Nrf2激活剂抑制慢性阻塞性肺疾病小鼠NLRP3炎症小体的活化

Nrf2 activator inhibits the activation of NLRP3 inflammasome in chronic obstructive pulmonary disease mice

目的探讨核因子E-2-相关因子2(Nrf2)-ARE通路的激活对慢性阻塞性肺疾病(COPD)模型小鼠中核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎症小体活性及其下游炎症因子白细胞介素1β(IL-1β)和IL-18表达的影响。方法采用鼻腔滴入脂多糖联合熏烟的方法构建COPD小鼠模型,腹腔注射萝卜硫素(SF,Nrf2激活剂)的方式进行药物干预。成组设计实验分为对照(NS)组、正常小鼠萝卜硫素干预(NS+SF)组、COPD造模(COPD)组和COPD小鼠萝卜硫素干预(COPD+SF)组。从小鼠的一般情况、肺组织的病理形态学观察以及支气管肺泡灌洗液(BALF)细胞分类计数、肺功能检测多个指标评价COPD小鼠模型以及Nrf2激活剂对COPD小鼠炎症细胞水平和病理改变的影响。运用酶联免疫吸附试验(ELISA)法检测小鼠外周血血清和BALF上清细胞因子IL-6、肿瘤坏死因子α(TNF-α)、IL-1β及IL-18的分泌。运用蛋白印迹(Western blot)法检测肺组织内Nrf2、NLRP3和半胱氨酸天冬氨酸蛋白酶1 p10亚基(Caspase-1 p10)蛋白的表达。结果与NS组比较,COPD组和COPD+SF组小鼠体质量差异均有统计学意义(P值均<0.01);BALF中白细胞以及各类炎症细胞数量增加(P值均<0.05);肺功能检测指标吸气峰流速、呼气峰流速、每分钟通气量、动态肺顺应性及第50毫秒用力呼气容积/用力肺活量均显著下降(P值均<0.01),气道阻力显著升高(P<0.01),符合COPD的临床特点。经药物干预2周后,与COPD组比较,COPD+SF组小鼠体质量增加(P<0.05);BALF中各类炎症细胞浸润均减少(P值均<0.05)。Western blot法证实干预2周后,萝卜硫素刺激Nrf2核蛋白表达增加,可明显抑制小鼠NLRP3和Caspase-1 p10蛋白的表达。ELISA法证实干预2周后,萝卜硫素可明显抑制小鼠IL-6、TNF-α、IL-1β及IL-18分泌。结论(1)Nrf2激活剂能够抑制COPD小鼠BALF中炎症细胞渗出和抑制COPD小鼠血清和BALF中TNF-α和IL-6炎症因子的分泌,减轻COPD小鼠肺组织病理损害。(2)Nrf2激活剂通过抑制NLRP3蛋白的表达来抑制炎症小体的活化,抑制血清和BALF中IL-1β和IL-18的分泌,减轻COPD小鼠炎症反应。

Objective To explore the effects of Nrf2-ARE signaling pathway on the activity of NLR pyrin domain-containing protein 3(NLRP3)inflammasome and the expression of downstream inflammatory cytokines interleukin-1β(IL-1β)and IL-18 in chronic obstructive pulmonary disease(COPD)model mice.Methods The experiment was divided into four groups:normal control(NS)group,normal mice with sulforaphane intervention(NS+SF)group,COPD model(COPD)group,and COPD mice with sulforaphane intervention(COPD+SF)group.The use of cigarettes smoked instillation of lipopolysaccharide combined method was used to establish a mouse model of COPD and the drug intervention was performed by intraperitoneal injection of sulforaphane(Nrf2-ARE pathway activator).Four indexes,the general condition of the mice,the pathological morphology of lung tissues,the bronchoalveolar lavage fluid(BALF)cells,and the pulmonary function testing,were used to evaluate the effects of COPD mouse model and Nrf2 activator on the changes of inflammatory cell level and pathology.The secretion levels of cytokines IL-1β,IL-18,IL-6 and tumor necrosis factor-α(TNF-α)in peripheral blood serum and alveolar lavage fluid were determined by enzyme linked immunosorbent assay.The protein contents of Nrf2,NLRP3 and Caspase-1 p10 in lung tissue were detected by Western blot.Results Compared with NS group,there were significant differences in body weight between COPD and COPD+SF group(both P<0.01).The number of white blood cells and various inflammatory cells increased in BALF(all P<0.05).Peak inspiratory flow,peak expiratory flow,minute volume,dynamic lung compliance and forced expiratory volume in 50 millisecond/forced vital capacity decreased significantly(all P<0.01),and resistance index value increased significantly(P<0.01),consistent with the clinical characteristics of COPD.After two weeks of drug intervention,compared with COPD group,COPD+SF group mice gained weight(P<0.05).The infiltration of various inflammatory cells in BALF was reduced(P<0.05).Western blot confirmed that after two weeks of intervention,sulforaphane stimulated the expression of Nrf2 nuclear protein and significantly inhibited the expression of NLRP3 and Caspase-1 p10 in mice.After two weeks of intervention,sulforaphane significantly inhibited the secretion of IL-6,TNF-α,IL-1β and IL-18.Conclusions(1)Nrf2 activator can inhibit the exudation of inflammatory cells in BALF of COPD mice and the secretion of TNF-α and IL-6 inflammatory factors in serum and BALF of COPD mice,thus reducing the pathological damage of lung tissue in COPD mice.(2)Nrf2 activator inhibits the activation of NLRP3 inflammasome by inhibiting the expression of NLRP3 protein and the secretion of IL-1β and IL-18 in serum and BALF,thus reducing the inflammatory response in COPD mice.