Overexpression of pregnane X and glucocorticoid receptors and the regulation of cytochrome P450 in human epileptic brain endothelial cells.
Overexpression of pregnane X and glucocorticoid receptors and the regulation of cytochrome P450 in human epileptic brain endothelial cells.
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DOI：
10.1111/epi.13703
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发表时间：
2017-04
期刊：
影响因子：
5.6
通讯作者：
Janigro D
中科院分区：
文献类型：
--
作者：
Ghosh C;Hossain M;Solanki J;Najm IM;Marchi N;Janigro D
最近的证据表明，细胞色素P450酶(Cyps)的代谢对人类癫痫的耐药表型有贡献。然而，癫痫脑中CYP的上游分子调控机制仍未得到充分研究。因此，我们研究了孕烷异源受体(PXR)和糖皮质激素(GR)核受体在癫痫手术后脑标本建立的内皮细胞中的表达和功能。PXR/GR的定位通过免疫组织化学方法在接受颞叶切除以缓解耐药癫痫的受试者的标本中进行评估。我们使用了从癫痫脑组织获得的内皮细胞(EPI-ECs；n=8)、商用人脑微血管内皮细胞(HBMECs；n=8)和人肝细胞(n=3)的原代培养。脑内皮细胞的PXR/GR信使RNA(MRNA)水平最初通过互补DNA(CDNA)芯片进行检测。Western印迹法检测PXR/GR蛋白的表达。在EPI-ECs(n=4)中进行PXR和GR沉默，并检测其对下游CYP表达的影响。免疫荧光法检测PXR/GR在人颞叶内皮细胞和神经元中的表达。与对照组相比，EPI-ECs中PXR和GR的mRNA和蛋白水平均升高。以肝细胞为阳性对照，PXR/GR表达水平最高。我们证实了PXR/GR在胞浆-核亚细胞中的表达，与对照组相比，EPI-ECs中PXR/GR显著增加。与对照组相比，EPI-ECs中高表达的细胞色素P3A4、细胞色素P450 2C9和细胞色素P450 2 1，而细胞色素P450 2 D6和细胞色素P2 2 19表达下调或缺失。外周静脉内皮细胞GR沉默导致细胞色素P450 3A4、细胞色素P2C9和PXR表达降低。外周静脉内皮细胞的PXR沉默导致细胞色素P3A4表达的特异性下调。我们的结果表明，人类癫痫脑来源的原代内皮细胞中PXR和GR增加。PXR或GR可能与CYP调节异常所维持的局部药物脑代谢有关。
Recent evidence suggests a metabolic contribution of cytochrome P450 enzymes (CYPs) to the drug‐resistant phenotype in human epilepsy. However, the upstream molecular regulators of CYP in the epileptic brain remain understudied. We therefore investigated the expression and function of pregnane xenobiotic (PXR) and glucocorticoid (GR) nuclear receptors in endothelial cells established from post‐epilepsy surgery brain samples. PXR/GR localization was evaluated by immunohistochemistry in specimens from subjects who underwent temporal lobe resections to relieve drug‐resistant seizures. We used primary cultures of endothelial cells obtained from epileptic brain tissues (EPI‐ECs; n = 8), commercially available human brain microvascular endothelial cells (HBMECs; n = 8), and human hepatocytes (n = 3). PXR/GR messenger RNA (mRNA) levels in brain ECs was initially determined by complementary DNA (cDNA) microarrays. The expression of PXR/GR proteins was quantified by Western blot. PXR and GR silencing was performed in EPI‐ECs (n = 4), and the impact on downstream CYP expression was determined. PXR/GR expression was detected by immunofluorescence in ECs and neurons in the human temporal lobe samples analyzed. Elevated mRNA and protein levels of PXR and GR were found in EPI‐ECs versus control HBMECs. Hepatocytes, used as a positive control, displayed the highest levels of PXR/GR expression. We confirmed expression of PXR/GR in cytoplasmic‐nuclear subcellular fractions, with a significant increase of PXR/GR in EPI‐ECs versus controls. CYP3A4, CYP2C9, and CYP2E1 were overexpressed in EPI‐ECs versus control, whereas CYP2D6 and CYP2C19 were downregulated or absent in EPI‐ECs. GR silencing in EPI‐ECs led to decreased CYP3A4, CYP2C9, and PXR expression. PXR silencing in EPI‐ECs resulted in the specific downregulation of CYP3A4 expression. Our results indicate increased PXR and GR in primary ECs derived from human epileptic brains. PXR or GR may be responsible for a local drug brain metabolism sustained by abnormal CYP regulation.