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Hydrophilic bile acids protect human blood-brain barrier endothelial cells from disruption by unconjugated bilirubin: an in vitro study.

ABSTRACT: Ursodeoxycholic acid and its main conjugate glycoursodeoxycholic acid are bile acids with neuroprotective properties. Our previous studies demonstrated their anti-apoptotic, anti-inflammatory, and antioxidant properties in neural cells exposed to elevated levels of unconjugated bilirubin (UCB) as in severe jaundice. In a simplified model of the blood-brain barrier, formed by confluent monolayers of a cell line of human brain microvascular endothelial cells, UCB has shown to induce caspase-3 activation and cell death, as well as interleukin-6 release and a loss of blood-brain barrier integrity. Here, we tested the preventive and restorative effects of these bile acids regarding the disruption of blood-brain barrier properties by UCB in in vitro conditions mimicking severe neonatal hyperbilirubinemia and using the same experimental blood-brain barrier model. Both bile acids reduced the apoptotic cell death induced by UCB, but only glycoursodeoxycholic acid significantly counteracted caspase-3 activation. Bile acids also prevented the upregulation of interleukin-6 mRNA, whereas only ursodeoxycholic acid abrogated cytokine release. Regarding barrier integrity, only ursodeoxycholic acid abrogated UCB-induced barrier permeability. Better protective effects were obtained by bile acid pre-treatment, but a strong efficacy was still observed by their addition after UCB treatment. Finally, both bile acids showed ability to cross confluent monolayers of human brain microvascular endothelial cells in a time-dependent manner. Collectively, data disclose a therapeutic time-window for preventive and restorative effects of ursodeoxycholic acid and glycoursodeoxycholic acid against UCB-induced blood-brain barrier disruption and damage to human brain microvascular endothelial cells.

SUBMITTER: Palmela I

PROVIDER: S-EPMC4358072 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Hydrophilic bile acids protect human blood-brain barrier endothelial cells from disruption by unconjugated bilirubin: an in vitro study.

Palmela Inês I Correia Leonor L Silva Rui F M RF Sasaki Hiroyuki H Kim Kwang S KS Brites Dora D Brito Maria A MA

Frontiers in neuroscience 20150313

Ursodeoxycholic acid and its main conjugate glycoursodeoxycholic acid are bile acids with neuroprotective properties. Our previous studies demonstrated their anti-apoptotic, anti-inflammatory, and antioxidant properties in neural cells exposed to elevated levels of unconjugated bilirubin (UCB) as in severe jaundice. In a simplified model of the blood-brain barrier, formed by confluent monolayers of a cell line of human brain microvascular endothelial cells, UCB has shown to induce caspase-3 acti ...[more]

PMID: 25821432

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Project description:Host-gut microbiota metabolic interactions are closely associated with health and disease. A manifestation of such co-metabolism is the vast structural diversity of bile acids (BAs) involving both oxidative stereochemistry and conjugation. Herein, we describe the development and validation of a LC-MS-based method for the analysis of human C24 BA metabolome in serum and urine. The method has high throughput covering the discrimination of oxidative stereochemistry of unconjugated species in a 15-min analytical cycle. The validated quantitative performance provided an indirect way to ascertain the conjugation patterns of BAs via enzyme-digestion protocols that incorporated the enzymes, sulfatase, β-glucuronidase, and choloylglycine hydrolase. Application of the method has led to the detection of at least 70 unconjugated BAs including 27 known species and 43 newly found species in the post-prandial serum and urine samples from 7 nonalcoholic steatohepatitis patients and 13 healthy volunteers. Newly identified unconjugated BAs included 3α, 12β-dihydroxy-5β-cholan-24-oic acid, 12α-hydroxy-3-oxo-5β-cholan-24-oic acid, and 3α, 7α, 12β-trihydroxy-5β-cholan-24-oic acid. High-definition negative fragment spectra of the other major unknown species were acquired to facilitate future identification endeavors. An extensive conjugation pattern is the major reason for the "invisibility" of the newly found BAs to other common analytical methods. Metabolomic analysis of the total unconjugated BA profile in combination with analysis of their conjugation patterns and urinary excretion tendencies have provided substantial insights into the interconnected roles of host and gut microbiota in maintaining BA homeostasis. It was proposed that the urinary total BA profile may serve as an ideal footprint for the functional status of the host-gut microbial BA co-metabolism. In summary, this work provided a powerful tool for human C24 BA metabolome analysis that bridges the gap between GC-MS techniques in the past age and LC-MS techniques currently prevailing in biomedical researches. Further applications of the present method in clinical, translational research, and other biomedical explorations will continue to boost the construction of a host-gut microbial co-metabolism network of BAs and thus facilitate the decryption of BA-mediated host-gut microbiota crosstalk in health and diseases. Graphical abstract ᅟ.

Project description:Background: The pathophysiology of bilirubin neurotoxicity in course of hypoxic-ischemic encephalopathy (HIE) in term and preterm infants is still poorly understood. We hypothesized that oxidative stress may be a common mechanism that link hyperbilirubinemia and HIE. Objectives: The objective of the present study was to evaluate whether unconjugated bilirubin (UCB) may enhance the HI brain injury by increasing oxidative stress and to test pioglitazone and allopurinol as new antioxidant therapeutic drugs in vitro. Methods: The effects of UCB were tested on organotypic hippocampal slices subjected to 30 min oxygen-glucose deprivation (OGD), used as in vitro model of HIE. The experiments were performed on mature (14 days in culture) and immature (7 days in culture) slices, to mimic the brains of term and preterm infants, respectively. Mature and immature slices were exposed to UCB, human serum albumin (HSA), pioglitazone, and/or allopurinol for 24 h, immediately after 30 min OGD. Neuronal injury was assessed using propidium iodide (PI) fluorescence. ROS formation was quantified by using the 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA) method. Results: In mature slices, we found that the neurotoxicity, as well as oxidative stress, induced by OGD were enhanced by UCB. HSA significantly prevented UCB-increased neurotoxicity, but had a slight reduction on ROS production. Allopurinol, but not pioglitazone, significantly reduced UCB-increased neurotoxicity induced by OGD. In immature slices exposed to OGD, no increase of neuronal death was observed, whereas oxidative stress was detected after UCB exposure. HSA, pioglitazone and allopurinol have no protective effects on both OGD-induced neuronal death and on UCB-induced oxidative stress. For this reason, UCB, pioglitazone and allopurinol was also tested on ischemic preconditioning protocol. We found that UCB abolished the neuroprotection induced by preconditioning and increased oxidative stress. These effects were restored by allopurinol but not pioglitazone. Conclusions: UCB characterized a different path of neuronal damage and oxidative stress in mature and immature hippocampal slice model of HIE. Management of hyperbilirubinemia in a complex pathological condition, such as HIE and hyperbilirubinemia, should be very careful. Allopurinol could deserve attention as a novel pharmacological intervention for hyperbilirubinemia and HIE.

Dataset Information

Hydrophilic bile acids protect human blood-brain barrier endothelial cells from disruption by unconjugated bilirubin: an in vitro study.

Publications

Hydrophilic bile acids protect human blood-brain barrier endothelial cells from disruption by unconjugated bilirubin: an in vitro study.

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