UHPLC-Orbitrap-Fusion-TMS-Based Metabolomics Study of Phenylpropionamides in the Seed of Cannabis sativa L. against Alzheimer's Disease.

Phenylpropionamides in the seed of Cannabis sativa L. (PHS) have a protective effect on neuroinflammation and antioxidant activity. In this study, the UHPLC-Orbitrap-fusion-TMS-based metabolomics approach was used to analyze the serum samples and identify potential biomarkers in Streptozotocin (STZ) induced Alzheimer's disease (AD) rats. The results revealed that primary bile acid biosynthesis and taurine and hypotaurine metabolism were significantly correlated with STZ-induced AD rats. In addition, the key enzymes in these two pathways were verified at the protein level. The levels of cysteine dioxygenase type I (CDO1), cysteine sulfinic acid decarboxylase (CSAD), cysteamine (2-aminoethanethiol) dioxygenase (ADO), 7α-hydroxylase (CYP7A1), and sterol 12α-hydroxylase (CYP8B1) were the key enzymes affecting the two pathways in AD rats compared with the control group (CON). Furthermore, after a high-dose group of phenylpropionamides in the seed of Cannabis sativa L. (PHS-H) was administrated, the levels of CDO1, CSAD, CYP7A1, and CYP8B1 were all callback. These findings demonstrate for the first time that the anti-AD effect of PHS is associated with the regulation of primary bile acid biosynthesis and taurine and hypotaurine metabolism in STZ-induced AD rats.

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.

Chemical profile of Swertia mussotii Franch and its potential targets against liver fibrosis revealed by cross-platform metabolomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Swertia mussotii Franch (SMF) is a well-known Tibetan medicine for the treatment of liver disease in China. However, the chemical profile and molecular mechanism of SMF against hepatic fibrosis are not yet well explored. AIM OF THE STUDY: This work aimed to elucidate the chemical profile of SMF and investigate the action mechanisms of SMF against carbon tetrachloride (CCl4)-induced hepatic fibrosis. MATERIALS AND METHODS: Ultra performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOFMS) and UNIFI platform was firstly employed to reveal the chemical profile of SMF. Cross-platform serum metabolomics based on gas chromatography/liquid chromatography-mass spectrometry were performed to characterize the metabolic fluctuations associated with CCl4-induced hepatic fibrosis in mice and elucidate the underlying mechanisms of SMF. Western blotting was further applied to validate the key metabolic pathways. RESULTS: A total of 31 compounds were identified or tentatively characterized from SMF. Twenty-seven differential metabolites were identified related with CCl4-induced liver fibrosis, and SMF could significantly reverse the abnormalities of seventeen metabolites. The SMF-reversed metabolites were involved in arachidonic acid metabolism, glycine, serine and threonine metabolism, tyrosine metabolism, arginine and proline metabolism, primary bile acid biosynthesis, glycerophospholipid metabolism and TCA cycle. The results of western blotting analysis showed that SMF could alleviate liver fibrosis by increasing the levels of CYP7A1, CYP27A1 and CYP8B1 and decreasing the level of LPCAT1 to regulate the metabolic disorders of primary bile acid biosynthesis and glycerophospholipid. CONCLUSION: It could be concluded that primary bile acid biosynthesis and glycerophospholipid metabolism were the two important target pathways for SMF-against liver fibrosis, which provided the theoretical foundation for its clinical use.

Hepatoprotection of auraptene from the peels of citrus fruits against 17α-ethinylestradiol-induced cholestasis in mice by activating farnesoid X receptor.

Cholestatic liver injury induced by estrogen is a common clinical syndrome in women undergoing oral administration of contraceptives, pregnancy or hormone replacement therapy. Estrogen-induced cholestasis is associated with the accumulation of endogenous bile acids, which play critical roles in the disease progression and symptoms. In the present study, we described the protective effect of auraptene, a simple coumarin present in the peels of citrus fruits, such as grapefruit, against 17α-ethinylestradiol (EE)-induced cholestasis, and further elucidated the involvement of farnesoid X receptor (FXR) in the hepatoprotective effect. Auraptene treatment alleviated EE-induced cholestasis through increasing the bile flow and biliary bile acid output. The mechanism underlying the alleviated cholestasis by auraptene was associated with the increased efflux and inhibited hepatic uptake of bile acids via an induction of efflux transporters (Bsep and Mrp2) and downregulation of Ntcp. Furthermore, auraptene reduced the bile acid synthesis through repressing Cyp7a1 and Cyp8b1, and increased the bile acid metabolism through an induction in the gene expression of Sult2a1. The mentioned genes involved in the bile acid homeostasis were modulated by FXR. We further demonstrated that the changes in transporters and enzymes, as well as ameliorated liver histology by auraptene, were abrogated by the FXR antagonist guggulsterone. In conclusion, auraptene alleviated EE-induced cholestasis due to FXR-mediated gene regulation.

Beneficial Effects of Vitamin D Treatment in an Obese Mouse Model of Non-Alcoholic Steatohepatitis.

Serum vitamin D levels negatively correlate with obesity and associated disorders such as non-alcoholic steatohepatitis (NASH). However, the mechanisms linking low vitamin D (VD) status to disease progression are not completely understood. In this study, we analyzed the effect of VD treatment on NASH in mice. C57BL6/J mice were fed a high-fat/high-sugar diet (HFSD) containing low amounts of VD for 16 weeks to induce obesity, NASH and liver fibrosis. The effects of preventive and interventional VD treatment were studied on the level of liver histology and hepatic/intestinal gene expression. Interestingly, preventive and to a lesser extent also interventional VD treatment resulted in improvements of liver histology. This included a significant decrease of steatosis, a trend towards lower non-alcoholic fatty liver disease (NAFLD) activity score and a slight non-significant decrease of fibrosis in the preventive treatment group. In line with these changes, preventive VD treatment reduced the hepatic expression of lipogenic, inflammatory and pro-fibrotic genes. Notably, these beneficial effects occurred in conjunction with a reduction of intestinal inflammation. Together, our observations suggest that timely initiation of VD supplementation (preventive vs. interventional) is a critical determinant of treatment outcome in NASH. In the applied animal model, the improvements of liver histology occurred in conjunction with reduced inflammation in the gut, suggesting a potential relevance of vitamin D as a therapeutic agent acting on the gut⁻liver axis.

Pectin penta-oligogalacturonide reduces cholesterol accumulation by promoting bile acid biosynthesis and excretion in high-cholesterol-fed mice.

Haw pectin penta-oligogalacturonide (HPPS) has important role in improving cholesterol metabolism and promoting the conversion of cholesterol to bile acids (BA) in mice fed high-cholesterol diet (HCD). However, the mechanism is not clear. This study aims to investigate the effects of HPPS on cholesterol accumulation and the regulation of hepatic BA synthesis and transport in HCD-fed mice. Results showed that HPPS significantly decreased plasma and hepatic TC levels but increased plasma high-density lipoprotein cholesterol (HDL-C) and apolipoprotein A-I (apoA-I) levels, compared to HCD. BA analysis showed that HPPS markedly decreased hepatic and small intestine BA levels but increased the gallbladder BA levels, and finally decreased the total BA pool size, compared to HCD. Studies of molecular mechanism revealed that HPPS promoted hepatic ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1), and scavenger receptor BI (SR-BI) expression but did not affect ATB binding cassette transporter G5/G8 (ABCG5/8) expression. HPPS inactivated hepatic farnesoid X receptor (FXR) and target genes expression, which resulted in significant increase of cholesterol 7α-hydroxylase 1 (CYP7A1) and sterol 12α-hydroxylase (CYP8B1) expression, with up-regulations of 204.2% and 33.5% for mRNA levels, respectively, compared with HCD. In addition, HPPS markedly enhanced bile salt export pump (BSEP) expression but didn't affect the sodium/taurocholate co-transporting polypeptide (NTCP) expression. In conclusion, the study revealed that HPPS reduced cholesterol accumulation by promoting BA synthesis in the liver and excretion in the feces, and might promote macrophage-to-liver reverse cholesterol transport (RCT) but did not liver-to-fecal RCT.

Lingonberries reduce atherosclerosis in Apoe(-/-) mice in association with altered gut microbiota composition and improved lipid profile.

SCOPE: To investigate the efficacy of lingonberries in prevention of atherosclerosis, using atherosclerosis-prone Apoe(-/-) mice and to clarify whether effects were associated with changes in the gut microbiota, gut metabolites, and lipid metabolism. METHODS AND RESULTS: Male Apoe(-/-) mice were fed either low-fat diet, high-fat diet, or high-fat diet with 44% lingonberries for 8 weeks. Blood lipid profiles, hepatic gene expression, atherosclerotic plaques in the aortic root region of the heart, bacterial 16S rRNA gene profiles, and cecal short-chain fatty acids (SCFAs) were analyzed. Triglyceride levels and amount of atherosclerotic plaques decreased in the group fed lingonberries in comparison to the high-fat group. Hepatic expression of the bile acid synthesis gene Cyp7a1 was significantly upregulated in the lingonberry group. Lingonberries increased the cecal relative abundance of bacterial genera Bacteroides, Parabacteroides and Clostridium. The cecal levels of total SCFAs were significantly lower in the lingonberry group, while the cecal proportion of propionic acid was higher in mice fed lingonberries. CONCLUSION: Intake of lingonberries resulted in decreased triglyceridemia and reduced atherosclerosis. The altered gut microbiota composition and SCFA profile was associated with increased hepatic bile acid gene expression in mice fed lingonberries.

Synergic hypocholesterolaemic effect of n-3 PUFA and oestrogen by modulation of hepatic cholesterol metabolism in female rats.

n-3 PUFA such as EPA and DHA as well as oestrogen have been reported to decrease blood levels of cholesterol, but their underlying mechanism is unclear. The purpose of this study was to determine the effects of the combination of n-3 PUFA supplementation and oestrogen injection on hepatic cholesterol metabolism. Rats were fed a modified AIN-93G diet with 0, 1 or 2 % n-3 PUFA (EPA+DHA) relative to the total energy intake for 12 weeks. Rats were surgically ovariectomised at week 8, and, after 1-week recovery, rats were injected with 17ß-oestradiol-3-benzoate (E2) or maize oil for the last 3 weeks. Supplementation with n-3 PUFA and E2 injection significantly increased the ratio of the hepatic expression of phosphorylated AMP activated protein kinase (p-AMPK):AMP activated protein kinase (AMPK) and decreased sterol regulatory element-binding protein-2, 3-hydroxy-3-methylglutaryl coenzyme A reductase and proprotein convertase subtilisin/kexin type 9. Supplementation with n-3 PUFA increased hepatic expression of cholesterol 7α-hydroxylase (CYP7A1), sterol 12α-hydroxylase (CYP8B1) and sterol 27-hydroxylase (CYP27A1); however, E2 injection decreased CYP7A1 and CYP8B1 but not CYP27A1. Additionally, E2 injection increased hepatic expression of oestrogen receptor-α and ß. In conclusion, n-3 PUFA supplementation and E2 injection had synergic hypocholesterolaemic effects by down-regulating hepatic cholesterol synthesis (n-3 PUFA and oestrogen) and up-regulating bile acid synthesis (n-3 PUFA) in ovariectomised rats.

Cellular Accumulation and Toxic Effects of Bile Acids in Cyclosporine A-Treated HepaRG Hepatocytes.

Alteration of bile acid (BA) profiles and secretion by cholestatic drugs represents a major clinical issue. Species differences exist in BA composition, synthesis, and regulation; however presently, there is no in vitro reproducible cell model to perform studies on BAs in humans. We have evaluated the capacity of the human HepaRG cell line to synthesize, conjugate, and secrete BAs, and analyzed changes in BA content and profile after cyclosporine A (CsA) treatment. Our data show that HepaRG cells produced normal BAs at daily levels comparable, though in different proportions, to those measured in primary human hepatocytes. A 4-h treatment with CsA led to BA accumulation and profile changes associated with occurrence of cholestatic features, while after 24 h BAs were decreased in cell layers and increased in media. The latter effects resulted from reduced function of BA uptake transporter (Na(+)-taurocholate cotransporting polypeptide), reduced expression of BA metabolizing enzymes, including cytochrome P4507A1, cytochrome P4508B1, and cytochrome P45027A1, and induction of alternative basolateral transporters. Noteworthy, HepaRG cells incubated in a 2% serum-supplemented medium showed dose-dependent accumulation of the cytotoxic BA lithocholic acid in a nonsulfoconjugated form associated with early inhibition of the canalicular transporter MRP2 and sulfotransferase 2A1. In summary, our data bring the first demonstration that an in vitro human liver cell line is able to produce and secrete conjugated BAs, and to accumulate endogenous BAs transiently, concomitantly to occurrence of various other cholestatic features following CsA treatment. Retention of the hydrophobic lithocholic acid supports its toxic role in drug-induced cholestasis. Overall, our results argue on the suitability of HepaRG cells for investigating mechanisms involved in the development of the disease.

Comparative regulation of major enzymes in the bile acid biosynthesis pathway by cholesterol, cholate and taurine in mice and rats.

These enzymes play important roles in the biosynthesis of bile acids. They are cholesterol 7alpha-hydroxylase (CYP7A1), the rate limiting enzyme in the classic pathway, sterol 12alpha-hydroxylase (CYP8B1), the key enzyme for synthesis of cholic acid (CA), and sterol 27-hydroxylase (CYP27), the initial enzyme in the alternative pathway. In the present study, the susceptibility of these three enzymes to dietary cholesterol and cholate, and the cholesterol lowering effect of taurine were determined in male C57BL/6 mice and Wistar rats. Both mice and rats were divided into 6 groups: control group (N), high cholesterol diet group (C), high cholesterol and cholate diet group (CB), and their 1% taurine-supplemented groups (NT, CT, CBT, respectively). After animals were fed with the respective diets for one week, the mRNA levels of CYP7A1 increased in the C-group compared with those of the N-group, and decreased in the CB-group compared with those of the C-group in both mice and rats. But the extent of decrease is different between the two species. CYP8B1 was also markedly repressed by cholate in mice, but not in rats. These results are consistent with the changes in serum and liver cholesterol concentrations. Taurine significantly increased CYP7A1 mRNA levels in the CBT-group compared with the CB-group in both animal models, with a subsequent decrease in serum and liver cholesterol levels and increase in fecal bile acid excretion. Up-regulated CYP8B1 was also observed after taurine supplementation in the CBT-group in mice. No increase in CYP7A1 was produced by taurine in the CT-group compared with that of the C-group in mice, although the changes of serum and liver cholesterol and fecal bile acids indicated taurine showed an efficient cholesterol lowering effect. In addition, CYP27 was induced in both C- and CB-groups of rats but not of mice, and no changes were produced by taurine. The overall results suggest that there are differences between mice and rats in susceptibility of the three enzymes to dietary cholesterol and cholate, and taurine induced CYP7A1 to produce its cholesterol-lowering effect only in the presence of cholate in the cholesterol diet.