Background
The
Conclusion
Caveolin-1 (Cav-1) related Akt/mTOR signaling probably contributes to BBB dysfunction by activating NMDAR on human brain microvascular cells.
Methods
The cell localization of NMDAR GluN1 subunit and Cav-1 was observed on human brain microvascular HBEC-5i cells after immunofluorescence double staining. The transendothelial resistance (TEER) of BBB in vitro was measured by Millicell-ERS cell resistance meter. Sodium fluorescein (SF) was used to measure the permeability of BBB in vitro. A stable Cav-1-silenced HBEC-5i cell line was established by infecting the cells with a lentivirus encoding Cav-1 shRNA. The changes of the protein and mRNA of MMP9 and Occludin induced by NMDA were detected by Western blot (WB) and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), respectively. The phosphorylated proteins of Cav-1, Akt, and mTOR were detected by WB.
Results
NMDAR GluN1 was expressed in the cytoplasm and part of the cell membrane of the HBEC-5i cell line. NMDAR activation decreased TEER and increased the SF of BBB in vitro. HBEC-5i cells incubated with NMDA enhanced the phosphorylation of Cav-1, Akt, and mTOR, also promoting the expression of MMP9 along with the degradation of Occludin. These effects could be reversed by pretreatment with NMDAR antagonist (MK801) or Cav-1 antagonist (Daidzein), or Akt antagonist (LY294002), respectively. Further silencing Cav-1 with LV-Cav-1-RNAi also played a similar protective effect.