Mechanistic studies on the alleviation of ANIT-induced cholestatic liver injury by Polygala fallax Hemsl. polysaccharides.

ETHNOPHARMACOLOGICAL RELEVANCE: Polygala fallax Hemsl. is a traditional folk medicine commonly used by ethnic minorities in the Guangxi Zhuang Autonomous Region, and has a traditional application in the treatment of liver disease. Polygala fallax Hemsl. polysaccharides (PFPs) are of interest for their potential health benefits. AIM OF THIS STUDY: This study explored the impact of PFPs on a mouse model of cholestatic liver injury (CLI) induced by alpha-naphthyl isothiocyanate (ANIT), as well as the potential mechanisms. MATERIALS AND METHODS: A mouse CLI model was constructed using ANIT (80 mg/kg) and intervened with different doses of PFPs or ursodeoxycholic acid. Their serum biochemical indices, hepatic oxidative stress indices, and hepatic pathological characteristics were investigated. Then RNA sequencing was performed on liver tissues to identify differentially expressed genes and signaling pathways and to elucidate the mechanism of liver protection by PFPs. Finally, Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to verify the differentially expressed genes. RESULTS: Data analyses showed that PFPs reduced the levels of liver function-related biochemical indices, such as ALT, AST, AKP, TBA, DBIL, and TBIL. PFPs up-regulated the activities of SOD and GSH, down-regulated the contents of MDA, inhibited the release of IL-1ß, IL-6, and TNF-α, or promoted IL-10. Pathologic characterization of the liver revealed that PFPs reduced hepatocyte apoptosis or necrosis. The RNA sequencing indicated that the genes with differential expression were primarily enriched for the biosynthesis of primary bile acids, secretion or transportation of bile, the reactive oxygen species in chemical carcinogenesis, and the NF-kappa B signaling pathway. In addition, the results of qRT-PCR and Western blotting analysis were consistent with those of RNA sequencing analysis. CONCLUSIONS: In summary, this study showed that PFPs improved intrahepatic cholestasis and alleviated liver damage through the modulation of primary bile acid production, Control of protein expression related to bile secretion or transportation, decrease in inflammatory reactions, and inhibition of oxidative pressure. As a result, PFPs might offer a hopeful ethnic dietary approach for managing intrahepatic cholestasis.

Recent advances in age-related metabolic dysfunction-associated steatotic liver disease.

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects approximately 25% of the world's population and has become a leading cause of chronic liver disease. In recent years, an increasing amount of data suggests that MASLD is associated with aging. As the population ages, age-related MASLD will become a major global health problem. Targeting an aging will become a new approach to the treatment of MASLD. This paper reviews the current studies on the role of aging-related factors and therapeutic targets in MASLD, including: Oxidative stress, autophagy, mitochondrial homeostasis, bile acid metabolism homeostasis, and dysbiosis. The aim is to identify effective therapeutic targets for age-related MASLD and its progression.

Distinct bile acid alterations in response to a single administration of PFOA and PFDA in mice.

Per- and polyfluoroalkyl substances (PFASs), a group of synthetic chemicals that were once widely used for industrial purposes and in consumer products, are widely found in the environment and in human blood due to their extraordinary resistance to degradation. Once inside the body, PFASs can activate nuclear receptors such as PPARα and CAR. The present study aimed to investigate the impact of perfluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA) on liver structure and functions, as well as bile acid homeostasis in mice. A single administration of 0.1 mmole/kg of PFDA, not PFOA, elevated serum ALT and bilirubin levels and caused cholestasis in WT mice. PFDA increased total and various bile acid species in serum but decreased them in the liver. Furthermore, in mouse livers, PFDA, not PFOA, down-regulated mRNA expression of uptake transporters (Ntcp, Oatp1a1, 1a4, 1b2, and 2b1) but induced efflux transporters (Bcrp, Mdr2, and Mrp2-4). In addition, PFDA, not PFOA, decreased Cyp7a1, 7b1, 8b1, and 27a1 mRNA expression in mouse livers with concomitant hepatic accumulation of cholesterol. In contrast, in PPARα-null mice, PFDA did not increase serum ALT, bilirubin, or total bile acids, but produced prominent hepatosteatosis; and the observed PFDA-induced expression changes of transporters and Cyps in WT mice were largely attenuated or abolished. In CAR-null mice, the observed PFDA-induced bile acid alterations in WT mice were mostly sustained. These results indicate that, at the dose employed, PFDA has more negative effects than PFOA on liver function. PPARα appears to play a major role in mediating most of PFDA-induced effects, which were absent or attenuated in PPARα-null mice. Lack of PPARα, however, exacerbated hepatic steatosis. Our findings indicate separated roles of PPARα in mediating the adaptive responses to PFDA: protective against hepatosteatosis but exacerbating cholestasis.

Allocholic acid protects against α-naphthylisothiocyanate-induced cholestasis in mice by ameliorating disordered bile acid homeostasis.

Cholestasis is a pathological condition characterized by disruptions in bile flow, leading to the accumulation of bile acids (BAs) in hepatocytes. Allocholic acid (ACA), a unique fetal BA known for its potent choleretic effects, reappears during liver regeneration and carcinogenesis. In this research, we investigated the protective effects and underlying mechanisms of ACA against mice with cholestasis brought on by α-naphthylisothiocyanate (ANIT). To achieve this, we combined network pharmacology, targeted BA metabolomics, and molecular biology approaches. The results demonstrated that ACA treatment effectively reduced levels of serum AST, ALP, and DBIL, and ameliorated the pathological injury caused by cholestasis. Network pharmacology analysis suggested that ACA primarily regulated BA and salt transport, along with the signaling pathway associated with bile secretion, to improve cholestasis. Subsequently, we examined changes in BA metabolism using UPLC-MS/MS. The findings indicated that ACA pretreatment induced alterations in the size, distribution, and composition of the liver BA pool. Specifically, it reduced the excessive accumulation of BAs, especially cholic acid (CA), taurocholic acid (TCA), and ß-muricholic acid (ß-MCA), facilitating the restoration of BA homeostasis. Furthermore, ACA pretreatment significantly downregulated the expression of hepatic BA synthase Cyp8b1, while enhancing the expression of hepatic efflux transporter Mrp4, as well as the renal efflux transporters Mdr1 and Mrp2. These changes collectively contributed to improved BA efflux from the liver and enhanced renal elimination of BAs. In conclusion, ACA demonstrated its potential to ameliorate ANIT-induced liver damage by inhibiting BA synthesis and promoting both BA efflux and renal elimination pathways, thus, restoring BA homeostasis.

Modulating intestinal barrier function by sphingosine-1-phosphate receptor 1 specific agonist SEW2871 attenuated ANIT-induced cholestatic hepatitis via the gut-liver axis.

Bile acid (BA) homeostasis throughout the enterohepatic circulation system is a guarantee of liver physiological functions. BA circulation disorders is one of the characteristic clinical manifestations of cholestasis, and have a closely relationship with intestinal barrier function, especially ileum. Here, our in vivo and in vitro studies showed that intestinal tight junctions (TJs) were disrupted by α-naphthylisothiocyanate (ANIT), which also down-regulated the protein expression of sphingosine-1-phosphate receptor 1 (S1PR1) in the top of villus of mice ileum. Activating S1PR1 by specific agonist SEW2871 could improve TJs via inhibiting ERK1/2/LKB1/AMPK signaling pathway in the ileum of ANIT-treated mice and ANIT-cultured Caco-2 cells. SEW2871 not only regained ileum TJs by activating S1PR1 in the epithelial cells of ileum mucosa, but also recovered ileum barrier function, which was further verified by the recovered BA homeostasis in mice ileum (content and tissue) by using of high-performance liquid chromatographytandem mass spectrometry (LC-MS/MS). Subsequently, the improved intestinal injury and inflammation further strengthened that SEW2871 modulated intestinal barrier function in ANIT-treated mice. Finally, our data revealed that along with the down-regulated levels of serum lipopolysaccharides (LPS), SEW2871 improved liver function and relieved hepatitis via blocking TLR4/MyD88/NF-kB signaling pathway in ANIT-treated mice. In conclusion, these results demonstrated that activating intestinal S1PR1 by SEW2871 could modulate intestinal barrier function, leading to the improvement of cholestatic hepatitis in ANIT-treated mice via the "gut-liver" axis.

GLP-2 Improves Hepatic Inflammation and Fibrosis in Mdr2-/- Mice Via Activation of NR4a1/Nur77 in Hepatic Stellate Cells and Intestinal FXR Signaling.

BACKGROUND & AIMS: Glucagon-like peptide (GLP)-2 may exert antifibrotic effects on hepatic stellate cells (HSCs). Thus, we aimed to test whether application of the GLP-2 analogue teduglutide has hepatoprotective and antifibrotic effects in the Mdr2/Abcb4-/- mouse model of sclerosing cholangitis displaying hepatic inflammation and fibrosis. METHODS: Mdr2-/- mice were injected daily for 4 weeks with teduglutide followed by gene expression profiling (bulk liver; isolated HSCs) and immunohistochemistry. Activated HSCs (LX2 cells) and immortalized human hepatocytes and human intestinal organoids were treated with GLP-2. mRNA profiling by reverse transcription polymerase chain reaction and electrophoretic mobility shift assay using cytosolic and nuclear protein extracts was performed. RESULTS: Hepatic inflammation, fibrosis, and reactive cholangiocyte phenotype were improved in GLP-2-treated Mdr2-/- mice. Primary HSCs isolated from Mdr2-/- mice and LX2 cells exposed to GLP-2 in vitro displayed significantly increased mRNA expression levels of NR4a1/Nur77 (P < .05). Electrophoretic mobility shift assay revealed an increased nuclear NR4a1 binding after GLP-2 treatment in LX2 cells. Moreover, GLP-2 alleviated the Tgfß-mediated reduction of NR4a1 nuclear binding activity. In vivo, GLP-2 treatment of Mdr2-/- mice resulted in increased intrahepatic levels of muricholic acids (accordingly Cyp2c70 mRNA expression was significantly increased), and in reduced mRNA levels of Cyp7a1 and FXR. Serum Fgf15 levels were increased in Mdr2-/- mice treated with GLP-2. Accordingly, GLP-2 treatment of human intestinal organoids activated their FXR-FGF19 signaling axis. CONCLUSIONS: GLP-2 treatment increased NR4a1/Nur77 activation in HSCs, subsequently attenuating their activation. GLP-2 promoted intestinal Fxr-Fgf15/19 signaling resulting in reduced Cyp7a1 and increased Cyp2c70 expression in the liver, contributing to hepatoprotective and antifibrotic effects of GLP-2 in the Mdr2-/- mouse model.

Geniposide alleviated bile acid-associated NLRP3 inflammasome activation by regulating SIRT1/FXR signaling in bile duct ligation-induced liver fibrosis.

BACKGROUND: Geniposide (GE), the active compound derived from Gardeniae Fructus, possesses valuable bioactivity for liver diseases, but GE effects on bile duct ligation (BDL)-induced cholestasis remain unclear. This study aimed to elucidate the influence of GE on BDL-induced liver fibrosis and to investigate the underlying mechanisms. METHODS: GE (25 or 50 mg/kg) were intragastrical administered to C57BL/6 J mice for two weeks to characterize the hepatoprotective effect of GE on BDL-induced liver fibrosis. NLRP3 inflammasome activation was detected in vivo, and BMDMs were isolated to explore whether GE directly inhibited NLRP3 inflammasome activation. Serum bile acid (BA) profiles were assessed utilizing UPLC-MS/MS, and the involvement of SIRT1/FXR pathways was identified to elucidate the role of SIRT1/FXR in the hepaprotective effect of GE. The veritable impact of SIRT1/FXR signaling was further confirmed by administering the SIRT1 inhibitor EX527 (10 mg/kg) to BDL mice treated with GE. RESULTS: GE treatment protected mice from BDL-induced liver fibrosis, with NLRP3 inflammasome inhibition. However, development in vitro experiments revealed that GE could not directly inhibit NLRP3 activation under ATP, monosodium urate, and nigericin stimulation. Further mechanistic data showed that GE activated SIRT1, which subsequently deacetylated FXR and restored CDCA, TUDCA, and TCDCA levels, thereby contributing to the observed hepaprotective effect of GE. Notably, EX527 treatment diminished the hepaprotective effect of GE on BDL-induced liver fibrosis. CONCLUSION: This study first proved the hepaprotective effect of GE on liver fibrosis in BDL mice, which was closely associated with the restoration of BA homeostasis and NLRP3 inflammasome inhibition. The activation of SIRT1 and the subsequent FXR deacetylation restored the BA profiles, especially CDCA, TUDCA, and TCDCA contents, which was the main contributor to NLRP3 inhibition and the hepaprotective effect of GE. Overall, our work provides novel insights that GE as well as Gardeniae Fructus might be the potential attractive candidate for ameliorating BDL-induced liver fibrosis.

Yinchen decoction protects against cholic acid diet-induced cholestatic liver injury in mice through liver and ileal FXR signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Cholestasis is a pathophysiological syndrome characterized by the accumulation of bile acids (BAs) that leads to severe liver disease. Artemisia capillaris is documented in Chinese Pharmacopoeia as the authentic resources for Yinchen. Although Yinchen (Artemisia capillaris Thunb.) decoction (YCD) has been used in China for thousands of years to treat jaundice, the underlying mechanisms to ameliorate cholestatic liver injury have not been elucidated. AIM OF THE STUDY: To investigate the molecular mechanism of how YCD protects against 1% cholic acid (CA) diet-induced intrahepatic cholestasis through FXR signaling. MATERIALS AND METHODS: Wild-type and Fxr-deficient mice were fed a diet containing 1% CA to establish the intrahepatic cholestasis model. The mice received low-, medium-, or high-dose YCD for 10 days. Plasma biochemical markers were analyzed, liver injury was identified by histopathology, and hepatic and plasma BA content was analyzed. Western blot was used to determine the expression levels of transporters and enzymes involved in BA homeostasis in the liver and intestine. RESULTS: In wild-type mice, YCD significantly improved plasma transaminase levels, multifocal hepatocellular necrosis, and hepatic and plasma BA contents, upregulated the expression of hepatic FXR and downstream target enzymes and transporters. Meanwhile, YCD significantly induced the expressions of intestinal FXR and FGF15 and hepatic FGFR4. In contrast, the hepatic protective effect of YCD on cholestasis was abolished in Fxr-deficient mice. CONCLUSION: YCD protects against cholestatic liver injury induced by a CA diet by restoring the homeostasis of BAs via activation of the liver FXR/SHP and ileal FXR/FGF15 signaling pathways. Furthermore, chlorogenic acid and caffeic acid may be the pharmacological agents in YCD responsible for protecting against cholestatic liver injury.

Protective mechanism of Yinchenzhufu decoction against cholestatic liver injury induced by lithic acid based on network pharmacology / 药学学报

Yinchenzhufu decoction (YCZFD) is a classic formula for treating Yin Huang syndrome, which can improve liver injury caused by cholestasis. However, the mechanism of action of YCZFD still remains unclear. This article used network pharmacology, molecular docking, animal experiments, and molecular biology methods to explore the mechanism of YCZFD in treating liver injury caused by cholestasis. A mouse model of acute cholestasis induced by lithocholic acid was used to investigate the effects of YCZFD on liver injury. The experimental procedures described in this paper were reviewed and approved by the Ethical Committee at the Shanghai University of Traditional Chinese Medicine (approval NO. PZSHUTCM190823002). The results showed that YCZFD could reduce the levels of blood biochemical indicators and improve hepatocyte damage of cholestatic mice. Then, multiple databases were used to predict the corresponding targets of YCZFD active components on cholestatic liver injury. An intersection target protein-protein interaction (PPI) networks based on String database and Cytoscape software was used to demonstrate the possible core targets of YCZFD against cholestatic liver injury. The results indicated that core targets of YCZFD include tumor necrosis factor, interleukin-1β, non-receptor tyrosine kinase Src, interleukin-6, etc. GO (gene ontology) and KEGG (kyoto encyclopedia of genes and genomes) enrichment analysis indicated that YCZFD may regulate the tumor necrosis factor signaling pathway, nuclear factor-κB signaling pathway, bile secretion, and other related factors to ameliorate the cholestatic liver injury. AutoDockTools software was used to perform molecular docking verification on the core targets and components of YCZFD. To verify the results of network pharmacology, UPLC-MS/MS method was used to determine the effect of YCZFD on levels of bile acid profiles in mouse liver tissues. It was found that treatment with YCZFD significantly reduced the content of free bile acids, taurine bound bile acids, and total bile acids in the liver tissues of cholestatic mice. Then, results from real time PCR and Western blot also found that YCZFD can upregulate the expression of hepatic nuclear receptor farnesoid X receptor, metabolizing enzyme (UDP glucuronidase transferase 1a1), and efflux transporters (bile salt export pump, multidrug resistance-associated protein 2, multidrug resistance-associated protein 3, etc) in cholestasis mice, promote bile acid metabolism and excretion, and improve bile acid homeostasis. Moreover, YCZFD can also inhibit pyroptosis and inflammation by regulating NOD-like receptors 3 pathway, thereby inhibiting cholestatic liver injury.

Gut microbiota involved in desulfation of sulfated progesterone metabolites: A potential regulation pathway of maternal bile acid homeostasis during pregnancy.

Abnormally raised circulating bile acids (BA) during pregnancy threat fetal and offspring health. Our previous study has identified sulfated progesterone metabolites (PMSs) in part account for dysregulation of maternal BA homeostasis during pregnancy, however, limited intervention strategies to remedy increased serum BA through PMSs during pregnancy are available. The purpose of this study is to test the feasibility of manipulating BA homeostasis and progesterone metabolism through steering gut microbiota. A total of 19 pregnant sows were randomly treated with standard diet or vancomycin-supplemented diet, to investigate the intercorrelation of PMSs, intestinal microbiota, and maternal BA metabolism from day 60 of gestation (G60) until farrowing (L0). Pregnant mice orally gavaged with epiallopregnanolone sulfate (PM5S) or vehicle and nonpregnant mice were sampled and further analyzed to verify the effect of PM5S on maternal BA metabolism. The present study revealed that oral vancomycin reduced maternal fasting serum total BA (TBA) levels and postprandial serum TBA levels at day 90 of gestation (G90). BA profile analysis showed the decreased TBA after vancomycin treatment was attributed to the decrease of primary BA and secondary BA, especially hyodeoxycholic acid (HDCA). By using newly developed UPLC-MS/MS methods, we found vancomycin increased fecal excretion of allopregnanolone sulfate (PM4S) and PM5S during late gestation and thus maintaining the relative stability of serum PM4S and PM5S, which play an important role in BA metabolism. Further study in mice showed that pregnant mice have higher serum and liver TBA levels compared with nonpregnant mice, and PM5S administration induced higher gallbladder TBA levels and TBA pool in pregnant mice. In addition, after oral vancomycin, the continuously decreased Parabacteroides genus, potentially enriched with genes encoding steroids sulfatase, may explain the increased fecal PMSs excretion in pregnant sows. Taken together, our study provides the evidence that pregnancy-induced elevation of BA levels in sow is likely regulated by manipulation of gut microbiota, which offer new insights into the prevention and treatment of disrupted BA homeostasis during pregnancy by targeting specific microbiota.

Idiosyncratic liver injury induced by bolus combination treatment with emodin and 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucopyranoside in rats.

Polygoni Multiflori Radix (PMR) is a commonly used traditional Chinese medicine in clinical practice, while adverse effects of hepatotoxicity related to PMR have been frequently reported. The clinical case reports indicated that PMR hepatotoxicity could occur under both overdose medication/long-term exposure and low doses with short-duration (idiosyncratic) conditions. The combination treatment with emodin and 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucopyranoside (TSG), two major PMR components, was reported to contribute to PMR hepatotoxicity after long-term treatment. However, the role of the combination treatment of these two components in PMR-induced idiosyncratic liver injury has not been clearly clarified. In this study, the LPS-mediated inflammatory stress model rats were adopted to explore the idiosyncratic liver injury induced by the bolus combination treatment with emodin and TSG. After a bolus oral administration with TSG (165 mg/kg), emodin (5 mg/kg) or their combination in both normal and LPS-mediated inflammatory stress model rats, the systemic/hepatic concentrations of emodin, emodin glucuronides and bile acids were determined; the hepatotoxicity assessments were conducted via monitoring histopathological changes and liver injury biomarkers (ALT and AST). Moreover, the protein expressions of bile acid homeostasis- and apoptosis-related proteins were examined. No liver damage was observed in the normal rats after a bolus dose with the individual or combination treatment, while the bolus combination treatment with emodin and TSG induced liver injury in the LPS-mediated inflammatory stress model rats, evidenced by the elevated plasma levels of alanine aminotransferase (∼66%) and aspartate aminotransferase (∼72%) accompanied by severe inflammatory cell infiltration and apoptotic hepatocytes in liver tissue. Moreover, such combination treatment at a bolus dose in the LPS-mediated inflammatory stress model rats could significantly elevate the hepatic TBA levels by about 45% via up-regulating the hepatic protein expression levels of bile acid synthesis enzymes and inhibiting that of bile acid efflux transporters and the expression levels of apoptosis-related proteins. Our study for the first time proved the major contribution of the combination treatment with emodin and TSG in PMR-induced idiosyncratic liver injury.

Intratracheal exposure to polyhexamethylene guanidine phosphate disrupts coordinate regulation of FXR-SHP-mediated cholesterol and bile acid homeostasis in mouse liver.

A public health crisis in the form of a significant incidence of fatal pulmonary disease caused by repeated use of humidifier disinfectants containing polyhexamethylene guanidine phosphate (PHMG) recently arose in Korea. Although the mechanisms of pulmonary fibrosis following respiratory exposure to PHMG are well described, distant-organ effect has not been reported. In this study, we investigated whether intratracheal administration of PHMG affects liver pathophysiology and metabolism. Our PHMG mouse model showed a significant decrease in liver cholesterol level. An mRNA-seq analysis of liver samples revealed an alteration in the gene expression associated with cholesterol biosynthesis and metabolism to bile acids. The expression of genes involved in cholesterol synthesis was decreased in a real-time PCR analysis. To our surprise, we found that the coordinate regulation of cholesterol and bile acid homeostasis was completely disrupted. Despite the decreased cholesterol synthesis and low bile acid levels, the farnesoid X receptor/small heterodimer partner pathway, which controls negative feedback of bile acid synthesis, was activated in PHMG mice. As a consequence, gene expression of Cyp7a1 and Cyp7b1, the rate-limiting enzymes of the classical and alternative pathways of bile acid synthesis, was significantly downregulated. Notably, the changes in gene expression were corroborated by the hepatic concentrations of the bile acids. These results suggest that respiratory exposure to PHMG could cause cholestatic liver injury by disrupting the physiological regulation of hepatic cholesterol and bile acid homeostasis.

In vitro models to detect in vivo bile acid changes induced by antibiotics.

Bile acid homeostasis plays an important role in many biological activities through the bile-liver-gut axis. In this study, two in vitro models were applied to further elucidate the mode of action underlying reported in vivo bile acid changes induced by antibiotics (colistin sulfate, tobramycin, meropenem trihydrate, and doripenem hydrate). 16S rRNA analysis of rat fecal samples anaerobically incubated with these antibiotics showed that especially tobramycin induced changes in the gut microbiota. Furthermore, tobramycin was shown to inhibit the microbial deconjugation of taurocholic acid (TCA) and the transport of TCA over an in vitro Caco-2 cell layer used as a model to mimic intestinal bile acid reuptake. The effects induced by the antibiotics in the in vitro model systems provide novel and complementary insight explaining the effects of the antibiotics on microbiota and fecal bile acid levels upon 28-day in vivo treatment of rats. In particular, our results provide insight in the mode(s) of action underlying the increased levels of TCA in the feces upon tobramycin exposure. Altogether, the results of the present study provide a proof-of-principle on how in vitro models can be used to elucidate in vivo effects on bile acid homeostasis, and to obtain insight in the mode(s) of action underlying the effect of an antibiotic, in this case tobramycin, on bile acid homeostasis via effects on intestinal bile acid metabolism and reuptake.

New insights into the bile acid-based regulatory mechanisms and therapeutic perspectives in alcohol-related liver disease.

Cholestasis is a key causative factor in alcohol-related liver disease (ALD) and variable degrees of cholestasis occur in all stages of ALD. However, the pathogenetic mechanisms and biomarkers associated with cholestasis are not well characterized. Cholestatic disease is marked by the disruption of bile acids (BA) transport and homeostasis. Consequently, in both human and experimental ALD, the disease shows a direct correlation with an imbalance in BA equilibrium, which in turn may also affect the severity of the disease. Modulation of BA metabolism or signaling pathways is increasingly considered as a potential therapeutic strategy for ALD in humans. In this paper, we highlight the key advances made in the past two decades in characterizing the molecular regulatory mechanisms of BA synthesis, enterohepatic circulation, and BA homeostasis. We summarize recent insights into the nature of the linkage between BA dysregulation and ALD, including the abnormal expression of genes involved in BA metabolism, abnormal changes in receptors that regulate BA metabolism, and disturbance in the gut flora engaged in BA metabolism caused by alcohol consumption. Additionally, we provide novel perspectives on the changes in BAs in various stages of ALD. Finally, we propose potential pharmacological therapies for ALD targeting BA metabolism and signaling.

Modulation of Disordered Bile Acid Homeostasis and Hepatic Tight Junctions Using Salidroside against Hepatocyte Apoptosis in Furan-Induced Mice.

Furan, a processing-induced food contaminant, has attracted great attention due to its hepatotoxicity. To further investigate the underlying mechanism of salidroside (SAL) alleviating furan-induced liver damage, we divided Balb/c mice into the control group, the furan (8 mg/kg/day) group, and three groups of three different doses of SAL (10/20/40 mg/kg/day) in the current research. The shifted serum profile was observed through untargeted metabonomics, to which the bile acid metabolism was related, and the alleviating effect of SAL against furan-induced apoptosis was caused by the metabolism. Target bile acid quantification for the liver and serum showed that SAL positively regulated the homeostasis of bile acids disturbed by furan. Meanwhile, SAL significantly upregulated the synthesis genes of bile acids (Cyp7a1, Cyp7b1, Cyp8b1, and Cyp27a1) and the uptake transport genes (Ntcp and Oatps) and downregulated the efflux transport genes (Bsep, Ost-α, Ost-ß, Mrp2, and Mrp4). Transmission electron microscopy of the bile canaliculi and tight junctions and the levels of tight junction marker proteins (ZO-1, occludin, and claudin-1) confirmed that the disruption of the hepatic tight junction was inhibited by SAL. The connection between the apoptosis- and tight junction-related proteins was observed through the construction of a protein-protein interaction network. SAL suppressed the furan-induced hepatocyte apoptosis evidenced by the detection of TUNEL and Bax, Bcl-2, and caspase-3 levels. Taken together, SAL alleviated furan-induced hepatocyte apoptosis via altering the disordered homeostasis of bile acids and hepatic tight junctions.

Gut microbiota mediates methamphetamine-induced hepatic inflammation via the impairment of bile acid homeostasis.

Methamphetamine (Meth), an addictive psychostimulant of abuse worldwide, has been a common cause of acute toxic hepatitis in adults. Gut microbiota has emerged as a modulator of host immunity via metabolic pathways. However, the microbial mechanism of Meth-induced hepatic inflammation and effective therapeutic strategies remain unknown. Here, mice were intraperitoneally (i.p.) injected with Meth to induce hepatotoxicity. Cecal microbiome and bile acids (BAs) composition were analyzed after Meth administration. Fecal microbiota transplantation (FMT) technology was utilized to investigate the role of microbiota. Additionally, the protective effects of obeticholic acid (OCA), an agonist of farnesoid X receptor (FXR), were evaluated. Results indicated that Meth administration induced hepatic cholestasis, dysfunction and aroused hepatic inflammation by stimulating the TLR4/MyD88/NF-κB pathway in mice. Meanwhile, Meth disturbed the cecal microbiome and impaired the homeostasis of BAs. Interestingly, FMT from Meth administered mice resulted in serum and hepatic BA accumulation and transferred similar phenotypic changes into the healthy recipient mice. Finally, OCA normalized Meth-induced BA accumulation in both serum and the liver, and effectively protected against Meth-induced hepatic dysfunction and inflammation by suppressing the TLR4/MyD88/NF-κB pathway. This study established the importance of microbial mechanism and its inhibition as a potential therapeutic target to treat Meth-related hepatotoxicity.

2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucopyranoside enhances the hepatotoxicity of emodin in vitro and in vivo.

Herb-induced liver injury results from the interplay between the herb and host with the herbal components serving as the major origin for hepatotoxicity. Although Polygoni Multiflori Radix (PMR) has been frequently reported to induce liver injury, contributions of its major components such as emodin, emodin-8-O-ß-D-glucopyranoside, physcion and 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucopyranoside (TSG) towards its hepatotoxicity have not been clearly identified. Our initial cytotoxicity screenings of the major PMR components using rat hepatocytes identified emodin as the most toxic. Subsequently, the bile acid homeostasis-related mechanisms of emodin and its combination treatment with TSG in PMR-associated liver injury were explored in sandwich-cultured rat hepatocytes (SCRH) and verified in rats. In SCRH, emodin was found to be able to induce total bile acid accumulation in a dose-dependent manner. In both SCRH and rats, the presence of TSG significantly enhanced the hepatotoxicity of emodin via i) increasing its hepatic exposure by inhibiting its glucuronidation mediated metabolism; ii) enhancing its disruption on bile acid homeostasis through amplifying its inhibition on bile acid efflux transporters and its up-regulation on bile acids synthesis enzymes; iii) enhancing its apoptosis. Our study for the first time demonstrated the critical role of the combination treatment with emodin and TSG in PMR-induced liver injury.

Maternal and Fetal Bile Acid Homeostasis Regulated by Sulfated Progesterone Metabolites through FXR Signaling Pathway in a Pregnant Sow Model.

Abnormally elevated circulating bile acids (BA) during pregnancy endanger fetal survival and offspring health; however, the pathology and underlying mechanisms are poorly understood. A total of nineteen pregnant sows were randomly assigned to day 60 of gestation, day 90 of gestation (G60, G90), and the farrowing day (L0), to investigate the intercorrelation of reproductive hormone, including estradiol, progesterone and sulfated progesterone metabolites (PMSs), and BA in the peripheral blood of mother and fetuses during pregnancy. All data were analyzed by Student's t-test or one-way ANOVA of GraphPad Prism and further compared by using the Student-Newman-Keuls test. Correlation analysis was also carried out using the CORR procedure of SAS to study the relationship between PMSs and BA levels in both maternal and fetal serum at G60, G90, and L0. Allopregnanolone sulphate (PM4S) and epiallopregnanolone sulphate (PM5S) were firstly identified in the maternal and fetal peripheral blood of pregnant sows by using newly developed ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) methods. Correlation analysis showed that pregnancy-associated maternal BA homeostasis was correlated with maternal serum PM4S levels, whereas fetal BA homeostasis was correlated with fetal serum PM5S levels. The antagonist activity role of PM5S on farnesoid X receptor (FXR)-mediated BA homeostasis and fibroblast growth factor 19 (FGF19) were confirmed in the PM5S and FXR activator co-treated pig primary hepatocytes model, and the antagonist role of PM4S on FXR-mediated BA homeostasis and FGF19 were also identified in the PM4S-treated pig primary hepatocytes model. Together with the high relative expression of FGF19 in pig hepatocytes, the pregnant sow is a promising animal model to investigate the pathogenesis of cholestasis during pregnancy.

Long-term oral administration of Epimedii Folium induced cholestasis in mice by interfering with bile acid transport.

ETHNOPHARMACOLOGICAL RELEVANCE: Epimedii Folium (EF) is a common traditional Chinese medicine that functions as a tonifying kidney yang to strengthen bones and muscles and dispel wind dampness (limb pain, lethargy, nausea, anorexia, and loose stools). Several studies have reported the potential risk of cholestatic liver damage from EF use; however, there have been few investigations of EF-induced cholestasis, particularly the underlying mechanisms. AIMS OF THE STUDY: The purpose of this study was to evaluate the risk of EF-induced cholestasis in vivo and to explore the mechanisms of action. MATERIALS AND METHODS: ICR mice were orally administered a water extract of EF (WEF) in doses of 6.5 and 19.5 g/kg/day for 14 weeks. Liver-to-body weight ratios, body weight, histopathological examination, and biochemical analyses were performed to assess WEF-induced cholestasis in the mice. Genes associated with bile acid (BA) metabolism and transport, including sodium taurocholate cotransporting polypeptide (NTCP), cytochrome P450 8B1 (CYP8B1), bile-salt export pump (BSEP), multidrug resistance P-glycoproteins 1 (MDR1), and farnesoid X receptor (FXR), were measured at the transcript and protein levels to investigate the potential mechanisms through which cholestasis is aroused by EF. RESULTS: After administration of WEF for 14 weeks, mice in the high-dose WEF group showed poor health with an increased liver-to-body weight ratio as well as higher serum aminotransferase, alkaline phosphatase, direct bilirubin, and total BA levels. Compared with the control group, mRNA expression of NTCP and cholesterol 7a-hydroxylase (CYP7A1) increased, and levels of BSEP, MDR1, multidrug resistance-associated protein 2, and multidrug resistance-associated protein 3 decreased in the WEF-treated group. NTCP, BSEP, MDR1, and CYP8B1 showed similar mRNA and protein expression trends. CONCLUSION: We demonstrated that the long-term oral administration of WEF causes cholestatic liver injury in mice, which is consistent with reported clinical cases. Furthermore, we found that the destruction of BA metabolism and transport is involved in WEF-induced cholestasis. The fine-scale molecular mechanisms of WEF-induced cholestasis and the active compounds of EF need further study.

Da-Chai-Hu-Tang Protects From Acute Intrahepatic Cholestasis by Inhibiting Hepatic Inflammation and Bile Accumulation via Activation of PPARα.

Cholestasis is caused by intrahepatic retention of excessive toxic bile acids and ultimately results in hepatic failure. Da-Chai-Hu-Tang (DCHT) has been used in China to treat liver and gallbladder diseases for over 1800 years. Here, we demonstrated that DCHT treatment prevented acute intrahepatic cholestasis with liver injury in response to α-naphthylisothiocyanate (ANIT) not to bile duct ligation (BDL) induced-extrahepatic cholestasis. ANIT (80 mg/kg) increased serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), direct bilirubin (DBiL), total bilirubin (TBiL), and total bile acids (TBA) which was attenuated by DCHT treatment in a dose-dependent manner. DCHT treatment at high dose of 1.875 g/kg restored bile acid homeostasis, as evidenced by the recovery of the transcription of genes implicated in bile acid biosynthesis, uptake and efflux. DCHT treatment (1.875 g/kg) reversed ANIT-evoked disordered glutathione homeostasis (as determined by GSH/GSSG ratio) and increased in the mRNA levels for Il6, Il1b and Tnfa associated with liver inflammation. Using network pharmacology-based approaches, we identified 22 putative targets involved in DCHT treatment for intrahepatic cholestasis not extrahepatic cholestasis. In addition, as evidenced by dual-luciferase reporter assays, compounds from DCHT with high affinity of PPARα increased luciferase levels from a PPARα-driven reporter. PPARα agonist fenofibrate was able to mimic the cytoprotective effect of DCHT on intrahepatic cholestasis, which was abolished by the PPARα antagonist GW6471. KEGG enrichment and western blot analyses showed that signaling axes of JNK/IL-6/NF-κB/STAT3 related to PPARα might be the principal pathway DCHT affects intrahepatic cholestasis. Taken together, the present study provides compelling evidence that DCHT is a promising formula against acute intrahepatic cholestasis with hepatotoxicity which works via PPARα activation.