The Impact Of KLF2/RelA Imbalance On Neutrophil Apoptosis Of Neutrophilic Asthma Guinea Pig Model

Objective: By observing the expression of lung Krüppel-like transcriptionfactor (KLF2), RelA and Bcl-xl in the neutrophil of neutrophilic asthma guinea pigs,preliminary study the role and participation mechanisms of KLF2and RelA inneutrophil apoptosis in neutrophilic asthma, providing new ideas for the preventionand treatment of asthma.Methods: Forty adult male guinea pigs were ramdomly divided into threegroups:control group (group A), neutrophilic asthma group(group B), neutrophilicasthma treatment group(group C).The guinea pigs in group B and C were sensitizedwith ovalbumin(0VA) and Freund’s complete adjuvant(FCA) together,then werecontinuously stimulated with0VA atomization inhalation until appearing asthmasymptom to establish neutrophilic asthma model.The guinea pigs of the treatmentgroup were intraperitoneal injected with dexamethasone before challenge.To evaluatethe asthma model by observing the change of behavior and bronchial lung tissuepathology of guinea pigs after challenge,and by detecting the total number of cell anddifferential cell counts in brochial alveolar lavage fiuid(BALF),and also by detectingairway resistance. Flow cytometry was used to detect apoptosis rate ofneutrophils,and RT-PCR was used to detect the expression of KLF2and RelA mRNAin inflammatory cells of bronchoalveolar lavage fluid and blood neutrophils. Westernblot was used to detect the protein expression levels of KLF2, RelA and Bcl-xl inneutrophils of blood.Result:(1) The airway resistance of the guinea pigs in group B was increased with theincreasing of inhaled concentration of methacholine,which were significantly higher than group A（P<0.01) in different concentrations of methacholine challenge. Theairway resistance level of the guinea pigs in group C was lower than which of group B（P<0.05).(2) During the atomization of OVA, the guinea pigs of group B shown typicalsymptoms of asthma such as shortness of breath, coughing, sneezing and abdominalmuscle twitching, the guinea pigs of group C also appeared the above symptoms butwas significantly eased. the guinea pigs of group A did not appear the abovesymptoms.(3) The pathological study of group A showed that bronchial and lung tissue wasnormal, bronchial epithelial was integrity and inflammatory cells was few. The lungsection of group B showed typical changes of asthma that bronchial luminal wasnarrow,mucosal epithelial was shed and with inflammatory cell infiltration.In theguinea pigs of group C,the bronchial mucosa was still have obvious edema, but theinfiltration of inflammatory cell was better than which in group B.(4)The total number of cells and the proportion of neutrophil of BALF in groupB were significantly higher than group A（The total number of cells105.75±12.36VS37.25±9.37,N%18.97±4.59(%) VS4.28±0.95(%), P<0.01). Compared with group B,the total number of cells and the proportion of neutrophil of BALF in group C wassignificantly lower（The total number of cells58.72±10.98VS105.75±12.36,N%11.75±2.51(%) VS18.97±4.59(%),P<0.01).(5) The neutrophil apoptosis rate in blood of guinea pigs in group A was12.21±2.79（%）,which in group B was significantly lower than that in group A(2.68±1.25(%) VS12.21±2.79（%）,P <0.01). The neutrophil apoptosis rate of group Cwas higher than which in group B(5.30±1.63(%) VS2.68±1.25(%),P<0.05) but stilllower than which in group A.(6) The protein level of Bcl-xl in group B was lower than group A （0.50±0.12VS0.82±0.14P <0.01), Compared with group B, the protein level of Bcl-xlin groupC（0.67±0.11VS0.50±0.12）increased but still lower than which in group A.(7) The proportion of KLF2/RelA of BALF and blood neutrophils in the guineapigs of group B in RNA level was decreased than group A(BALF0.57±0.15VS 1.17±0.21，血0.57±0.11VS2.14±0.23,P <0.01), and the protein levels ofKLF2/RelA in blood neutrophils of group B was also decreased than group A(0.65±0.17VS1.54±0.23,P <0.01); the indicators of C which were mentioned aboveimproved significantly compared with which in group B respectively (P<0.01).(8) The correlation analysis showed that neutrophils apoptosis rate was positivelycorrelated with the KLF2/RELA protein ratio (r=0.8826，P＜0.001).Conclusion:1.The neutrophil apoptosis rate in neutrophilic asthma guinea pigs was reduced;2.The imbalance of KLF2/RelA may play an important role in the delay ofneutrophil apoptosis in neutrophilic asthma，which may be a potential mechanism ofthe increased number of neutrophils.3. The regulation of neutrophil apoptosis by KLF2and RelA was not through theway of Bcl-xl.