| Objective To investigate peripheral blood of people,A549 and BEAS-2B cells in Cigarette smoke extract(CSE)treated PDE4 D,PGE2 and other proteins were examined to investigate the role of PDE4 D gene in the development of COPD.Methods According to the 2017 GOLD,69 COPD patients and 62 healthy people who were examined during the same period were selected from our hospital from June 2018 to January 2019.In A549 and BEAS-2B,cells were divided into a treatment group and an untreated group with 3% CSE and 10% CSE,respectively.After treatment for 48 hours,1 ?M and 2 n M of roflumilast were added to the treatment group,respectively.Detection of phosphate in peripheral blood and cells by Real-time Quantitative PCR Detecting System(q PCR),enzyme linked immunosorbent assay(ELISA),and Western Blot The expression of diesterase 4D(phosphodiesterase 4D,PDE4D),prostaglandin E2(Prostaglandin E2,PGE2),adenosine-3 ',5'-cyclized monophosphate(c AMP).Results Compared with the control group,the PDE4 D gene expression in COPD patients increased at the blood level(1.098±0.3033 N = 62 vs 2.463 ± 0.2592 N = 69,p<0.001).The ELISA test results showed that PDE4D(2.763± 0.3616 N = 62 vs 5.312±1.034 N = 69,p<0.05),PGE2(998.1±78.96 N = 39 vs 1631±224.9 N = 43,p<0.05)expression increased,c AMP expression decreased(1975±437.2 N = 39 vs 975.4 ± 119.9 N = 43,p<0.05);at the cell level,the A549 group: the q PCR detection treatment group increased the expression of PDE4 D gene(1.015±0.1278 vs 12.39±1.158,p<0.001),PDE4 D gene expression decreased after after adding 2n M roflumilast(12.39±1.158 vs 3.360±0.4531,p<0.01),and adding 1 ?M roflumilast(12.39±1.158 vs 1.200 ± 0.2212,p<0.001),PDE4 D gene expression decreased;Compared with the untreated group,the expression of PDE4 D protein in the 3% CSE treated group was increased by ELISA(10.07±0.01522 vs 13.12 ± 0.3932,p<0.01).Compared with the 3% CSE treated group,after adding 2n M roflumilast,there was no significant change in the expression of PDE4 D protein(13.12±0.3932 vs 11.99±0.7162,p>0.05).The expression of PDE4 D protein decreased after adding 1 ?M roflumilast(13.12±0.3932 vs 9.812±0.1830,p<0.01);3% CSE The expression of PGE2 increased in the treatment group(587.3±65.66 vs920.9±58.62,p<0.01),adding 2n M roflumilast(920.9±58.62 vs 720.4±35.65,p<0.05)and 1?M roflumilast(920.9±58.62 N = 12 vs 538.7±22.55 N = 9,p<0.001),the expression of PGE2 all decreased;the expression of c AMP in the 3% CSE treatment group decreased(3.547±0.04364 vs 0.8125 ± 0.1084,p<0.001),and 2n M roflumilast was added After sterastre(0.8125±0.1084 vs 1.250±0.08699,p<0.05)and 1 ?M roflumilast(0.8125±0.1084 vs 5.719 ±0.08714,p<0.001),the expression of c AMP protein increased.BEAS-2B group: The expression of PDE4 D gene in the 10% CSE treatment group was increased by real-time quantitative PCR(1.030±0.1677 vs 7.890±1.117,p<0.01).Compared with the 10% CSE treatment group,after adding 2n M roflumilast,There was no significant change in the expression of PDE4 D gene(7.890±1.117 vs 7.950±0.7797,p<0.05).After adding 1 ?M roflumilast,the expression of PDE4 D gene decreased(7.890±1.117 vs 1.352±0.1448,p<0.01);ELISA test Compared with the untreated group,the PDE4 D protein expression increased in the 10% CSE treated group(10.31±0.5739 vs 16.72±1.590,p<0.05).After adding 2n M roflumilast,the PDE4 D protein expression did not change significantly(16.72±1.590 vs15.76±0.9673,p>0.05),PDE4 D protein expression decreased after adding 1 ?M roflumilast(16.72±1.590 vs 11.57±0.5055,p<0.05);PGE2 expression increased in the 10% CSE treatment group(502.3±43.98 vs 705.0±85.05,p<0.05).Compared with 10% CSE-treated PGE2,the addition of 2n M roflumilast did not significantly change the expression of PGE2(705.0±85.05 vs 808.2±61.87,p>0.05).After sterectomy,PGE2 expression decreased(705.0±85.05 vs 381.5±56.52,p<0.05);c AMP expression decreased in the 10% CSE treatment group(5.914±0.01393 vs 3.989±0.1717,p<0.001),after adding 2n M roflumilast(3.989±0.1717 vs 3.979±0.2480,p>0.05),there was no significant change in the expression of c AMP.After the addition of 1 ?M roflumilast,the expression of c AMP increased(3.989±0.1717 vs 5.558±0.1174,p<0.01).The expression of PDE4 D protein detected by Western blotting showed that the expression of PDE4 D protein in the A549 3% CSE treated group(1.252 ± 0.06915)was higher than that in the untreated group(0.9388 ± 0.08469)(p<0.05).The 2n M roflumilast treatment group(0.8970 ± 0.04827)and the 1?M roflumilast-treated group(0.7987 ± 0.09246)showed a decrease in the expression of PDE4 D protein(p<0.05).In BEAS-2B,the expression of PDE4 D in the 10% CSE-treated group(2.014 ± 0.1574)was not significantly different from the untreated group(1.951 ± 0.1353)(p> 0.05),After adding 2n M roflumilast(1.821 ± 0.01671)or 1 ?M roflumilast(1.594 ±0.2107),it failed to cause significant changes in PDE4 D protein expression(p>0.05).Conclusion(1)Compared with healthy controls,PDE4 D expression in peripheral blood of patients with COPD increased significantly.(2)CSE can provide evidence for the clinical use by simulating the COPD caused by smoking.(3)CSE induce up-regulation of PDE4 D and PGE2 expression,resulting in decreased c AMP expression,so CSE may beinvolved in the pathogenesis of COPD by regulating the expression of factors such as PGE2?c AMP on the pathway and related pathways.? Roflumilast is currently used as a PDE4 inhibitor in clinic.This study found that it can effectively inhibit CSE induce PDE4 D upregulation to treat COPD,so PDE4 D may become an important target for the treatment of COPD in the future,but further research is needed to explore the more precise pathway PDE4 D expression changes leading to the pathogenesis of COPD,so that the effective treatment of COPD can reduce the side effects and adverse reactions caused by drugs at the same time. |
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