In Vitro Study And Discussion Of The Formation Of Neutrophil Extracellular Traps By Tobacco Extracts On Copd Patients During The Stable Period,and Erythromycin Intervention

Objective Discuss the difference between NETosis,under the effect of tobacco extracts,on stable-period chronic obstructive pulmonary disease(COPD)patients and that on normal people,plus the effect of erythromycin internvention.Method Recruit 10 healthy volunteers and 10 stable-period COPD patients whose average age is 64.After separating and purifying peripheral blood neutrophils,stimulate them according to groups.(1)Use 0.1%,0.3%,0.5%,1% and 2% CSEs to stimulate neutrophils.Use the fluorescence DNA quantitative method to observe the NETosis under the stimulations of CSEs of different concentrations.(2)Stimulate the sample with PMA and CSE.Measure the formation of neutrophils in each group by fluorescence DNA quantitative method.Use immunofluorescence staining to observe the formations of NETs on each sample,as well as the morphological features and structures of NETs induced by CSE on COPD sample..(3)Under the precondition of using 0.1% CSE to stimulate unanimously,use erythromycins of concentrations 1ug/mL,5ug/mL and 10ug/mL to intervene with the samples,then use the fluorescence DNA quantitative method to observe the inhibitory effect by erythromycin on NETosis of neutrophils with the induction of CSE,under each concentration of erythromycin.(4)While stimulating with CSE,intervene with erythromycin and DPI separately and then discuss the impact of erythromycin on the formation of NETs by neutrophils under the stimulus of CSE.Result(1)0.1%-0.5% CSE group stimulates polymorphonuclear leukocytes to produce NETs concentrations(526.1±182.7 ng/ml,424.8±103.1 ng/ml,381.2±82.2 ng/ml)compared with normal control group(299.8±72.0ng/ml)increased,P<0.05;the concentration of NETs(362.1±117.5 ng/ml,351.8±117.6 ng/ml)in 1% CSE and 2% CSE stimulation group is not obviously different from that in the normal group(299.8±72.0 ng/ml),P>0.05;(2)CSE can stimulate the production of NETs in human neutrophils,。NETs produced by neutrophils induced by cigarette smoke stimuli also contain NE and HMGB1,in the absence of any stimulation,there is no apparent difference in the concentration of NETs produced(265.7±74.1 ng/ml)in the normal person compared with the COPD group(323.8±79.7 ng/ml)P>0.05;under the stimulation of PMA,the COPD(433.5±98ng/ml)and the normal group(314.5±74.1ng/ml)generate obviously more NETs than the normal control group,in which the difference is statistically significant,P<0.05;under the stimulation of CSE,the amount of NETs produced in the COPD group(439.7±121.8 ng/ml)compared with the normal group(333.3±84.2 ng/ml)is statistically different,P<0.05;(3)There is no significant difference in NETs-DNA content(282.2±117.8ng/ml)produced by 1μg/ml erythromycin compared with the normal group(364.8±145ng/ml),P>0.05;compared with the normal group(364.8±145ng/ml),the release of NETs is significantly decreased in the 5μg/ml erythromycin group(248.9±86ng/ml)and the 10μg/ml erythromycin group(257.4±90.3ng/ml).The difference is statistically significant,P<0.05;(4)10μg/ml erythromycin(249.2±82.5ng/ml)is added in the normal group and DPI intervention(251.5±67.7ng/ml)of CSE stimulation group are compared with the stimulation control group(333.3±84.2ng/ml),in terms of the number of both NETs release decreasing,the difference is statistically significant,P<0.05;likewise,the NETs-DNA concentration in the erythromycin intervention group(337.9±75.4ng/ml)and the DPI intervention group(303.4±44.6ng/ml)in the COPD group is also lower than that in the simple stimulation group(439.7±121.8ng/ml),with statistical significance,P<0.05.Conclusion(1)CSE can stimulate the production of NETs in human neutrophils,and the concentration of NETs produced by different concentrations of CSE stimulation first increases and then decreases.(2)Neutrophils in patients with COPD are more likely to be NETosis than normal people after CSE stimulation.Neutrophils in patients with COPD are more likely to be NETosis than normal people after CSE stimulation.(3)There is a dose-effect relationship between erythromycin and its inhibition of tobacco smoke induced NETs production.(4)Erythromycin inhibits the progression of NETosis induced by CSE stimulation of COPD neutrophils.