慢性阻塞性肺疾病(COPD)模型大鼠肺组织中NLRP3炎症小体表达水平的变化及意义

Changes and Significance of NLRP3 Inflammasome in the Rats Model of Chronic Obstructive Pulmonary Disease(COPD)

目的探讨NOD样受体热蛋白结构相关蛋白3(NLRP3)炎症小体在慢性阻塞性肺疾病(COPD)模型中的变化及意义。方法将30只雄性大鼠随机分为2组,正常对照组(n=10只)给予自由饮水和摄食,喂养75天;吸烟组(n=20只)采用单纯吸入香烟烟雾的方法制造大鼠COPD模型,连续75天后再根据大鼠最大呼气流速(PEF)及气道肺组织病理变化分为吸烟COPD组(n=9只)和吸烟非COPD组(n=11只)。收集支气管肺泡灌洗液(BALF)计算巨噬细胞(AMC)、中性粒细胞(NEU)和淋巴细胞(LYM)的绝对计数。采用酶联免疫吸附试验(ELISA)、逆转录-聚合酶链反应(RT-PCR)技术分别测定BALF,肺组织中NLRP3炎症小体的浓度以及NLRP3mRNA的相对表达量。结果与正常对照组比较,吸烟非COPD组、吸烟COPD组BALF中NEU(×106/L)(524.2±73.1,916.9±84.8vs 106.6±31.8),AMC(×106/L)(1 570.9±273.5,2 307.8±410.4vs 496.6±61.7)和LYM(×106/L)(72.1±14.7,115.4±23.8vs 21.6±4.2),计数明显升高,吸烟COPD组BALF中NEU,AMC和LYM计数亦较吸烟非COPD组明显升高(F=350.59,100.57,82.63,均P<0.05)。与正常对照组比较,吸烟非COPD组、吸烟COPD组BALF(pg/ml)(107.8±23.5,126.7±34.9vs 76.7±15.1),肺组织(pg/ml)(118.8±33.3,147.4±40.2vs 84.1±21.5)中NLRP3炎症小体的浓度明显升高,与吸烟非COPD组比较,吸烟COPD组BALF,肺组织中NLRP3炎症小体的浓度亦明显升高(F=9.53,9.17,均P<0.05)。吸烟非COPD组和吸烟COPD组肺组织NLRP3mRNA相对表达量均显著高于正常对照组(P<0.05),吸烟COPD组肺组织NLRP3mRNA相对表达量显著高于吸烟非COPD组(P<0.05)。BALF,肺组织中NLRP3炎症小体浓度、肺组织NLRP3mRNA相对表达量与NEU,AMC,LYM计数均呈显著正相关(P<0.05)。结论 NLRP3炎症小体参与了COPD慢性炎症的发生、发展,与肺组织炎症细胞以及气道病理改变密切相关,阻断NLRP3炎性小体的活化,有望成为治疗COPD新的靶点和方向。

Objective To explore the changes and significance of NLRP3 inflammasome in the rats of chronic obstructive pulmonary disease(COPD).Methods 30 male rats were divided randomly into two groups.Control group(n=10)was given with drink and eat freely for 75 days.Smoking group(n=20)was given with inhaling cigarette smoke to establish rat model of COPD.After 75 days,the rats in smoking group were divided into smoking COPD group(n=9)and smoking non-COPD group(n=11)according to the maximum expiratory velocity(PEF)and pathologic changes of airway lung tissue.Bronchoalveolar lavage fluid(BALF)was collected to calculate the count of macrophages(AMC),neutrophils(NEU),and lymphocyte(LYM).Enzyme-linked immunosorbent(ELISA),reverse transcription-polymerase chain reaction(RT-PCR)was used to detect the NLRP3 inflammasome in BALF and lung tissue,and the relative expression of NLRP3 mRNA.ResultsCompared with control group,the count of NEU(×106/L)(524.2±73.1,916.9±84.8 vs 106.6±31.8),AMC(×106/L)(1 570.9±273.5,2 307.8±410.4 vs 496.6±61.7)and LYM(×106/L)(72.1±14.7,115.4±23.8 vs 21.6±4.2)in BALF of smoking COPD group and smoking non-COPD group were increased significantly,these indicators were also increased significantly in smoking COPD group compared with smoking non-COPD groups(F=350.59,100.57 and 82.63,all P<0.05).Compared with control group,the count of NEU,AMC,LYM in BALF of smoking COPD group and smoking non-COPD group were increased significantly,these indicators were also increased significantly in smoking COPD group compared with smoking non-COPD groups(P<0.05).Compared with control group,the concentration of NLRP3 inflammasome in BALF(pg/ml)(107.8±23.5,126.7±34.9 vs 76.7±15.1),lung tissue(118.8±33.3 pg/ml,147.4±40.2 pg/ml vs 84.1±21.5 pg/ml)of smoking COPD group and smoking non-COPD group was increased significantly,and these indicators were also increased significantly in smoking COPD group compared with smoking non-COPD groups(F=9.53,9.17,all P<0.05).The NLRP3 mRNA relative expression in lung tissue of smoking COPD group and smoking non-COPD group was significantly lower than that of control group,and the NLRP3 mRNA relative expression in lung tissue of smoking COPD group was significantly lower than that of smoking non-COPD group(P<0.05).The concentration of NLRP3 inflammasome in BALF,lung tissue,and NLRP3 mRNA relative expression in lung tissue were significantly positive correlated with NEU,AMC and LYM count(P<0.05).Conclusion NLRP3 inflammasome involves in the occurrence and development of chronic inflammation of COPD,and closely related with inflammation cells of lung tissue and airway pathologic change,blocking the activation of NLRP3 inflammasome is expected to be as a new target and direction in the treatment of COPD.