Effects Of Pregnane X Receptor/nuclear Factor-?B On Glutamate-induced Expression And Activity Of P-glycoprotein In The Brain Microvascular Endothelial Cells

Excessive expression of P-glycoprotein(P-gp)in epilepsy lesions is one of the main resistance mechanisms of drug-resistant epilepsy(DRE).It is one of the research hotspots to explore the regulation mechanism of P-gp overexpression in epilepsy brain.Pregnane X receptor(PXR),a transcription factor containing a ligand-binding domain and a DNA-binding domain,regulates expression of critical genes in xenobiotic detoxification system,involved in elimination,metabolism and transport of xenobiotics.The experiments have demonstrated that PXR-mediated drug interactions can affect patients with some diseases,such as cancer,HIV and so on.Especially a series of chemotherapeutics can induce expression of drug metabolizing enzymes and drug transporters in tumor cells,leading to tumor drug resistance through PXR activation.Could PXR inhibition decrease P-gp overexpression in the blood-brain barrier induced by seizures?It remains unclear.The experiments showed that elevated glutamate during seizures induces P-gp overexpression in the blood-brain barrier through activation of cyclooxygenase-2 and nuclear factor-?B(NF-?B)pathway.Our previous Experiments have proved that NF-?B regulates P-gp expression in the brain of kainic acid-induced seizure rats.Further study has found that PXR up-regulated P-gp expression in the chronic epileptic brain of rats induced by kainic acid.It is no report that whether PXR/NF-?B are jointly involved in seizure-induced P-gp overexpression in the blood-brain barrier.Increased concentration of excitatory neurotransmitter glutamate in the microenvironment of brain is a common pathological feature among different etiological epilepsies.This in-vitro experiment was aimed to explore the effects of inhibited PXR/NF-?B on the expression and function of P-gp in mouse brain microvascular endothelial bEnd.3 cells treated with glutamate mimicking brain microenvironment during seizures,which might provide a theoretical basis for studying the regulation mechanism of seizure-induced P-gp overexpression.Part? Effects of downregulated PXR on glutamate-induced expression and activity of P-glycoprotein in the mouse brain microvascular endothelial cellsObjective: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(bEnd.3 cells)exposed to glutamate mimicking conditions in vivo during seizures.Methods:The bEnd.3 cells,an immortalized mouse brain microvascular endothelial cell line,were cultured in vitro.The cells were treated with culture medium containing 0,10 ?M,50 ?M,100 ?M glutamate for 30 min and exposed to 100 ?M glutamate for different durations(0,15 min,30 min).Protein expressions and mRNA levels of P-gp were determined in bEnd.3 cells followed PXR knockdown using small interfering RNA(siRNA)and exposed to glutamate.Western blot was used to detect the protein expressions of P-gp and PXR in each group.In immunofluorescence assay,bEnd.3 cells were treated with different concentration glutamate for 30min before detection.The expression of P-gp mRNA was detected by RT-qPCR.Rhodamine 123(P-gp probe substrate,Rh123)accumulation assay was used to study the activity of the P-gp in cells.Results:The protein expressions of PXR were 1.00±0.20,1.05±0.12,1.43±0.16,1.62±0.12 and the P-gp protein expressions were 1.00±0.24,1.04±0.26,1.50±0.11,2.15±0.10 in the 0(the control group),10,50,100 ?M glutamate groups respectively.PXR and P-gp protein expressions in the 50 and 100 ?M glutamate group were significantly higher than that in the blank group(P<0.05),especially maximal expressions occurred in the 100 ?M glutamate group.The protein expressions of PXR were 1.00±0.14,1.37±0.17,1.80±0.19 and the protein expressions of P-gp were 1.00±0.14,1.20±0.06,2.18±0.24 in different treatment duration groups(treated with culture medium containing 100 ?M glutamate for 0,15,30 min)respectively.Glutamate exposures as short as 15 min and 30 min significantly increased PXR expressions(P<0.05);P-gp expression in the 30 min groups was higher than that in the control group(P<0.05).The data of immunofluorescence analysis suggested that glutamate promoted the PXR accumulation in the nuclear.Compared with the control group,nuclear PXR level was significantly increased in the glutamate group(1.00±0.09 vs 1.43±0.22;t=-3.11,P=0.04).PXR siRNA treatment induced significant reduction of PXR protein by approximately 37 percent,compared to NC siRNA(1.00±0.00 vs 0.63±0.18;t=3.41,P=0.02).The protein level of P-gp was decreased by approximately 43 percent(1.00±0.00 vs 0.57±0.09;t=7.94,P=0.00)and the mRNA level of P-gp was decreased by approximately 52 percent(1.00±0.04 vs 0.48±0.08;t=10.98,P=0.00)in bEnd.3 cells followed PXR knockdown and exposed to glutamate.The fluorescence intensity of intracellular Rhl23 in the glutamate group(0.72±0.01)was lower than that in the control group(1.00±0.03;t=9.66,P=0.00).The fluorescence intensity of Rh123 in the glutamate plus verapamil(P-gp inhibitor)group(1.07±0.04)was higher than that in the glutamate group(t=-11.93,P=0.00).The fluorescence intensity of Rh123 in the PXR siRNA plus glutamate group(0.91±0.03)was higher than that in the NC siRNA plus glutamate 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 mouse brain microvascular endothelial cells exposed to glutamate 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.Part ? Effects of inhibited PXR/NF-?B on glutamate-induced expression and activity of P-glycoprotein in the mouse brain microvascular endothelial cellsObjective:To investigate the effects of inhibited PXR/NF-?B on expression and activity of P-gp in bEnd.3 cells treated with glutamate mimicing microenviroment of seizures.Methods:The bEnd.3 cells were cultured in vitro and divided into control group,glutamate treated-group(glutamate group),and glutamate treated-group pretreated with NC siRNA(NC siRNA+glutamate group)or PXR siRNA(PXR siRNA+glutamate group)or NF-kB p65 siRNA(NF-?B p65 siRNA+glutamate group)or PXR siRNA and NF-?B p65 siRNA(PXR siRNA+NF-?B p65 siRNA+glutamate group).Except control group was exposed to normal culture medium,all groups were exposed to culture medium containing 100 ?M glutamate for 30 min.The protein expressions of PXR,NF-?B p65,P-gp were detected by Western blot.Rh123 accumulation assay was used to study the activity of P-gp in bEnd.3 cells.Results:Compared with control group,nuclear NF-?B p65 protein level was increased in glutamate group(1.01±0.07 vs 2.14±0.18;t=-10.37,P=0.00).The protein expressions of PXR and P-gp in the PXR siRNA+glutamate group were significantly decreased compared with NC siRNA+glutamate group(t=13.76,P=0.00;t=7.77,P=0.00);the protein expressions of NF-?B p65 and P-gp in the NF-?B p65 siRNA+glutamate group were significantly decreased compared with NC siRNA+glutamate group(t=10.76,P=0.00;t=7.25,P=0.00);the protein expressions of PXR,NF-?B p65,P-gp in the PXR siRNA+glutamate group were significantly decreased compared with NC siRNA+glutamate group(t=15.84,P=0.00;t=10.84,P=0.00;t=10.60,P=0.00);the protein expression of P-gp in the PXR siRNA+NF-?B p65 siRNA+glutamate group was significantly lower than that in the PXR siRNA+glutamate group and NF-?B p65 siRNA+glutamate group(t=2.83,P=0.02;t=3.35,P=0.01);The protein expression of NF-?B p65 in the PXR siRNA+ glutamate group was significantly decreased compared with NC siRNA+glutamate group(t=2.52,P=0.04);the protein expression of PXR in the NF-?B p65 siRNA+glutamate group was significantly decreased compared with NC siRNA+glutamate group(t=15.66,P=0.00).Compared with NC siRNA+glutamate group,the fluorescence intensity of Rh123 was significantly increased in the PXR siRNA+glutamate group(1.34±0.13 vs 1.00±0.00;t=-4.11,P=0.00),NF-?B p65 siRNA+glutamate group(1.50±0.11 vs 1.00±0.00;t=-6.11,P=0.00)and PXR siRNA+NF-?B p65 siRNA+glutamate group(1.90±0.10 vs 1.00±0.00;t=-10.91,P=0.00),respectively;the fluorescence intensity of Rh123 of PXR siRNA+NF-?B p65 siRNA+glutamate group was significantly higher than that of PXR siRNA+glutamate group and NF-?B p65 siRNA+glutamate group(t=-6.81,P=0.00;t=-4.80,P=0.00),respectively.Conclusions:PXR and NF-?B p65 may jointly involved in the regulation of seizure-induced P-gp expression and activity in endothelial cells of brain microvascular.