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Ruscogenin Attenuates Cerebral Ischemia-Induced Blood-Brain Barrier Dysfunction by Suppressing TXNIP/NLRP3 Inflammasome Activation and the MAPK Pathway

View Article: PubMed Central - PubMed

ABSTRACT

Ruscogenin, an important steroid sapogenin derived from Ophiopogon japonicus, has been shown to inhibit cerebral ischemic injury. However, its potential molecular action on blood-brain barrier (BBB) dysfunction after stroke remains unclear. This study aimed to investigate the effects of ruscogenin on BBB dysfunction and the underlying mechanisms in middle cerebral artery occlusion/reperfusion (MCAO/R)-injured mice and oxygen–glucose deprivation/reoxygenation (OGD/R)-injured mouse brain microvascular endothelial cells (bEnd.3). The results demonstrated that administration of ruscogenin (10 mg/kg) decreased the brain infarction and edema, improved neurological deficits, increased cerebral brain flow (CBF), ameliorated histopathological damage, reduced evans blue (EB) leakage and upregulated the expression of tight junctions (TJs) in MCAO/R-injured mice. Meanwhile, ruscogenin (0.1–10 µM) treatment increased cell viability and trans-endothelial electrical resistance (TEER) value, decreased sodium fluorescein leakage, and modulated the TJs expression in OGD/R-induced bEnd.3 cells. Moreover, ruscogenin also inhibited the expression of interleukin-1β (IL-1β) and caspase-1, and markedly suppressed the expression of Nucleotide-binding domain (NOD)-like receptor family, pyrin domain containing 3 (NLRP3) and thiredoxin-interactive protein (TXNIP) in vivo and in vitro. Furthermore, ruscogenin decreased reactive oxygen species (ROS) generation and inhibited the mitogen-activated protein kinase (MAPK) pathway in OGD/R-induced bEnd.3 cells. Our findings provide some new insights into its potential application for the prevention and treatment of ischemic stroke.

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Effects of ruscogenin on the cell viability and barrier function in bEnd.3 cells subjected to OGD/R. (A,B) The bEnd.3 cells were treated with ruscogenin at various concentrations (0.01–40 µM) and the cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in normal conditions, or after 6 h of OGD and 18 h reoxygenation (n = 6); (C,D) the bEnd.3 cells were treated with ruscogenin (0.1–10 µM) and PDTC (10 µM), and subsequently exposed to 6 h of OGD and 18 h reoxygenation. The barrier-protection effect of ruscogenin was detected using TEER and sodium fluorescein assays. (n = 3). The data are expressed as means ± SD. ##p < 0.01 vs. Control, * p < 0.05, ** p < 0.01 vs. Model.
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ijms-17-01418-f005: Effects of ruscogenin on the cell viability and barrier function in bEnd.3 cells subjected to OGD/R. (A,B) The bEnd.3 cells were treated with ruscogenin at various concentrations (0.01–40 µM) and the cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in normal conditions, or after 6 h of OGD and 18 h reoxygenation (n = 6); (C,D) the bEnd.3 cells were treated with ruscogenin (0.1–10 µM) and PDTC (10 µM), and subsequently exposed to 6 h of OGD and 18 h reoxygenation. The barrier-protection effect of ruscogenin was detected using TEER and sodium fluorescein assays. (n = 3). The data are expressed as means ± SD. ##p < 0.01 vs. Control, * p < 0.05, ** p < 0.01 vs. Model.

Mentions: Briefly, OGD/R was induced in a hypoxia chamber in RPMI 1640 culture medium without glucosein, in an atmosphere of 5% CO2, 94% N2 and 1% O2 for 6 h in the bEnd.3 cells, after which the cells were cultured under normoxia conditions for 18 h. We first investigated the various concentrations of ruscogenin on bEnd.3 cells under normoxia conditions in normal culture medium. The result suggested that pretreatment with ruscogenin (0.01–40 µM) had no obvious effect in normal bEnd.3 cells (Figure 5A). Then, we further investigated the effect of various concentration of ruscogenin (0.1–10 µM) pretreatment in bEnd.3 cells subjected to 6 h of OGD and 18 h reoxygenation. The results demonstrated that OGD/R-induced reduction of bEnd.3 cell viability was significantly recovered by the pretreatment with ruscogenin in various concentrations (Figure 5B). Meanwhile, the results of the trans-endothelial electrical resistance (TEER) value and fluorescein sodium permeability demonstrated that pretreatment with ruscogenin at various concentrations could increase the TEER value and inhibit the sodium fluorescein permeability compared with the model case (Figure 5C,D). Pyrrolidine dithiocarbamate (PDTC, 10 µM, a NF-κB inhibiter) also showed similar protection in bEnd.3 cells subjected to OGD/R (Figure 5B–D).


Ruscogenin Attenuates Cerebral Ischemia-Induced Blood-Brain Barrier Dysfunction by Suppressing TXNIP/NLRP3 Inflammasome Activation and the MAPK Pathway
Effects of ruscogenin on the cell viability and barrier function in bEnd.3 cells subjected to OGD/R. (A,B) The bEnd.3 cells were treated with ruscogenin at various concentrations (0.01–40 µM) and the cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in normal conditions, or after 6 h of OGD and 18 h reoxygenation (n = 6); (C,D) the bEnd.3 cells were treated with ruscogenin (0.1–10 µM) and PDTC (10 µM), and subsequently exposed to 6 h of OGD and 18 h reoxygenation. The barrier-protection effect of ruscogenin was detected using TEER and sodium fluorescein assays. (n = 3). The data are expressed as means ± SD. ##p < 0.01 vs. Control, * p < 0.05, ** p < 0.01 vs. Model.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5037697&req=5

ijms-17-01418-f005: Effects of ruscogenin on the cell viability and barrier function in bEnd.3 cells subjected to OGD/R. (A,B) The bEnd.3 cells were treated with ruscogenin at various concentrations (0.01–40 µM) and the cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in normal conditions, or after 6 h of OGD and 18 h reoxygenation (n = 6); (C,D) the bEnd.3 cells were treated with ruscogenin (0.1–10 µM) and PDTC (10 µM), and subsequently exposed to 6 h of OGD and 18 h reoxygenation. The barrier-protection effect of ruscogenin was detected using TEER and sodium fluorescein assays. (n = 3). The data are expressed as means ± SD. ##p < 0.01 vs. Control, * p < 0.05, ** p < 0.01 vs. Model.
Mentions: Briefly, OGD/R was induced in a hypoxia chamber in RPMI 1640 culture medium without glucosein, in an atmosphere of 5% CO2, 94% N2 and 1% O2 for 6 h in the bEnd.3 cells, after which the cells were cultured under normoxia conditions for 18 h. We first investigated the various concentrations of ruscogenin on bEnd.3 cells under normoxia conditions in normal culture medium. The result suggested that pretreatment with ruscogenin (0.01–40 µM) had no obvious effect in normal bEnd.3 cells (Figure 5A). Then, we further investigated the effect of various concentration of ruscogenin (0.1–10 µM) pretreatment in bEnd.3 cells subjected to 6 h of OGD and 18 h reoxygenation. The results demonstrated that OGD/R-induced reduction of bEnd.3 cell viability was significantly recovered by the pretreatment with ruscogenin in various concentrations (Figure 5B). Meanwhile, the results of the trans-endothelial electrical resistance (TEER) value and fluorescein sodium permeability demonstrated that pretreatment with ruscogenin at various concentrations could increase the TEER value and inhibit the sodium fluorescein permeability compared with the model case (Figure 5C,D). Pyrrolidine dithiocarbamate (PDTC, 10 µM, a NF-κB inhibiter) also showed similar protection in bEnd.3 cells subjected to OGD/R (Figure 5B–D).

View Article: PubMed Central - PubMed

ABSTRACT

Ruscogenin, an important steroid sapogenin derived from Ophiopogon japonicus, has been shown to inhibit cerebral ischemic injury. However, its potential molecular action on blood-brain barrier (BBB) dysfunction after stroke remains unclear. This study aimed to investigate the effects of ruscogenin on BBB dysfunction and the underlying mechanisms in middle cerebral artery occlusion/reperfusion (MCAO/R)-injured mice and oxygen&ndash;glucose deprivation/reoxygenation (OGD/R)-injured mouse brain microvascular endothelial cells (bEnd.3). The results demonstrated that administration of ruscogenin (10 mg/kg) decreased the brain infarction and edema, improved neurological deficits, increased cerebral brain flow (CBF), ameliorated histopathological damage, reduced evans blue (EB) leakage and upregulated the expression of tight junctions (TJs) in MCAO/R-injured mice. Meanwhile, ruscogenin (0.1&ndash;10 &micro;M) treatment increased cell viability and trans-endothelial electrical resistance (TEER) value, decreased sodium fluorescein leakage, and modulated the TJs expression in OGD/R-induced bEnd.3 cells. Moreover, ruscogenin also inhibited the expression of interleukin-1&beta; (IL-1&beta;) and caspase-1, and markedly suppressed the expression of Nucleotide-binding domain (NOD)-like receptor family, pyrin domain containing 3 (NLRP3) and thiredoxin-interactive protein (TXNIP) in vivo and in vitro. Furthermore, ruscogenin decreased reactive oxygen species (ROS) generation and inhibited the mitogen-activated protein kinase (MAPK) pathway in OGD/R-induced bEnd.3 cells. Our findings provide some new insights into its potential application for the prevention and treatment of ischemic stroke.

No MeSH data available.


Related in: MedlinePlus