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.

Keywords: blood-brain barrier; glycoursodeoxycholic acid; human brain microvascular endothelial cells; interleukin-6; unconjugated bilirubin; ursodeoxycholic acid.

Figure 1

Ursodeoxycholic acid (UDCA) and glycoursodeoxycholic acid (GUDCA) protect human brain microvascular endothelial cells (HBMEC) from unconjugated bilirubin (UCB)-induced apoptosis. A cell line of HBMEC was incubated without (control) or with 100 μM UCB, in the presence of 100 μM human serum albumin, for 48 h. The bile acids (50 μM) were added prior to (1 h) or after (4 or 8 h) UCB addition. Quantification of apoptosis is shown as percentage of apoptotic nuclei per total number of cells and results are expressed as mean ± SEM from at least three independent experiments. ^***P < 0.001 vs. control; ^§P < 0.05, ^§§P < 0.01, and ^§§§P < 0.001 vs. UCB alone.

Figure 2

Glycoursodeoxycholic acid (GUDCA), but not ursodeoxycholic acid (UDCA), has a protective effect on unconjugated bilirubin (UCB)-induced caspase-3 activation in human brain microvascular endothelial cells (HBMEC). A cell line of HBMEC was incubated without (control) or with 100 μM UCB, in the presence of 100 μM human serum albumin, for 4 h. The bile acids (50 μM) were added 1 h prior to UCB incubation. Quantification of caspase-3 activity is shown as fold change from control values and results are expressed as mean ± SEM from at least three independent experiments. ^***P < 0.001 vs. control; ^§P < 0.05 vs. UCB alone.

Figure 3

Ursodeoxycholic acid (UDCA) and glycoursodeoxycholic acid (GUDCA) have protective effects on unconjugated bilirubin (UCB)-induced ultrastructural changes in human brain microvascular endothelial cells (HBMEC). A cell line of HBMEC was incubated without (control) or with 100 μM UCB, in the presence of 100 μM human serum albumin, for 48 h. The bile acids (50 μM) were added 1 h prior to the addition of UCB. Representative ultrastructure observations by transmission electron microscopy are shown. In UCB-treated cells note the decrease in mitochondrial cristae (arrows) and ribosomes (arrowhead) and the appearance of detaching cellular fragments (asterisk), which are abrogated by UDCA and GUDCA.

Figure 4

Decrease in unconjugated bilirubin (UCB)-induced interlkeukin (IL)-6 mRNA expression and cytokine release in human brain microvascular endothelila cells (HBMEC) is higher with ursodeoxycholic acid (UDCA) than with glycoursodeoxycholic acid (GUDCA). A cell line of HBMEC was incubated without (control) or with 100 μM UCB, in the presence of 100 μM human serum albumin, for 1 or 4 h. The bile acids (50 μM) were added 1 h prior to the addition of UCB. Changes in IL-6 mRNA after 1 h (A) and protein release after 4 h of incubation (B) are shown. Quantification is shown as fold change from control values and results are expressed as mean ± SEM from at least three independent experiments. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 vs. control; ^§P < 0.05 and ^§§§P < 0.001 vs. UCB alone; ^#P < 0.05 and ^##P < 0.01 UDCA vs. GUDCA.

Figure 5

Ursodeoxycholic acid (UDCA), but not glycoursodeoxycholic acid (GUDCA), abrogates paracellular permeability impairment to sodium fluorescein (SF) in human brain microvascular endothelial cells (HBMEC). A cell line of HBMEC was incubated without (control) or with 100 μM UCB, in the presence of 100 μM human serum albumin, for 48 h. The bile acids (50 μM) were added prior to (1 h) or after (4 or 8 h) UCB addition. Quantification of permeability to SF is expressed as fold change from control values and results are shown as mean ± SEM from at least three independent experiments. ^***P < 0.001 vs. control; ^§P < 0.05 and ^§§P < 0.01 vs. UCB alone; ^#P < 0.05 UDCA vs. GUDCA.