Modulation of glucocorticoid receptor in human epileptic endothelial cells impacts drug biotransformation in an in vitro blood-brain barrier model.
Modulation of glucocorticoid receptor in human epileptic endothelial cells impacts drug biotransformation in an in vitro blood-brain barrier model.
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作者：
Ghosh C;Hossain M;Mishra S;Khan S;Gonzalez-Martinez J;Marchi N;Janigro D;Bingaman W;Najm I
核受体和细胞色素P450（CYP）调节多种药物的肝脏代谢。核受体在耐药性癫痫患者的神经血管单位表达。我们研究了糖皮质激素受体（GR）沉默或抑制人癫痫脑内皮细胞（EPI-EC）是否通过β-多药转运蛋白（多药耐药蛋白1，MDR 1）机制在功能上影响药物在血脑屏障（BBB）体外模型中的生物利用度。使用了来自耐药癫痫患者的手术切除脑标本、原代EPI‐ EC和对照人脑微血管内皮细胞（HBMEC）。在EPI‐ EC中，分析GR沉默前后GR、CYP 3A 4和MDR 1的表达。将内皮细胞与星形胶质细胞共培养，并接种在体外基于流动的BBB模型（DIV-BBB）中。或者，将GR抑制剂米非司酮加入到EPI-EC DIV-BBB中。通过测量跨内皮电阻来监测BBB的完整性。通过葡萄糖-乳酸水平评估细胞活力。对[3 H]蔗糖和[14 C]苯妥英的渗透性进行定量。通过测量试卤灵形成和奥卡西平（OXC）代谢来确定抗抑郁功能。在EPI-EC中GR的沉默和抑制导致胆甾烷X受体、CYP 3A 4和MDR 1表达降低。GR沉默或抑制并不影响体外血脑屏障的性质，因为跨内皮电阻和P蔗糖没有改变，葡萄糖代谢得以维持。GR EPI-EC沉默或抑制导致（1）与对照相比，苯妥英BBB渗透性增加;（2）通过试卤灵形成间接评价的抗氧化功能降低;（3）与对照相比，OXC生物利用度提高，近腔（脑侧）OXC水平增加。我们的研究结果表明，在BBB调节EPI-ECs中的GR表达通过包括P450和外排转运蛋白的机制改变药物代谢和渗透。后者可用于未来的药物设计和克服抗药性。
Nuclear receptors and cytochrome P450 (CYP) regulate hepatic metabolism of several drugs. Nuclear receptors are expressed at the neurovascular unit of patients with drug‐resistant epilepsy. We studied whether glucocorticoid receptor (GR) silencing or inhibition in human epileptic brain endothelial cells (EPI‐ECs) functionally impacts drug bioavailability across an in vitro model of the blood–brain barrier (BBB) by CYP‐multidrug transporter (multidrug resistance protein 1, MDR1) mechanisms. Surgically resected brain specimens from patients with drug‐resistant epilepsy, primary EPI‐ECs, and control human brain microvascular endothelial cells (HBMECs) were used. Expression of GR, pregnane X receptor, CYP3A4, and MDR1 was analyzed pre‐ and post‐GR silencing in EPI‐ECs. Endothelial cells were co‐cultured with astrocytes and seeded in an in vitro flow‐based BBB model (DIV‐BBB). Alternatively, the GR inhibitor mifepristone was added to the EPI‐EC DIV‐BBB. Integrity of the BBB was monitored by measuring transendothelial electrical resistance. Cell viability was assessed by glucose‐lactate levels. Permeability of [3H]sucrose and [14C]phenytoin was quantified. CYP function was determined by measuring resorufin formation and oxcarbazepine (OXC) metabolism. Silencing and inhibition of GR in EPI‐ECs resulted in decreased pregnane X receptor, CYP3A4, and MDR1 expression. GR silencing or inhibition did not affect BBB properties in vitro, as transendothelial electrical resistance and Psucrose were unaltered, and glucose metabolism was maintained. GR EPI‐EC silencing or inhibition led to (1) increased Pphenytoin BBB permeability as compared to control; (2) decreased CYP function, indirectly evaluated by resorufin formation; (3) improved OXC bioavailability with increased abluminal (brain‐side) OXC levels as compared to control. Our results suggest that modulating GR expression in EPI‐ECs at the BBB modifies drug metabolism and penetration by a mechanism encompassing P450 and efflux transporters. The latter could be exploited for future drug design and to overcome pharmacoresistance.
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影响因子：
5.6
作者：
Ghosh C;Gonzalez-Martinez J;Hossain M;Cucullo L;Fazio V;Janigro D;Marchi N
通讯作者：
Marchi N
影响因子：
4.9
作者：
Hartz AM;Pekcec A;Soldner EL;Zhong Y;Schlichtiger J;Bauer B
通讯作者：
Bauer B
CYP3A4 在人类癫痫脑中的细胞定位和功能意义。
影响因子：
5.6
作者：
Ghosh C;Marchi N;Desai NK;Puvenna V;Hossain M;Gonzalez-Martinez J;Alexopoulos AV;Janigro D
通讯作者：
Janigro D
影响因子：
5.8
作者：
GHERSIEGEA, JF;PERRIN, R;MINN, A
通讯作者：
MINN, A
影响因子：
5.6
作者：
Cucullo, Luca;Hossain, Mohammed;Janigro, Damir
通讯作者：
Janigro, Damir