Protective Effect Of Dexmedetomidine Against Lung Injury In COPD Via MiRNA-146a

BackgroundChronic obstructive pulmonary disease(COPD)is a kind of lung tissue and airway injury,characterized by persistent airflow limitation in chronic bronchitis and emphysema(or),can progress to chronic pulmonary disease pulmonary heart disease and respiratory failure,and airway and lung tissue of toxic gases or harmful particles resulting in abnormal inflammatory reaction,high incidence,morbidity and mortality,causing serious social and economic burden.Dexmedetomidine(Dex)is a new type of highly selective alpha 2 adrenergic receptor agonist.Dexmedetomidine is commonly used in clinical anesthesia drugs.More and more clinical studies showed that dexmedetomidine can inhibit cell apoptosis and inflammatory reaction and oxidative stress reaction mechanism for the brain,heart,kidney,liver and lung and other organs,play a protective role,especially in the lung protective,the effect is most obvious.MiR-146a is a research hotspot in recent years,miR-146a targeting TLR4/dependent MyD88 pathway is an important part of TNF receptor associated factor 6(TRAF6)and interleukin 1 receptor associated kinase 1(IRAKI),the activation of TLR4 downstream signaling molecule of NF-kappa B,induced by inflammatory factors,such as the release of the expression of IL-6,IL-1 beta,TNF-alpha so,promote the inflammatory reaction cascade,involved in the pathogenesis of inflammation related COPD.Therefore,we put forward the hypothesis that dexmedetomidine can regulate the protective effect of miRNA-146a against lung injury in COPD,and its mechanism is not clear.This is the focus of this research,but also innovation.The objectives of this study include establishing the chronic obstructive pulmonary disease(COPD)in rat models for investigating the pathological basis,and clarifying the protective effects of dexmedetomidineagainst lung injury in COPD via miR-146a.PART 1 Establishment of COPD rat model and detection of lung function and histological examinationObjectiveThe establishment of animal model of COPD in rat lung,by observing the rats general condition,pulmonary function test,blood gas analysis,bronchoalveolar lavage fluid detection and pathological examination of lung tissue,clear pathological basis of lung injury in COPD.Methods16 SD rats were randomly divided into two groups:normal control group(n = 8)and COPD group(n = 8).The model of COPD lung injury in rats was established by smoke inhalation method.The pulmonary functions,including tidal volume(TV),peak expiratory flow(PEF),forced expiratory flow at 50%(EF 50),forced expiratory volume in 0.3 seconds(FEV0.3)and FEV0.3 and forced vital capacity ratio(FEV0.3/FVC),were detected.Analysis of arterial oxygen partial pressure(PaO2)and partial pressure of carbon dioxide(PaCO2)were performed by arterial blood gas analyzer.Rat bronchial lavage fluid was collected,and the cells were counted and classified.The lung tissue was taken and the wet/dry weight ratio was measured,the tissue sections were examined and the pathological examination was performed.Results1.General conditionThe rats in the control group had normal diet and weight gain.smooth hair and no obvious respiratory symptoms;rats in COPD group gradually appeared anorexia,weight loss,hair dark,dull,reduced activity,and the emergence of respiratory symptoms such as sneezing,wheezing and increased respiratory rate.2.Pulmonary functionThe TV of rats in control groupwas 2.65±0.21 mL,the value of PEF was 38.55±0.24mL/s,the value of EF 50 was 1.81±0.06 mL/s,the value of FEV0.3 was 4.44±0.26 mL,the value of FEV0.3/FVCwas 88.4510.34%;the TV of COPD ratswas 1.26±0.17 mL,the value of PEF was 17.61±0.35 mL/s,the value of EF 50 was 1.20±0.14 mL/s,the value of FEV0.3 was 2.52±0.28 mL,the value of FEV0.3/FVC was 63.39±0.22%.The lung function of rats in COPD group was higher than that of control group.The TV,PEF,EF 50,FEV0.3 and FEV0.3/FVC of rats in COPD group were significantly lower than those in control group(p<0.05).3.Arterial blood gas analysisThe value of PaO2in the control groupwas 89.35±4.30 mmHg,and the value of PaO2in the COPD groupwas 73.12±5.11 mmHg.The value of PaO2in the COPD ratswas significantly lower than the value of PaO2in the control group(p<0.05).The value of PaCO2 in the control groupwas 43.22±5.19 mmHg,and the value of PaCO2jn the COPD groupwas 56.36±6.71 mmHg.The value of PaCO2in the COPD groupwas significantly higher than that in the control group(p<0.05).4.Bronchoalveolar lavage fluid(BALF)cell count and protein testCompared to the control group,the total number of white blood cells in the BALF of COPD rats were significantly increased(2.33±1.19 ×108/L vs.1.45±0.41×108/L,p<0.05),and the percentage of neutrophils was significantly higher(17.4±7.2%vs.8.6±3.4%,p<0.05),the percentageof macrophage decreased significantly(73.3±2.6%vs.83.4±1.1%.P<0.05)and there were no significant differences between the percentages of lymphocytes(8.1±2%vs.7.8±2.7%,p>0.05).The protein content of BALF in the control group rats was 193.19±33.21 mg/L,the protein content of BALF inthe COPD group was 363.93±41.38 mg/L.The protein content of BALF inthe COPD group was significantly higher than the protein content of BALF inthe control group(p<0.05).5.Wet/dry weight ratio(W/D)The color oflungsin the control group was pink,the W/D of lung was 4.02±0.39,however,the color of lungs in the COPD group was pale,and there were several pulmonary bulla on the surface of lung with different sizes,and the W/D of lung in the COPD group was 5.41±1.03.The W/D of COPD group was significantly higher than that of control group(p<0.05).6.Pathological testLung tissue sections in control group were microscopically normal trachea and bronchial epithelial cells of cilia is rich,neat,no shedding,at all levels of the bronchial wall is neat,no thickening and infiltration of inflammatory cells in the lumen,no inflammatory exudate,alveolar structural integrity,no obvious pathology expansion.The histological sections of lung tissues in COPD group exhibited the sawtooth-like thickening and hyperplasia of bronchial ciliated columnar epithelial,cilia lodging,submucosal glands hyperplasia,inflammatory cells infiltration and hyperplasia of goblet cells.The mucus was accumulated in the bronchial lumen.The connective tissues of the bronchial lumen were significantly proliferated and thickened with the infiltration of monocyte and lymphocyte.The alveolus exhibited different sizes and disorderded structure.The alveolar wall changed to be thin and disrupted.The alveolar space enlarged and some alveolus merged into a larger cavity.ConclusionThe smoke suction method can effectively establish a rat model of lung injury in COPD.The model can be used to clarify the pathological basis of typical lung injury in COPD.PART 2 Dexmedetomidine inhibited COPD apoptosis of alveolar epithelial cells in rats via reducing expression of miRNA-146aObjectiveThe study of dexmedetomidine reducing expression of miRNA-146a inhibits the apoptosis of alveolar epithelial cells of COPD rats,using flow cytometry,real-time quantitative PCR detection of miRNA-146a and apoptosis related factor p53 and Bcl-2 expression level change,in order to clear the effect of dexmedetomidine on the protective mechanism of COPD lung.Methods24 SD rats were randomly divided into three groups:blank control group(n = 8),COPD without dexmedetomidine treatment group(n=8)and COPD with dexmedetomidine treatment group(n=8),isolation,purification and cultivation of rat alveolar epithelial cells.Rat alveolar epithelial cells of blank control group are from normal control group.Rat alveolar epithelial cells of COPD without dexmedetomidine treatment group and COPD with dexmedetomidine treatment group are from COPD rat model group.Rat alveolar epithelial cells of COPD with dexmedetomidine treatment group were administered with 5?M dexmedetomidine and cultured for 3 days.Rat alveolar epithelial cells of blank control group and COPD without dexmedetomidine treatment group were administered with saline and cultured for 3 days.Cell apoptosis detection and real-time quantitative PCR detection were detecting to clear cell apoptosis and the expression of miRNA-146a and apoptosis related factor p53 and Bcl-2.Results1.Dexmedetomidine inhibits the apoptosisofalveolar epithelial cell in the lung injury COPDThe percentages of injury cells,necrotic cells and apoptotic cells in COPD without dexmedetomidine treatment group and COPD with dexmedetomidine treatment group were significantly higher than that in the normal control group(p<0.05),while thepercentages of normal survival cells decreased significantly(p<0.05);the percentages of injury cells and apoptotic cells in COPD with dexmedetomidine treatment groupwere significantly lower than that in COPD without dexmedetomidine treatment group(p<0.05),however,the percentage of normal cellsin the COPD with dexmedetomidine treatment group was significantly higher than that in the COPD without dexmedetomidine treatment group(p<0.05).The rate of apoptotic cells in alveolar epithelial of COPD with dexmedetomidine treatment group was significantly lower than that in COPD without dexmedetomidine treatment group(11.15±0.51 vs 30.19±1.61%,p<0.05).2.Dexmedetomidine inhibits the expression of miRNA-146aThe results of real-time quantitative PCR detection showed that the expression of miRNA-146a in the blank control group was significantly lower than that in the COPD with dexmedetomidine treatment group and COPD without dexmedetomidine treatment group(p<0.05),but the expression of miRNA-146a in the COPD with dexmedetomidine treatment groupwas significantly lower than that of COPD without dexmedetomidine treatment group(p<0.05).3.Dexmedetomidine inhibits the expression of p53Real-time quantitative PCR detection results showed that the expression of p53 in the blank control group was significantly lower than that in the COPD with dexmedetomidine treatment group and the COPD without dexmedetomidine treatment group(p<0.05),however,the expression of p53 in the COPD with dexmedetomidine treatment group was significantly lower than that in the COPD without dexmedetomidine treatment group(p<0.05).4.Dexmedetomidine inhibits the expression of Bcl-2Real-time quantitative PCR detection results showed that the expression of Bcl-2 in the blank control group was significantly lower than that in the COPD with dexmedetomidine treatment group and,the COPD without dexmedetomidine treatment group(p<0.05),however,the expression of Bcl-2 in the COPD with dexmedetomidine treatment group was significantly lower than that in the COPD without dexmedetomidine treatment group(p<0.05).ConclusionMiRNA-146a mediats the apoptosis of alveolar epithelial cells by p53 and Bcl-2.Dexmedetomidine can effectively reduce the expression of miRNA-146a to inhibitthe apoptosis of lung parenchyma cells of COPD rat model for protecting the lung tissue.