Phosphatidylcholine attenuates aggregation of nonsteroidal anti-inflammatory drugs with bile acid.

Prolonged usage of nonsteroidal anti-inflammatory drugs (NSAIDs) causes gastrointestinal injury. Bile acids and phospholipids have been shown to exasperate and attenuate NSAIDs' toxicity, respectively. However, the molecular mechanisms underlying these effects remain undetermined. We have investigated the molecular interactions in various mixtures of indomethacin (Indo), a commonly used NSAID, and cholic acid (CA), a bile acid, in the presence and absence of palmitoyloleylphosphatidylcholine (POPC) lipids. We found that CA and Indo spontaneously form mixed micelles, with the hydrophobic face of CA and hydrophobic region of Indo forming the core. Increasing the Indo concentration resulted in more stable and larger aggregates that contain a progressively larger number of Indo molecules. More dynamic aggregates with a maximum size of 15 were obtained when the relative concentration of CA was higher. The mixture of CA, Indo, and POPC also led to ternary mixed micelles in which CA and Indo distribute almost uniformly on the surface such that intra-CA, intra-Indo, and CA/Indo interactions are minimized. A number of previous reports have shown that Indo perforates the cell membrane in the presence of bile acids (e.g., Petruzzelli et al., (2006) Dig. Dis. Sci., 51, 766-774). We propose that this may be related to the stable, highly charged, large CA/Indo binary micelles observed in our simulations. Similarly, the diminished ability of the CA/Indo mixture to aggregate in the presence of POPC may partly explain the lower toxicity of PC-conjugated NSAIDs.

On the mechanism of accumulation of cholestanol in the brain of mice with a disruption of sterol 27-hydroxylase.

The rare disease cerebrotendinous xanthomatosis (CTX) is due to a lack of sterol 27-hydroxylase (CYP27A1) and is characterized by cholestanol-containing xanthomas in brain and tendons. Mice with the same defect do not develop xanthomas. The driving force in the development of the xanthomas is likely to be conversion of a bile acid precursor into cholestanol. The mechanism behind the xanthomas in the brain has not been clarified. We demonstrate here that female cyp27a1(-/-) mice have an increase of cholestanol of about 2.5- fold in plasma, 6-fold in tendons, and 12-fold in brain. Treatment of cyp27a1(-/-) mice with 0.05% cholic acid normalized the cholestanol levels in tendons and plasma and reduced the content in the brain. The above changes occurred in parallel with changes in plasma levels of 7alpha-hydroxy-4-cholesten-3-one, a precursor both to bile acids and cholestanol. Injection of a cyp27a1(-/-) mouse with (2)H(7)-labeled 7alpha-hydroxy-4-cholesten-3-one resulted in a significant incorporation of (2)H(7)-cholestanol in the brain. The results are consistent with a concentration-dependent flux of 7alpha-hydroxy-4-cholesten-3-one across the blood-brain barrier in cyp27a1(-/-) mice and subsequent formation of cholestanol. It is suggested that the same mechanism is responsible for accumulation of cholestanol in the brain of patients with CTX.

Bile acids inhibit duodenal secretin expression via orphan nuclear receptor small heterodimer partner (SHP).

Small heterodimer partner (SHP) is an orphan nuclear receptor in which gene expression can be upregulated by bile acids. It regulates its target genes by repressing the transcriptional activities of other nuclear receptors including NeuroD, which has been shown to regulate secretin gene expression. Here, we evaluated the regulation on duodenal secretin gene expression by SHP and selected bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA). In vitro treatment of CDCA or fexaramine elevated the SHP transcript level and occupancy on secretin promoter. The increase in the SHP level, induced by bile acid treatment or overexpression, reduced secretin gene expression, whereas this gene inhibitory effect was reversed by silencing of endogenous SHP. In in vivo studies, double-immunofluorescence staining demonstrated the coexpression of secretin and SHP in mouse duodenum. Feeding mice with 1% CA-enriched rodent chow resulted in upregulation of SHP and a concomitant decrease in secretin transcript and protein levels in duodenum compared with the control group fed with normal chow. A diet enriched with 5% cholestyramine led to a decrease in SHP level and a corresponding increase in secretin expression. Overall, this study showed that bile acids via SHP inhibit duodenal secretin gene expression. Because secretin is a key hormone that stimulates bile flow in cholangiocytes, this pathway thus provides a novel means to modulate secretin-stimulated choleresis in response to intraduodenal bile acids.

Cellular uptake mechanism of molecular umbrella.

Molecular umbrella provided a promising avenue for the design of the intracellular delivery of hydrophilic therapeutic agents. However, the limited understanding of its cellular uptake would be a roadblock to its effective application. Herein, we investigate the ability and mechanism of cellular entry of a fluorescently labeled diwalled molecular umbrella, which was synthesized from cholic acid, spermine, and 5-carboxyfluorescein, into Hela cells, with the extent of uptake analyzed by confocal fluorescence microscopy and flow cytometry. It is found that the as-synthesized diwalled molecular umbrella can greatly facilitate cellular uptake of hydrophilic agent, 5-carboxyfluorescein. In vitro experiments with diffuse marker, endocytic marker, and inhibitors suggested that several distinct uptake pathways (e.g., passive diffuse, clathrin-mediated endocytosis, and caveolae/lipid-raft-dependent endocytosis) are involved in the internalization of diwalled molecular umbrella. These results, together with its low toxicity and good biocompatibility, thus demonstrate the suitability of molecular umbrella for application as vectors in drug delivery systems.

Cholic Acid Attenuates ER Stress-Induced Cell Death in Coxsackievirus-B3 Infection.

Coxsackievirus Type B3 (CVB3) is an enterovirus that belongs to the Picornaviridae and causes various diseases such as myocarditis and hand-foot-mouth disease. However, an effective antiviral drug is still not developed. In this study, we looked for potential inhibitors of CVB3 replication by examining the survival of CVB3-infected HeLa cells. We detected an antiviral effect by cholic acid and identified it as a candidate inhibitor of CVB3 replication. Cholic acid circulates in the liver and intestines, and it helps the digestion and absorption of lipids in the small intestine. HeLa cells were cultured in 12-well plates and treated with cholic acid (1 and 10 µg/ml) and 106 PFU/ml of CVB3. After 16 h post-infection, the cells were lysed and subjected to western blot analysis and RT-PCR. The production of the viral capsid protein VP1 was dramatically decreased, and translation initiation factor eIF4G1 cleavage was significantly inhibited by treatment with 10 µg/ml cholic acid. Moreover, cholic acid inhibited ERK signaling in CVB3-infected HeLa cells. RT-PCR showed that the amounts of the CVB3 RNA genome and mRNA for the ER stress-related transcription factor ATF4 were significantly reduced. These results showed that cholic acid strongly reduced ER stress and CVB3 proliferation. This compound can be developed as a safe natural therapeutic agent for enterovirus infections.

Potential Functional Byproducts from Guava Purée Processing.

The valorization of guava waste requires compositional and functional studies. We tested three byproducts of guava purée processing, namely refiner, siever, and decanter. We analyzed the chemical composition and quantified the prebiotic activity score and selected carbohydrates; we also determined the water holding (WHC), oil holding (OHC), cation exchange capacities, bile acid binding, and glucose dialysis retardation (GDR) of the solid fraction and the antioxidative and α-amylase inhibitory capacities (AIC) of the ethanolic extract. Refiner contained 7.7% lipid, 7.08% protein and a relatively high phytate content; it had a high prebiotic activity score and possessed the highest binding capacity with deoxycholic acid. Siever contained high levels of low molecular weight carbohydrates and total tannin but relatively low crude fiber and cellulose contents. It had the highest binding with chenodeoxycholic acid (74.8%), and exhibited the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity. Decanter was rich in cellulose and had a high prebiotic activity score. The WHC and OHC values of decanter were within a narrow range and also exhibited the highest binding with cholic acid (86.6%), and the highest values of GDR and AIC. The refiner waste could be included in animal feed but requires further processing to reduce the high phytate levels. All three guava byproducts had the potential to be a source of antioxidant dietary fiber (DF), a finding that warrants further in vivo study. PRACTICAL APPLICATION: To differing extents, the guava byproducts exhibited useful physicochemical binding properties and so possessed the potential for health-promoting activity. These byproducts could also be upgraded to other marketable products so the manufacturers of processed guava might be able to develop their businesses sustainably by making better use of them.

Structure, evolution, and liver-specific expression of sterol 12alpha-hydroxylase P450 (CYP8B).

The rat CYP8B cDNA encoding sterol 12alpha-hydroxylase was cloned and sequenced. The amino acid sequence of the heme-binding region of CYP8B was close to those of CYP7A (cholesterol 7alpha-hydroxylase) and CYP7B (oxysterol 7alpha-hydroxylase). Molecular phylogenetic analysis suggests that CYP8B and the CYP7 family derive from a common ancestor. The P450s of the CYP7 and CYP8 families, except for CYP8A (prostacyclin synthase), catalyze the oxygenation of sterols from an alpha surface in the middle of the steroid skeleton. These facts suggest that CYP8B is a P450 closely linked to those of the CYP7 family. CYP8B was expressed specifically in liver. Hepatic CYP8B mRNA level and the 12alpha-hydroxylase activity were altered by cholestyramine feeding, starvation, streptozotocin-induced diabetes mellitus, and administration of clofibrate, dexamethasone or thyroxin, indicating the pretranslational regulation of CYP8B expression. The enhanced CYP8B mRNA expression in streptozotocin-induced diabetic rats was significantly decreased by insulin within 3 h of its administration. These facts demonstrate a regulatory role of insulin in CYP8B expression as a suppressor.

Effect of cholesterol, cholic acid and cholestyramine administration on the intestinal mRNA expressions related to cholesterol and bile acid metabolism in the rat.

BACKGROUND AND AIM: To evaluate the importance of cholesterol and bile acid concentrations in the intestinal lumen to cholesterol homeostasis, we investigated the effect of cholesterol-, bile salt- or cholestyramine-administration on the regulation of intestinal mRNA related to cholesterol and bile acid metabolism. METHODS: Male Wistar rats fed on standard laboratory chow (AIN-93) were allocated into four experimental groups: (i) control group; (ii) cholesterol group (CH), which was fed cholesterol in diet (2% [w/w]) for 14 days; (iii) cholic acid (CA) group, which was fed CA in diet (1% [w/w]) for 14 days; (iv) cholestyramine (CT) group, which was fed CT in diet (5% [w/w]) for 14 days. Blood, liver and small intestine were obtained after 14 days. Serum lipids and bile acids were measured by colorimetric assays, and hepatic and intestinal mRNA relating to lipid and bile acid metabolism was studied by reverse transcription-polymerase chain reaction. RESULTS: Intestinal ABCG8, liver X receptor alpha, small heterodimer partner (SHP) and sterol regulatory element binding protein 1c (SREBP-1c) mRNA expressions were markedly increased in the CH group. Intestinal multidrug resistance associated protein (MRP) 2 and MRP3 mRNA expressions were markedly increased in the CA group. In the CT group intestinal MRP2, ABCG5, ABCG8, SHP and SREBP-1c mRNA expressions were markedly decreased. CONCLUSION: The bile acid availability in the intestinal lumen alters the expression of various intestinal mRNA relating to not only bile acid metabolism, but also lipid metabolism. Though the mechanism of the intestinal SHP elevation is unclear, cholesterol feeding may affect the intestinal bile acid metabolism via intestinal SHP expression.

Antilithiasic effect of beta-cyclodextrin in LPN hamster: comparison with cholestyramine.

Beta-Cyclodextrin (BCD), a cyclic oligosaccharide that binds cholesterol and bile acids in vitro, has been previously shown to be an effective plasma cholesterol lowering agent in hamsters and domestic pigs. This study examined the effects of BCD as compared with cholestyramine on cholesterol and bile acid metabolism in the LPN hamster model model for cholesterol gallstones. The incidence of cholesterol gallstones was 65% in LPN hamsters fed the lithogenic diet, but decreased linearly with increasing amounts of BCD in the diet to be nil at a dose of 10% BCD. In gallbladder bile, cholesterol, phospholipid and chenodeoxycholate concentrations, hydrophobic and lithogenic indices were all significantly decreased by 10% BCD. Increases in bile acid synthesis (+110%), sterol 27-hydroxylase activity (+106%), and biliary cholate secretion (+140%) were also observed, whereas the biliary secretion of chenodeoxycholate decreased (-43%). The fecal output of chenodeoxycholate and cholate (plus derivatives) was increased by +147 and +64%, respectively, suggesting that BCD reduced the chenodeoxycholate intestinal absorption preferentially. Dietary cholestyramine decreased biliary bile acid concentration and secretion, but dramatically increased the fecal excretion of chenodeoxycholate and cholate plus their derivatives (+328 and +1940%, respectively). In contrast to BCD, the resin increased the lithogenic index in bile, induced black gallstones in 34% of hamsters, and stimulated markedly the activities of HMG-CoA reductase (+670%), sterol 27-hydroxylase (+310%), and cholesterol 7alpha-hydroxylase (+390%). Thus, beta-cyclodextrin (BCD) prevented cholesterol gallstone formation by decreasing specifically the reabsorption of chenodeoxycholate, stimulating its biosynthesis and favoring its fecal elimination. BCD had a milder effect on lipid metabolism than cholestyramine and does not predispose animals to black gallstones as cholestyramine does in this animal model.