| BackgroundChronic obstructive pulmonary disease (COPD) is a serious damage to human health, and the death toll caused by its accounts for 5% of deaths worldwide, which contributes one of the important factors to a high morbidity and mortality in chronic disease. The world health organization (who) caused by chronic obstructive pulmonary disease (COPD) is expected to death over the next 10 years will increase by 30%. Our group has do some research about the pathogenesis from the lung epithelial cells, endothelial cells, such as the study of the pathogenesis of multiple Angle, chemokines and the course of illness of eosinophils and neutrophils (PMNs) raise and inflammation may also be caused by chronic obstructive pulmonary disease (COPD) to start and the important reason for deferment, on the other hand the COPD peripheral blood neutrophils (PMNs) migration also there are exceptions, show the migration velocity increased significantly reduce the number of which direction to chemotactic migration, resulting in PMNs in whole lung tissue African abnormal accumulation of orientation, the nonspecific inflammation of the lungs and damage leading to airway inflammation of lung tissue and emphysema.Polymorphonuclear leukocytes (PMNs) are the primary mediators of the innate host immune defense against most bacterial and fungal pathogens. In this rapid process, polarization is a basic prerequisite for neutrophils to effectively directly migration and bacterial killing.As an important signal molecule of cell physiological process, Ca2+ was confirmed to participate in polymorphonuclear (PMNs) polarity changes. As a Non-excitable cells excitable cells, neutrophils intracellular Ca2+ signal is mainly raised from the internal flow of Ca2+ caused by endoplasmic reticulum Ca2+ library empty caused s, which called Store Operated Calcium Entry (SOCE). This mechanism in the feelings of endoplasmic reticulum Ca2+ ions change matrix interaction between molecules (STIM) 1,2, and Orail molecules on the cell membrane, the effect of calcium library flow (SOCE) inside the maneuverability of calcium is regulating the non-excitable cells [Ca2+ ]j rise in the key mechanism. But at the moment, the polarity of neutrophils from peripheral venous blood of COPD is aberrant or not, and if this change is associated with SOCE or not, literature has not yet been elucidated.The harm of smoking on the pathogenesis of COPD has been confirmed in great quantities. The epidemiological studies about parts of parts of Guangdong province shows that only 15.1% of smokers with COPD, and there is no statistically significant effect about smoking on COPD prevalence. Previous literature has reported that Store-operated Ca2+ entry (SOCE) plays a role in the polarization of neutrophil-like HL-60 cells by regulating the activation of Akt, Src, and Rho family GTPases under the stimulus of N-formyl-methionyl-leucyl-phenylalanine (fMLP).so we raise a question that whether or not the polarization and chemotaxis of neutrophils is associate with SOCE, and if the phosphorylation of Akt and Src is related with it? Which has certain significance on smoking in the pathogenesis and pathogenic mechanism of COPD?Objective1. To observe the difference of polarization of neutrophils from peripheral venous blood of COPD patients with healthy control subjects and healthy smoking subjects:spontaneous polarization rate and directional polarization rate under the fMLP gradient.2. To detect the intracellur calcium leverage of neutrophils caused by SOCE of COPD patients with healthy control subjects and healthy smoking subjects.3. To detect the expression of important crosslinking functional component of SOCE:matrix molecules (STIM) 1,2 and Orail is different or not between COPD patients and HC, HS.4. To explore the polarity of neutrophils and whether the classic PI3K signaling pathways is involved in it when stimulated by cigarette smoke extract (CSE) by detecting the Akt phosphorylation level and Src phosphorylation, and whether the PI3K inhibitors and Akt inhibitors hold back the polarization of neutrophils triggered by CSE.5. To explore the polarization of neutrophils, Akt phosphorylation, Src phosphorylation levels and intracellular calcium signal when pretreatment by SOCE inhibitors.Methods1. The neutrophils were freshly isolated by standard isolation protocol. This protocol comprises three successive steps:dextran sedimentation, Ficol-Histopaque density (1.077g/L) centrifugation and hypotonic lysis. Most of erythrocytes were removed during dextran sedimentation; while Ficol-Histopaque density (1.077g/L) centrifugation separates mononuclear cells from neutrophils with remaining RBC removed using ddH2O. More than 95% of the cells isolated were neutrophils, as assessed by Wright-Giemsa staining. Viability, determined by trypan blue exclusion, was> 98%.2. Pretreatment With or without 2-APB, SKF96365, Ly249002 or Deguelin for 15 minutes, the polarization of neutrophils is observed on Zigmong Chamber when stimulated by linear concentration gradient of cigarette smoke extract (CSE) or fMLP.3. Confocal laser-scanning microcopy was used to observe the intracellular calcium of neutrophils when pretreated with 2-APB, SKF96365 which is then triggered by CSE or TG.4. The phosphorylation of Akt and Src was detected by Western blot when neutrophils is pretreated with 2-APB, SKF96365, Ly249002 or Deguelin for 30 min then stimulated by CSE for 15min.5. The results of experimental data with mean ± standard deviation(χ ± SD), using SPSS 13.0 statistical packages for statistical analysis. Group compared with one-way ANOVA and inter-group comparison was significant using LSD when the homogeneity of variances was meet, while, group compared with Welch and inter-group comparison was significant using Dennett T3 when the homogeneity of variances was not meet. P<0.05 means the significance.Results1. When compared with healthy control subjects and healthy smoking subjects, the spontaneous polarization rate from 4 groups of COPD patients is significantly higher[(15.70±8.01)%, P< 0.05, (27.50±11.99)%, (24.21±8.45)%, (18.13 ±6.56)% than (7.99±3.91)%, (5.76±4.81)%, P< 0.01].And there is no significant of spontaneous polarization rate between COPD patients with HC and HS.2. When stimulated by fMLP gradient concentration, neutrophils from COPD patient showed a lower directional polarization rate when compared with HC and HS [(2.75±1.27)%, (3.52±1.29)%, (3.83±1.82)%, (3.06±0.62)% than (7.44±3.31)%, (8.44±3.47)%, P< 0.01)]. And there is no significant of directional polarization rate between COPD patients with HC and HS.3. The intracellular calcium rise triggered by TG about endoplasmic reticulum calcium empty is lower about 30% in neutrophils from COPD patients when compared with HC and HS. And the second calcium peak with named SOCE is lower about 60% in COPD patients’ neutrophils.4. The expression of important components of SOCE:matrix crosslinked protein (STIM) 1,2, and Orail were significantly higher than HC and HS.5. The highest directional polarization rate of neutrophils caused by CSE is 0.5%. The rates of cell polarization were increased when neutrophils were stimulated with 0.5% CSE [(41.49±4.76)%], the rates of cell polarization in SKF96365 [(19.09±1.83)%, P= 0.000] or 2-APB [(14.06±1.62)%, P= 0.000] groups were lower than in CSE group.6. The basal Ca2±store were depleted with TG, then the level of cytoplasmic Ca2+ was restored by the supplementation of 2mM Ca2±to the medium, which revealed SOCE. SOCE was significantly inhibited by 30% and 45% by pretreatment of the cells with SKF96365 and 2-APB, respectively (P<005), with the Ca2±store depletion unaffected. CSE elicited an initial small increase in cytosolic Ca2+, in the presence or absence of SOCE inhibitors. The re-addition of extracellular Ca2+ resulted in a rapid Ca2+ influx into the cells, and this increase in cytoplasmic Ca2+ was reduced by 30～% and 60～% in the cells pretreated with SKF96365 and 2-APB, respectively(P<0.01), without affecting the Ca2+ store release.7. Neutrophils polarized toward CSE gradient directly, which were inhibited by pretreatment with Ly249002 and Deguelin (P=0.000; P=0.000).8. The levels of phosphorylation of Akt and Src were regulated when neutrophils were stimulated with CSE(F=6.376, P=0.004; F=41.57,P=0.000).The levels of phosphorylation of Akt and Src were not affected by Ly249002 or Deguelin.9. The levels of phosphorylation of Akt and Src were upregulated along with CSE stimulation(F=6.376, P=0.004;F=41.57, P=0.000).SKF96365 and 2-APB showed inhibitory effects on the activation of Src but not Akt(P=0.011; P=0.036).Conclusions1. The neutrophils from COPD patients showed a higher spontaneous polarization rate and a lower directional polarization rate when compared with healthy control subjects and healthy smoking subjects, means that neutrophils of COPD patients showed an excessive but inaccurate polarization.2. The expression of the important function component of SOCE:STIM1,2 and Orail is increased in COPD patients compared with HC and HS. And the intracellular calcium rise caused by SOCE was lower.3. Src and Akt phospholation plays an important role in CSE-induced polarization of neutrophils.4. SOCE is stimulated when CSE-induced neutrophils polarization.5. SOCE plays a role in neutrophil polarization by regulating the activation of Src. |
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