The Influence Of Brain Microvascular Endothelial Cells Permeability Induced By Extract Of LPS Pretreated PMNs

Objective The Lipopolysaccharide (LPS) stimulates monocytes /macrophages (monocytes, macrophages and lymphocytes included ) and vascular endothelial cells to excrete early inflammatory mediators. These inflammatory mediators composed of cell factors and cell active substances will activate polymorphonuclear neutrophils (PMNs) and other inflammatory effect cells, which promotes the occurrence and development of inflammatory diseases. Glutamate (Glu) is an excitatory amino acid in the center nerve system (CNS) which effects on the Glu receptors on neurons for physiological functions. The N-methyl-D-aspartate receptor (NMDAR) is an ionotropic Glu receptor which medicates the excitation function of Glu and other relative endogenous acidic amino acid. In recent researches, human PMNs preconditioned with formylmethionyl leucyl phenylalanine (fMLF) excreted Glu and adjusted the permeability of the brain microvascular endothelial cells (BMECs). However, currently there are no reports on whether LPS stimulates PMNs to excrete Glu which effects on NMDAR with the result of permeability change in the CNS infectious diseases. The research aims to investigate the mechanism of the permeability change in the blood brain barrier (BBB) in the early stage of infectious brain edema injuries in terms of understanding the PMNS pretreatment with LPS. Methods 1. PMNs in peripheral venous blood from healthy volunteers were isolated and purified with by discontinuous percoll gradient centrifugation, the morphornogy of PMNs was observed by Wright staining and cell activation of PMNs was detected with trypan blue staining. 2. 100μg/ml LPS after pretreatment was put in a 5% CO₂ incubator at 37℃and was centrifuged at the time points of 5 min, 15 min, 30 min, 45 min, 60 min and 90 min. The concentration of the Glu was measured by LC-6A High-performance Liquid Chromatographer in the supernatant without cells. 3. The rat BMECs were primarily isolated and cultured, and their property was identified with the VIII factor. 4. The single layer of BMECs cultured in vitro were divided into six groups, namely, the normal control group, the LPS preconditioning PMN extract group preconditioned, the LPS preconditioning group, the PMNs extract preconditioning group, the MK-801 preconditioning group, and the Glu preconditioning group. The permeability of BMECs was evaluated by measuring the passing rate of ¹²⁵I-BSA viaγray counter. 5. The expression of NMDAR1 on BMECs was evaluated by Western-blotting in the normal control group, the LPS preconditioning PMN extract group preconditioned, the LPS preconditioning group, the PMNs extract preconditioning group, the MK-801 preconditioning group, and the Glu preconditioning group.Results 1. Many segmental neutrophils were observed by Wright staining in the peripheral blood. The purity of PMNs was 97.6%±0.57%, the recovery rate of PMNs was 89.7%±7.4%, and the proportion of live PMNs by trupan blue staining was more than 98%,alteration of PMNs morphous less than 8%. 2. The concentration of Glu in the LPS preconditioning PMNs subgroup with 5 min time point was 6.319±1.454 (μmol/L), the highest and there was significant difference between it and the normal control group (P<0.01). 3. It was observed by the VIII factor immunohisto- chemistry method that 95% isolated cells showed yellow cytoplasm (DAB enhancement technique) and non- monoclonal antibody groups showed no change in cytoplasm, which demonstrated that more than 95% cultured cells were BMECs. 4. In each group conditioned for 240 min, the permeability index of Bake average of the normal control group, the LPS preconditioning PMN extract group preconditioned, the LPS preconditioning group, the PMNs extract preconditioning group, the MK-801 preconditioning group, and the Glu preconditioning group was 339.67±17.74, 831.17±10.15, 737.17±14.63, 495.5±10.56,423.83±10.63 and 903.67±40.63. Significant difference was observed between the LPS preconditioning PMNs extract group and the normal control group (P<0.01). S.It was showed that the expression of NMDAR1 increased in the LPS preconditioning PMNs extract group, with OD of 14.966±1.403 (The OD in the normal control group was 1 as the reference value), which was significant different from the that in the normal control group, the LPS preconditioning group, and the PMNs extract preconditioning group.Conclusions The research showed for the first time that the concentration of Glu increased after the LPS preconditioning and The Glu excreted by PMNs promoted the permeability of BMECs, which changed the function of BBB, possibly with a mechanism in terms of to the expression increase of NMDAR1 in BMECs.