The Role Of Caveolin-1/Akt/mTOR Pathway In Activation NMDAR Mediated Dysfunction Of Blood-brain Barrier In Vitro

Objective To study the role of Caveolin-1/Akt/m TOR pathway in activation NMDAR mediated dysfunction of blood-brain barrier in vitro at the level of signal transduction.Target on the function of NMDAR may provide a new idea for the treatment of central nervous system disease.Methods(1)After immunofluorescence double staining,the cell localization of NMDAR(mainly NR1 subunit)and Cav-1 on human brain microvascular HBEC-5i cells were observed by confocal laser scanning microscope;(2)After successfully establishing the blood-brain barrier(BBB)in vitro with HBEC-5i,the interventions were carried out according to the following groups: 1)NMDA group: NMDA was incubated with HBEC-5i alone for 24 hours;2)Blank control group;3)MK801/Daidzein/LY294002 group: MK801/Daidzein/LY294002 was incubated with HBEC-5i for 24 hours;4)MK801/Daidzein/LY294002 + NMDA group: HBEC-5i cells were pretreated with MK801/Daidzein/LY294002 for 2hours and then added with NMDA for 24 hours.The transendothelial resistance(TEER)of BBB in vitro was measured by Millicell-ERS cell resistance meter.Sodium fluorescein(SF)were used to measure the permeability of BBB in vitro.Western blot(WB)and real-time quantitative reverse transcription polymerase chain reaction(q RT-PCR)were used to detect the changes of protein and m RNA of MMP9 and Occludin induced by NMDA,and the phosphorylated proteins of Cav-1,Akt and m TOR were detected by WB;(3)HBEC-5i transfected with stable and low expression of LV-Cav-1 sh RNA and control sh RNA(LV-Ctrl sh RNA)were successfully screened.After treatment with NMDA for 24 hours,the TEER and permeability of BBB in vitro were measured,the changes of protein and m RNA of MMP9 and Occludin induced by NMDA were detected by WB and q RT-PCR,respectively.The phosphorylated proteins of Akt and m TOR were detected by WB.Results(1)NMDAR1 subunit(NR1)is expressed in the cytoplasm and part of the membrane of HBEC-5i,while Cav-1 is mainly expressed on the membrane of the cells.(2)In NMDA group,the TEER was decreased(P < 0.0001),while the permeability of fluorescein sodium SF increased(P < 0.0001),the m RNA and protein expression levels of MMP9 increased and the m RNA and protein expression levels of Occludin increased simultaneously(P < 0.01),the phosphorylation levels of Cav-1,Akt and m TOR protein increased(P < 0.05).Pretreatment with MK801/Daidzein/LY294002 can significantly reduce the phosphorylation of Cav-1,Akt and m TOR(P < 0.05),increase the expression of Occludin,as well as reduce the expression of MMP9(P < 0.05),so as to reduce the damage of NMDA to BBB in vitro;(3)Silencing the Cav-1 with sh RNA could increase the expression of Occludin,while reduce the expression of MMP9、pCav-1、p-Akt and p-m TOR(P < 0.05)induced by NMDA.Silencing the Cav-1with sh RNA also could reduce the decrease of TEER(P < 0.001)and the increase of sodium fluorescein SF permeability(P < 0.001)of BBB in vitro treated with NMDA.Conclusion(1)NMDAR subunit(NR1)is expressed in the cytoplasm and part of the cell membrane of HBEC-5i cell line.(2)Excessive activated NMDAR by NMDA can damage the BBB by upregulating the expression of p-Cav-1,p-Akt and p-m TOR,increasing MMP9,decreasing the expression of Occludin protein and m RNA.(3)Inhibiting Cav-1/Akt/m TOR pathway can reverse the damage of NMDA to the BBB.