| Inflammation is a protective reaction of living organisms with vascular system to invasive factors. It manifests as the damage of inflammation factor, the defensive reaction of organisms, and the recovery of tissue damage. Moderate inflammation reaction is a self-protective process, while extreme inflammation reaction can result in immunity damage of the organisms, and can even be fatal. Leucytoes are the most significant cell for inflammation. A hallmark of inflammation is the adhesion of leukocytes to vascular endothelial cell (VEC), and the following pass-through and migration to inflammatory sites. The binding of adhesive molecules expressed by leukocytes and VEC to their ligands is the essential molecular mechanism for this reaction.Selectin is one of the cell adhesive molecular families, and it mediates the adhesion of leukocytes to VEC, leukocytes to leukocytes, and leukocytes to platelets in inflammation. P-selectin, with relative molecular weight of about 140 KDa, is a transmembrane glycoprotein expressed on the surface of activated platelets and VEC. It plays a critical role in the initial inflammation reaction,and takes effects in cooperation with other selectins in the subsequent stages of inflammation. P-selectin can mediate leucocytes rolling on activated VEC by interacting with its major ligand PSGL-1. Therefore, the discovery of new equivalent to P-selectin ligands and the search or synthesis of the drug to influence P-selectin expression and function, will pave ways to reveal the mechanism of inflammation hence treat inflammation.Plant polysaccharide is a natural nontoxic substance with various biological features. Many plant polysaccharides, such as Radix astragali, Lentinus edodes, and Agaricus blazei Muri, have been shown to possess anti-acute inflammatory activities in clinical practices. But the anti-inflammatory mechanism of polysaccharide has been concealed due to the complicated structure and components or the difficulties of isolation and purification. Polysaccharide of Ginkgo Biloba Leave (PGBL) is one of the active components from Ginkgo Biloba leaves. It has been reported that PGBL has anti-inflammation activity though the mechanism is unclear.In our experiment, PGBL was obtained by boiling-water extraction, ethanol precipitation, and drying. To achieve purified PGBL (p-PGBL), PGBL was frozen and thawed repeatedly, ultrafiltrated, and gel chromatography. Then the fraction with the highest polysaccharide was collected, dialyzed, concentrated, and freeze-dried. Anti-inflammatory effects of p-PGBL were studied based on our acute peritonitis mouse model. The edema degree of mouse ear induced by xylol and the number of leucocytes and neutrophils were measured in acute peritonitis mouse.To research the anti-inflammation molecular mechanism of PGBL, the interaction between P-selectin and its ligands was first studied by using p-PGBL, P-selectin and its blocking mAb (9E1) in flow cytometry. Then the effects of p-PGBL in rolling and adhesion of HL-60 cells (neutrophils) on CHO-P, the CHO cells stably expressed P-selectin, were detected in flow chamber test that mimicks the physiological blood flow rate in micrangium. To further research the molecular mechanism of PGBL, Human Umbilical Vein Endothelial Cells (HUVEC)were isolated, and the interaction between neutrophils and HUVEC was observed in flow condition.Our results indicated that p-PGBL was water-soluble white power, with optical rotation as [a]20n = +43, relative molecular weight as about 10 KDa, and polysaccharide content as 98.6%. p-PGBL was composed of Gal, Man, Glu, Ara, Rha, and Galacturonic monosaccharide, whose molar ratio was 6.0: 2.4: 1.7: 4.6: 6.8: 1.0. In anti-inflammatory assay, we found that p-PGBL could inhibit the mouse ear edema degree induced by xylol and the neutrophil transmigration from blood vessel to mouse peritoneal cavity. Compared with those of PGBL, the inhibition effects were not significantly different. Therefore, p-PGBL could preserve anti-inflammatory activity, and its activity came from inhibiting neutrophils transmigration to mouse peritoneal cavity, such like heparin. In the flow cytometry experiments, we found that different concentrations of p-PGBL could block the binding of p-selectin to HL-60 cells (neutrophils). Such experimental results suggested that anti-inflammatory mechanism of PGBL was like that of p-PGBL which could act as the analogue of p-selectin ligand to block the binding of p-selectin to its ligands and inhibit leucocyte transmigration from blood vessel. In the flow chamber experiments simulating the physiological blood flow rate, when wall shear stress was 1.0 dyn/cm2, three concentrations of p-PGBL significantly decrease the rolling and adhesion to CHO-P from HL-60 cells (neutrophils). This suggested that under the condition close to physiological blood flow rate, p-PGBL could inhibit the adhesion of neutrophils to activated VEC and the transmigration from blood vessel. Such results were confirmed with HUVEC in the flow chamber assay. In addition, we found that P-selectin blocking mAb (9E1) almost completely blocked neutrophils rolling on CHO-P, however, it only partially blocked the rolling on HUVEC. This implies that other adhesion molecules or inflammatory factors on HUVEC also block the rolling process. How p-PGBL blocks other adhesion molecules or inflammatory factors needs further investigations.Our experimental results verified that PGBL had strong anti-inflammation effect. The molecular mechanism was that PGBL effectively blocked the interaction between the P-selectin and its ligands. Our research illustrated the anti-inflammatory mechanism of PGBL in P-selectin-mediated, supported the anti-adhesion theories of polysaccharide. It paves the way not only to discover new drugs with higher efficiency and lower toxicity, but also to utilize Ginkgo biloba resources.
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