KLF2/Re1A Imbalance In The Role Of Neutrophil Apoptosis In Patients Of Bronchial Asthma

Objective: Whether exist KLF2/RelA imbalances in the neutrophils of patients withbronchial asthma and the relationship with apoptosis.Method: Blood were collected from healthy,mild, moderate and severe patients withasthma, followed by separation of neutrophils.The apoptosis of neutrophils weretested by flow cytometry.The localiziton of KLF2and RelA of neutrophils were testedby immunofluorescence.The expression of mRNA and protein of KLF2,RelA andBcl-xl were tested by RT-PCR and Western Blot respectively.Result:1Neutrophil separation and purity determinationSuccessfully isolated neutrophils, by Wright-Giemsa staining，the neutrophilspurity more than95%.2Changes in the rate of neutrophil apoptosis in patients with asthmaneutrophil apoptosis rate： Control,mild, moderate, severe asthma groupneutrophil apoptosis rate declined(5.36±0.57,4.45±0.76,2.10±0.25,1.81±0.67), Thereis a statistical difference between control group and mild group and between the mildgroup and moderate group （P＜0.01）, but no significant difference between themoderate group and severe group（P>0.05）.3Changes of KLF2expression and localization in asthma patients neutrophilsand the relationship with apoptosis1）For the relative expressions of mRNA of KLF2in the neutrophils, mild asthmagroup（0.47±0.11）were lower than moderate asthma group(0.75±0.13)（P＜0.01）,moderate asthma group were lower than severe asthma group(1.17±0.28)（P ＜0.01）;mild and moderate asthma group lower than control group（1.52±0.37）（P＜0.01）, severe asthma group lower than control group（P＜0.05）;2）For the relative expressions of protein of KLF2in the neutrophils, mild asthmagroup（0.050±0.014）were lower than moderate asthma group(0.066±0.025)（P＜0.05）;moderate asthma group were lower than severe asthma group(0.091±0.023)（P＜0.01）;mild and moderate severe asthma group lower than controlgroup(0.124±0.021)（P＜0.01）,severe asthma group lower than controlgroup(0.124±0.021)（P＜0.05）;3） For the nuclear/cytoplasm mean optical density of KLF2in the neutrophils,control group（0.14±0.04）were lower than mild asthma group(0.33±0.10)（P＜0.01）;mild asthma group were lower than moderate asthma group(0.58±0.14)（P＜0.01）;moderate group lower than severe group(0.72±0.15)（P＜0.05）;4）Pearson correlation analysis of neutrophil apoptosis rate and protein of KLF2prompt no correlation correlation(r=0.1208，P>0.05）;5）Pearson correlation analysis of neutrophil apoptosis rate and KLF2nuclear/cytoplasm mean optical density prompt negative correlation（r=-0.7754，P＜0.001）.4Changes of RelA expression and localization in asthma patients neutrophilsand the relationship with apoptosis1) For the relative expressions of mRNA of RelA in the neutrophils,mild(1.11±0.44),moderate(2.33±0.56) and severe asthma group(2.65±0.44) higherthan control group(0.08±0.07)（P＜0.01）,moderate asthma group and severe asthmagroup were higher than mild asthma group（P＜0.01）, No significant differencebetween the moderate group and severe group（P>0.05）;2） For the relative expressions of protein of RelA in the neutrophils,mild(0.092±0.046),moderate(0.187±0.045) and severe asthma group(0.201±0.058)higher than control group(0.039±0.024)（P＜0.01）;,moderate asthma group andsevere asthma group were higher than mild asthma group（P＜0.01）, No significantdifference between the moderate group and severe group（P>0.05）;3） For the nuclear/cytoplasm mean optical density of RelA in the neutrophils,mild(0.28±0.13),moderate(0.63±0.26) and severe asthma group(0.74±0.24) higher than control group(0.09±0.04)（P＜0.01）;,moderate asthma group and severe asthmagroup were higher than mild asthma group（P＜0.01）, No significant differencebetween the moderate group and severe group（P>0.05）;4） Pearson correlation analysis of neutrophil apoptosis rate and protein of RelAprompt correlation correlation(r=-0.8079，P＜0.001）;5）Pearson correlation analysis of neutrophil apoptosis rate and RelA nuclear/cytoplasm mean optical density prompt negative correlation(（r=-0.7332，P＜0.001）.5Changes of KLF2/RelA in asthma patients neutrophils and the relationshipwith apoptosis1）For ratio of KLF2/RelA protein：mild, moderate, severe asthma group(9.8±4.4、2.7±1.3、7.8±2.9) all lower than control group（42.1±16.2）（P＜0.01）,butno significant difference between the mild,moderate and severe group（P>0.05）.2) Pearson correlation analysis of neutrophil apoptosis rate and KLF2/RelAprompt positive correlation correlation(（r=0.6689，P＜0.001）.6Changes of Bcl-xl in asthma patients neutrophilsFor the relative expressions of protein of Bcl-xl in the neutrophils,mild(0.481±0.257),moderate(0.047±0.031) and severe asthma group(0.019±0.015)lower than control group(0.870±0.252)（P＜0.01）,moderate asthma group and severeasthma group were lower than mild asthma group（P＜0.01）, No significantdifference between the moderate group and severe group（P>0.05）;Conclusion:1） Neutrophil apoptosis rate decreased with the aggravation of asthma severity.2）Peripheral blood neutrophils in patients with asthma exists the KLF2/RelAimbalance and the KLF2/RelA imbalance may result reduced neutrophil apoptosis inthe peripheral blood of patients with asthma.