Oxidative Stress And NF-κB Activation Of Rats Brain Microvascular Endothelial Cells

Objective: Vascular endothelial cells activation contributes to many deseasesformation and development.The dysfunction of brain microvascular endothelialcells(BMVEC) is deeply involved in many brain vascular diseases,and oxidativestress is one of the important causes.Nuclear factor kappa-B (NF-κB), aredox-sensitive,ubiquitous factor,is a kind of protein to regulate transcription.NF-κB activation promote the expression of some cell factors,adhesionmolecules.The mechanism of brain microvascular endothelial cells to oxidativestress is unclear,and lots need to be researched.We suppose that rats brainmicrovascular endothelial cells activation by oxidant LPS is characterized by thegeneration of reactive oxygen species (ROS) that enhance nuclear translocation ofthe transcription factor kappa-B (NF-κB), and antioxidant can inhibit NF-κBactivation.Methods: Four rats,about 100 grams weighed,were decapitated,and brain wasgot.The brains were dissected and minced.Microvessels were collected and placedinto culture plask with complete culture medium.The cells were incubated in 5%CO2 at 37℃.Rats brain microvascular endothelial cells were stimulated by strongoxidant LPS with different doses and different time.Then we can observe thechanges of NF-κB activation. Intracellular ROS production of endothelial cellsexposed to LPS is detected by 2',7'-Dichlorofluorescin diacetate(DCF-DA) andfluorescence microscope. NF-κB subunit translocation was assessed byelectrophoretic mobility shift assay(EMSA).Antioxidant NAC is used for verifyingthe ROS role in NF-κB activation.The result specificity and reliability were testedby using unlabeled oligonucleotide probe and antisera of P50 and P65.Results: The BMVEC with spindle shape grow by monolayer andattachment.Identification was made by factor VIII antigen. Endothelial-derived...