs in rat plasma, liver, bile and different intestinal section contents (duodenum, jejunum, ileum, cecum and colon) using high-performance liquid chromatography-tandem mass spectrometry (LC–MS/MS) to reveal the pattern of bile acid homeostasis in the enterohepatic circulation of bile acids in physiological situations. Dynamic changes in bile acid composition appeared throughout the enterohepatic circulation of the BAs; taurine- and glycine-conjugated BAs and free BAs had different dynamic homeostasis levels in the circulatory system. cholic acid (CA), beta-muricholic acid (beta-MCA), lithocholic acid (LCA), glycocholic acid (GCA) and taurocholic acid (TCA) greatly fluctuated in the bile acid pool under physiological conditions. Taurine- and glycine-conjugated bile acids constituted more than 90% in the bile and liver, whereas GCA and TCA accounted for more than half of the total bile acids and the secretion of bile mainly via conjugating with taurine. While over 80% of BAs in plasma were unconjugated bile acids, CA and HDCA were the most abundant elements. Unconjugated bile acids constituted more than 90% in the intestine, and CA, beta-MCA and HDCA were the top three bile acids in the duodenum, jejunum and ileum content, but LCA and HDCA were highest in the cecum and colon content. As the main secondary bile acid converted by microflora in the intestine, LCA was enriched in the cecum and DCA mostly in the colon. As endogenous substances, the concentrations of plasma BAs were closely related to time rhythm and diet. In conclusion, analyzing detailed BA profiles in the enterohepatic circulation of bile acids in a single run is possible using LC–MS/MS. Based on the physiological characteristics of the metabolic profiling of 19 BAs in the total bile acid pool and the time rhythm variation of the endogenous bile acids, this study provided a new valuable method and theoretical basis for the clinical research of bile acid homeostasis.

Synthesis of cholic-acid-carrying polymer and in-vitro evaluation of hepatoma-targeting nanoparticles decorated with the polymer

The specific interaction between bile acids and the bile acids transporters provides a promising way for hepatoma-targeted drug delivery. We synthesized an amphipathic polymer containing cholic acid (CA), the main bile acids in body, and prepared CA-functionalized nanoparticles to target hepatoma cells. Poly-[3-(4-vinylbenzonate)-7, 12-dihydroxy-5-cholan-24-oic acid] (PVBCA) was synthesized by introducing methyl cholate onto polyvinyl benzoate polymer backbone, and was characterized by 1H-NMR, FT-IR, and GFC. PVBCA can be incorporated onto PLGA nanoparticles surface via the emulsion-solvent evaporation procedure, resulting in the nanoparticles carrying CA moieties on their surface. The binding of CA moieties to the bile acids’ transporters on the cell membrane enhances the cellular uptake of the nanoparticles significantly. The SMMC-7721 cell uptake of PVBCA-decorated nanoparticles increases with amount of incorporated PVBCA and is 2- to 2.8-fold higher than that of the normal PLGA nanoparticles. By exclusion of specific endocytosis pathways using chemical inhibitors, we found that the uptake mechanism of PVBCA-decorated nanoparticles was mainly attributed to clathrin-and-caveolae-independent endocytosis, which was distinct from that of PLGA nanoparticles. The present study provides a simple and versatile method for hepatoma-targeted delivery of nanoparticles.

Ginseng alleviates cyclophosphamide-induced hepatotoxicity via reversing disordered homeostasis of glutathione and bile acid.

Cyclophosphamide (CP), a chemotherapeutic agent, is restricted due to its side effects, especially hepatotoxicity. Ginseng has often been clinically used with CP in China, but whether and how ginseng reduces the hepatotoxicity is unknown. In this study, the hepatoprotective effects and mechanisms under the combined usage were investigated. It was found that ginseng could ameliorate CP-induced elevations of ALP, ALT, ALS, MDA and hepatic deterioration, enhance antioxidant enzymes’ activities and GSH’s level. Metabolomics study revealed that 33 endogenous metabolites were changed by CP, 19 of which were reversed when ginseng was co-administrated via two main pathways, i.e., GSH metabolism and primary bile acids synthesis. Furthermore, ginseng could induce expression of GCLC, GCLM, GS and GST, which associate with the disposition of GSH, and expression of FXR, CYP7A1, NTCP and MRP 3, which play important roles in the synthesis and transport of bile acids. In addition, NRF 2, one of regulatory elements on the expression of GCLC, GCLM, GS, GST, NTCP and MRP3, was up-regulated when ginseng was co-administrated. In conclusion, ginseng could alleviate CP-induced hepatotoxicity via modulating the disordered homeostasis of GSH and bile acid, which might be mediated by inducing the expression of NRF 2 in liver.

Effect of colesevelam on faecal bile acids and bowel functions in diarrhoea-predominant irritable bowel syndrome.

About one-third of patients with IBS-diarrhoea (irritable bowel syndrome-D) have evidence of increased bile acid synthesis or excretion. To assess effects of the bile acid sequestrant, colesevelam, on faecal excretion of BAs, hepatic BA synthesis and diarrhoea in IBS-D; to appraise whether individual or random stool samples accurately reflect 48-h total faecal bile acid excretion and proportions of the main bile acids excreted and to study the faecal fat excretion in response to colesevelam. A single-centre, unblinded, single-dose trial of effects of colesevelam, 1875mg [3 tablets (625mg tablets)] orally, twice daily, for 10days on total 48-h faecal bile acid excretion and fasting serum C4 (7α-hydroxy-4-cholesten-3-one; surrogate of hepatic bile acid synthesis). Stool diaries documented bowel functions for 8days prior and 8days during colesevelam treatment. Stool 48-h samples and fasting serum were collected for faecal fat, faecal bile acid and serum C4. Colesevelam was associated with significantly increased faecal total bile acid excretion and deoxycholic acid excretion, increased serum C4 and more solid stool consistency. There was a significant inverse correlation between number of bowel movements per week and the total bile acid sequestered into stool during the last 48h of treatment. Random stool samples did not accurately reflect 48-h total or individual faecal bile acid excretion. Sequestration of bile acids by colesevelam did not increase faecal fat. Colesevelam increases delivery of bile acids to stool while improving stool consistency, and increases hepatic bile acid synthesis, avoiding steatorrhoea in patients with IBS-D. Overall effects are consistent with luminal bile acid sequestration by colesevelam.