The Mechanism Of P-glycoprotein High-expression Via Activation Of NMDA Receptor Signal Pathway In RBMECs

Part Ⅰ Establishment of high-expression P-glycoprotein model in RBMECs&Observation of NMDA receptor inhibitor acting on P-glycoprotein expressionObjective:To establish the model of high-expression P-glycoprotein in RBMECs. Methods:Cortex was obtained from newborn Wistar rat brains. After surface vessels and meninges were removed, cortex gray matter was minced and incubated. Then Fat, cell debris, and myelin were discarded by BSA. Finally, the pellet containing microvessels was resuspended and incubated at37℃in culture medium consisting of DMEM supplemented with1ng/ml bFGF,20%heated-inactivated fetal bovine serum, penicillin100kU/L and streptomycin100mg/L in culture dishes precoated with gelatin. Cytotoxicity was assessed by the MTT assay. P-gp expression was determined by Western Blot. Mdrl gene was analysed by RT-PCR. Results:The level of P-glycoprotein expression significantly increased in RBMECs after the treatment of100μM glutamate. At this concentration, glutamate also enhanced rat mdrla and mdrlb mRNA levels determined by RT-qPCR. MK801, a non-competitive N-methyl-D-aspartate(NMDA) receptor antagonist attenuated the changes of expression of P-glycoprotein induced by glutamate in RBMECs. Conclusion:Glutamate up-regulated P-glycoprotein expression by an NMDA receptor-mediated mechanism in RBMECs. Part II Determine the key modulating factors in glutamate up-regulated P-glycoprotein expression model in RBMECsObjective:To determine the key modulating factors in glutamate up-regulated P-glycoprotein expression model in RBMECs. Methods:Cortex was obtained from newborn Wistar rat brains. After surface vessels, meninges, fat, cell debris, and myelin were removed, the pellet containing microvessels was resuspended and incubated at37℃in culture medium consisting of DMEM supplemented with lng/ml bFGF,20%heated-inactivated fetal bovine serum, penicillin100kU/L and streptomycin100mg/L in culture dishes precoated with gelatin. The expression of eEF-2K, phospho-eEF-2, eEF-2and NF-κB was determined by Western Blot. NF-κB(p65) was analysed by ELISA. Results:The expression of eEF-2K was significantly increased in Glu+PBS group than which in control group at6h,24h,72h point. The expression of eEF-2K was decreased in MK801+Glu group than which in Glu+PBS group at6h,24h,72h point. The expression of phospho-eEF-2was decreased in MK801+Glu group than which in Glu+PBS group at5min,1h,3h point. The activity of NF-κB(p65) was significantly increased in Glu+PBS group than which in control group at1h and3h. The activity of NF-κB(p65) in Glu+PBS group was similar to which in Glu+PBS group at1h and3h. Conclusion:eEF-2K and eEF-2could have take part in the effect of glutamate up-regulated P-glycoprotein expression in RBMECs. MK801could inhibit NMDA receptor and then decrease the expression of eEF-2K, phospho-eEF-2and P-gp. The high activity of NF-κB(p65) might also link with the regulation of P-gp expression. Part III Effect of eEF-2K inhibitors to regulate glutamate up-regulated P-glycoprotein expression in RBMECsObjective:To determine the effect of eEF-2K inhibitors to regulate glutamate up-regulated P-glycoprotein expression in RBMECs. Methods: Cortex was obtained from newborn Wistar rat brains. After surface vessels, meninges, fat, cell debris, and myelin were removed, the pellet containing microvessels was resuspended and incubated at37℃in culture medium consisting of DMEM supplemented with lng/ml bFGF,20%heated-inactivated fetal bovine serum, penicillin100kU/L and streptomycin100mg/L in culture dishes precoated with gelatin. Cytotoxicity was assessed by the MTT assay. Mdrla, Mdrlb mRNA was checked by RT-qPCR. The expression of phospho-eEF-2and eEF-2was determined by Western Blot. Results:The expression of P-glycoprotein was decreased in NH125+Glu group than which in Glu+PBS group at3h,6h and24h. The expression of phospho-eEF-2was decresed in NH125+Glu group than which in control group at5min,1h,3h point. The expression of P-glycoprotein was markly decreased in NH125+MK801+Glu group according to Glu+PBS group. The expression of phospho-eEF-2was markly decreased in NH125+MK801+Glu group than which in Glu+PBS group. Conclusion:1μmol/L NH125could have down-regulated P-glycoprotein expression in glutamate up-regulated P-glycoprotein expression in RBMECs. eEF-2K inhibitor NH125could have decreased the phospholation of eEF-2and then decreased the expression of P-gp. NH125combined with MK801could have thoroughly decreased the expression of P-gp in high expression P-glycoprotein model in RBMECs. Conclusions1. Glutamate up-regulated P-glycoprotein expression by an NMDA receptor-mediated mechanism in RBMECs.2. eEF-2K and eEF-2could have take part in the effect of glutamate up-regulated P-glycoprotein expression in RBMECs.3. MK801could inhibit NMDA receptor and then decrease the expression of eEF-2K, phospho-eEF-2and P-gp.4. The high activity of NF-κB(p65) might also link with the regulation of P-gp expression, while MK801has little effect on the expression of NF-κB or the activity of NF-κB(p65).5.1μmol/L NH125could have down-regulated P-glycoprotein expression in glutamate up-regulated P-glycoprotein expression in RBMECs.6. eEF-2K inhibitor NH125could have decreased the phospholation of eEF-2and then decreased the expression of P-gp.7. NH125combined with MK801could have thoroughly decreased the expression of P-gp in high expression P-glycoprotein model in RBMECs.