Effects of downregulated pregnane X receptor on glutamate-induced expression and activity of P-glycoprotein in the mouse brain microvascular endothelial cells
10.3969/j.issn.1004-1648.2018.06.015
- VernacularTitle:下调孕烷X受体表达对谷氨酸诱导的小鼠脑微血管内皮细胞P-糖蛋白表达和功能的影响
- Author:
Yong-Fei CHENG
1
;
Nian YU
;
Qing DI
Author Information
1. 南京医科大学附属脑科医院神经内科
- Keywords:
pregnane X receptor;
P-glycoprotein;
glutamate;
brain microvascular endothelial cell;
Drug-resistant epilepsy
- From:
Journal of Clinical Neurology
2018;31(6):450-455
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the effects of downregulated pregnane X receptor (PXR)on expression and activity of P-glycoprotein(P-gp)in mouse brain microvascular endothelial cells (mBMEC)exposed to glutamate (GLU)to mimic conditions during seizures. Methods The bEnd.3 cells,were cultured in vitro and treated with culture medium containing 0 μmol,10 μmol,50 μmol,100 μmol GLU for 30 min and exposed to 100 μM GLU for different durations (0 min,15 min,30 min). With PXR knockdown using siRNA,the cells were divided into NC siRNA plus GLU group and siRNA-PXR plus GLU group. Western blotting was used to detect the protein expressions of P-gp and PXR in each group. Immunofluorescence assay was used to detect localization of PXR in cells. The expression of P-gp mRNA was detected by RT-qPCR. Rhodamine123 (Rh123)accumulation assay was used to study the activity of the P-gp in cells.Results PXR and P-gp protein expressions in the 50 μmol and 100 μmol GLU group were significantly higher than that in the blank group(P＜0.05),especially maximal expressions occurred in the 100 μmol GLU group. GLU exposures as short as 15 min and 30 min significantly increased PXR expressions(all P＜0.05);P-gp expression in the 30 min groups was higher than that in the blank group (P＜0.05 ). The data of immunofluorescence analysis suggested that the PXR nuclear accumulation increased in the 100 μM GLU group, compared with the blank group(P＜0.05). Compared with NC siRNA plus GLU group,the protein level of PXR was decreased by approximately 37%[(1.00 ± 0.00)vs(0.63 ± 0.18);t＝3.41,P＝0.02]and the levels of P-gp protein and mRNA were respectively decreased by 43%[(1.00 ± 0.00)vs (0.57 ± 0.09);t＝7.94,P＝0.00] and 52%[(1.00 ± 0.04)vs (0.48 ± 0.08);t ＝10.98,P＝0.00]in the siRNA-PXR plus GLU group. The fluorescence intensity of intracellular Rh123 in the GLU group (0.72 ± 0.01)was lower than that in the blank group [(1.00 ± 0.03);t ＝9.66,P＝0.00]. The fluorescence intensity of Rh123 in the GLU plus verapamil (P-gp inhibitor)group (1.07 ± 0.04)was higher than that in the GLU group (t＝ －11.93,P＝0.00). The fluorescence intensity of Rh123 in the siRNA-PXR plus GLU group (0.91 ± 0.03)was higher than that in the NC siRNA plus GLU group[(0.69 ± 0.05);t＝ －7.52,P＝0.00]. Conclusions Downregulation of PXR expression results in the inhibition of P-gp expression and activity in the mBMEC exposed to GLU to mimic seizures. PXR may play an important role in the regulation of seizure-induced expression and activity of P-gp in the blood-brain barrier.
