NLRP3抑制剂MCC950对中性粒细胞性哮喘气道炎症的影响及其作用机制

Effect of MCC950,an inhibitor of NLRP3 on neutrophilic asthma airway inflammation and relevant mechanism

目的探讨NLRP3抑制剂MCC950对中性粒细胞性哮喘气道炎症的影响及其作用机制。方法将BALB/C小鼠随机分为中性粒细胞性哮喘组(NA组)、干预组(MCC950组)和PBS对照组,NA组经皮下注射20μg卵清蛋白(ovalbumin,OVA)、75μL完全弗氏佐剂(complete Freund adjuvant,CFA)、25μL PBS的混合液,第21及22天均以1%OVA混悬液雾化吸入激发10 min;MCC950组处理同NA组,于雾化吸入前连续3 d经腹腔注射MCC950,50μg/g体重;PBS对照组于相同时间点给予相同剂量PBS经皮下/腹腔注射或雾化吸入。末次雾化后测定小鼠气道高反应性;末次雾化24 h后,制备小鼠肺泡灌洗液(bronchoalveolar lavage fluid,BALF),Wright-Giemsa染色法进行细胞分类计数,ELISA法测定IL-17A、IL-18和IL-1β的表达水平,比色法检测髓过氧化物酶(myeloperoxidase,MPO)活力;取小鼠肺组织,实时荧光定量PCR法检测NLRP3、caspase-1、IL-1β及RORγt mRNA的相对表达水平,并进行组织病理分析。结果与PBS对照组比较,NA组小鼠气道反应性明显增强(P<0.05);BALF中炎性细胞总数、中性粒细胞数量、巨噬细胞数量、IL-1β、IL-17A、IL-18的表达水平、MPO活力及肺组织NLRP3、IL-1β、RORγt mRNA相对表达量均显著升高(P均<0.01);支气管及小血管周围炎症细胞浸润明显增多,炎症指数明显升高(P<0.001)。与NA组比较,MCC950组小鼠气道阻力明显降低(P<0.05);BALF中炎性细胞总数、中性粒细胞数量、巨噬细胞数量、IL-1β、IL-17A及IL-18表达水平、MPO活力及肺组织IL-1β、RORγt mRNA相对表达量显著降低(P均<0.01),肺组织NLRP3、caspase-1 mRNA的相对表达量差异无统计学意义(P均>0.05);肺组织内炎性细胞渗出明显减轻,炎症指数明显降低(P<0.05)。结论MCC950可减轻中性粒细胞性哮喘气道炎症反应,其通过抑制NLRP3/IL-1β/IL-17A信号通路,减少下游炎症因子产生,降低气道高反应性而发挥作用。本实验为MCC950用于该类型哮喘的治疗提供了实验依据。

Objective To investigate the effect of MCC950 on neutrophilic asthma airway inflammation as well as the relevant mechanism.Methods BALB/c mice were divided randomly into three groups(PBS,NA and MCC950).The mice in NA group were injected s.c.with the mixture of 20μg ovalbumin(OVA),75μL complete Freund adjuvant(CFA)and 25μL PBS on day 0,and stimulated with PBS containing 1%OVA by inhalation for 10 min on days 21 and 22.The mice in MCC950 group were treated by the same method as that in NA group,while injected i.p.with MCC950 at a dosage of 50μg/g bodyweight for 3 consecutive days before inhalation.The mice in PBS group were treated with PBS at the same time points and the same dosages by subcutaneous/intraperitoneal injection or inhalation.The airway hyperresponsiveness of mice were measured after the last inhalation.The bronchoalveolar lavage fluid(BALF)of mice was prepared 24 h after the last inhalation,classifed and counted by Wright-Giemsa staining,determiend for the expression levels of IL-17A,IL-18 and IL-1βby ELISA and for myeloperoxidase(MPO)activity by colorimetry.The lung tissues of mice were collected,determined for the relative transcription levels of NLRP3,caspase-1,IL-1βand RORγt mRNAs by real-time fluorescent quantitative PCR,and analyzed for histopathology.Results Compared with those in PBS group,the airway responsiveness of mice in NA group was enhanced significantly(P<0.05),while the total number of inflammatory cells,numbers of neutrophils and macrophages,expression levels of IL-1β,IL-17A and IL-18 and MPO activity in BALF as well as the relative transcription levels of NLRP3,IL-1βand RORγt mRNAs in lung tissue incresaed significantly(each P<0.01);the inflammatory infiltration around bronchial tubes and small vessels as well as the inflammatory index increased significantly(P<0.001).However,compared with those in NA group,the airway hyperresponsiveness of mice in MCC950 group decreased significantly(P<0.05),while the total number of inflammatory cells,numbers of neutrophils and macrophages,expression levels of IL-1β,IL-17A and IL-18 and MPO activity in BALF as well as the relative transcription levels of IL-1βand RORγt mRNAs in lung tissue decreased significantly(each P<0.01),the relative transcription levels of NLRP3 and caspase-1 mRNAs showed no significant difference(each P>0.05);the inflammatory infiltration in lung tissue was relieved significantly,and inflammatory index decreased significantly(P<0.05).Conclusion MCC950 relieved the neutrophilic asthma airway inflammation by blocking the NLRP3/IL-1β/IL-17A signaling pathway and decreasing the production of downstream inflammatory factors and the airway hyperresponsiveness,which provided an experimental basis for the treatment of neutrophilic asthma.