Study On The Relation Between Neutrophils Intracellular Cortisol And Inflammatory Reaction

Objective:Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) induced by sepsis clinically is a critical illness seen commonly, and neutrophils are one of the most important inflammatory cells in its pathogenesis. However, due to the short life span neutrophils and the difficulties in cell separation, studies on the physiopathological changes of neutrophils in early phrase of sepsis and ALI/ARDS were relatively rare. In this study, after neutrophils were treated by endotoxin, the relation among the changes of its intracellular cortisol, glucocorticoid receptors (GR), some regulative factors and inflammatory reaction was be investigated, and the effects of dexamethasone and hydroxysafflor yellow A (HSYA) were also studied. The aim of this study is to further research the pathogenesis and pharmacotherapy of sepsis and ALI/ARDS.Methods:Neutrophils were be separated, purified and cultured by improved means. And neutrophils were divided randomly into endotoxin-time, endotoxin-dosage, dexamethasone, and hydroxysafflor yellow A (HSYA) groups. Each group includes 4 observation points, and each observation point includes 3 samples. After 4h treated by endotoxin, observation points in endotoxin-dosage group were established by endotoxin dosage (0μg/ml,0.1μg/ml, 1.0μg/ml, 10μg/ml). After treated by endotoxin, observation points were established by time (0,1,2,4h) in endotoxin-tim group, by dexamethasone dosage (0mmol/ml, 0.001mmol/ml, 0.01mmol/ml, 0.1mmol/ml) in dexamethasone group at 4h, and by HSYA dosage (Ommol/ml, 0.01mmol/ml, 0.1mmol/ml,1.0mmol/ml) in HSYA group at 4h. Means such as enzyme-linked immunosorbent assay were used to detect the following targets:tumor necrosis factor (TNF)-a and matrix metalloproteinase (MMP)-9 contents in cell culture fluid, intracellular cortisol, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), glucocorticoid receptors (GR), and so on.Results:1. The primitive modalities of retrieved neutrophils were protected finely, and the purified rate of neutrophils exceeded 90%.2. With time going, in cell culture fluid, TNF-a content significantly increased at lh (5.233±0.188 vs 4.370±0.444, p<0.05) and decreased at 4h (4.602±0.392 vs 5.233±0.188, p<0.05); MMP-9 content presented gradual increased trend. Intracellular cortisol content presented early decreased and latterly increased trend, and 11β-HSD1 activity presented increased trend. Intracellular GRa content decreased significantly at 2h and 4h (0.480±0.139,0.453±0.151 vs 0.637±0.065, p<0.05), and GRβcontent presented increased trend. GRa/GRP ratio decreased significantly at 4h (1.530±0.538 vs 3.330±1.984,p<0.05).3. With endotoxin dosage increasing, in cell culture fluid, TNF-a content significantly increased in low endotoxin-dosage group (5.453±0.655 vs 4.149±0.312; p<0.05), and after this, presented gradual decreased trend; MMP-9 content presented gradual increased trend. Intracellular cortisol content and 11β-HSD1 activity presented gradually increased trend. Intracellular GRa content decreased significantly in high endotoxin-dosage group (0.503±0.150 vs 0.723±0.075, p<0.05), and GRβcontent presented increased trend. GRa/GRP ratio decreased significantly in middle and high endotoxin-dosage group (2.027±0.292,1.707±0.541 vs 3.470±1.434, p<0.05).4. With dexamethasone dosage increasing, in cell culture fluid, TNF-a content decreased significantly in middle and high dexamethasone-dosage group (4.917±0.189,4.867±0.431 vs 5.510±0.150, p<0.05); MMP-9 content presented gradual increased trend. Intracellular cortisol content presented increased trend, 11β-HSD1 activity decreased significantly (0.240±0.392 vs 2.147±0.609, p<0.05). Intracellular GRa content presented gradual decreased trend, and GRβcontent increased significantly in high dexamethasone-dosage group (0.230±0.020 vs 0.177±0.021, p<0.05). GRα/GRβratio decreased significantly (1.340±0.139 vs 2.030±0.748,1.850±0.416,1.857±0.372, p<0.05).5. With HSYA dosage increasing, in cell culture fluid, TNF-αcontent presented gradual decreased trend; MMP-9 content decreased significantly in high HSYA-dosage group (28.763±6.007 vs 36.638±2.996, p<0.05). Intracellular cortisol content and 11β-HSD1 activity presented increased trend. Intracellular GRa content increased significantly in high HSYA-dosage group (0.560±0.101 vs 0.403±0.125, p<0.05), and GRβcontent presented decreased trend. GRα/GRβratio increased significantly in middle and high HSYA-dosage group (2.683±0.516, 3.207±0.542 vs 1.520±0.090, p<0.05).Conclusions:1. The improved means used in this study were helpful to protect cellular primitive modalities and to increase the purified rate of retrieved neutrophils.2. In order to rightly observe inflammatory reaction in experiment about neutrophils, different targets should be detected at the same time.3. The degree of neutrophils inflammatory reaction increased with the duration and degree of stimulation increasing. Removing unhealthy stimulator is a feasible measure to control neutrophils inflammation.4. In very early phrase of neutrophils inflammatory reaction, intracellular cortisol might be relatively absent. During this period, administering glucocorticoid might be helpful.5. With time going and stimulation degree increasing,11β-HSD1 activity increased, GRαcontent decreased and GRβcontent increased. These changes might induce intracellular cortisol surplus and glucocorticoid resistance.6. Exogenous glucocorticoid might increase cortisol content in neutrophils by biotransformation. On the other hand, it decreased 11β-HSD1 activity, which might be one of reasons that inhibit intracellular cortisol to excessively increase.7. During the period of neutrophils inflammatory reaction, exogenous glucocorticoid might dose-dependently make GRa content decrease and GRβcontent increase. These effects of exogenous glucocorticoid might reinforce neutrophils glucocorticoid resistance and might be harmful when high dosage exogenous glucocorticoid was administered.8. HSYA might dose-dependently make GRa content increase and GRβcontent decrease, which might attenuate glucocorticoid resistance and be helpful to inhibit neutrophils inflammatory reaction.