MCC950干预对哮喘小鼠气道NLRP3炎症小体及气道黏蛋白Muc5ac表达水平的影响

Effect of MCC950 intervention on the expression levels of NLRP3 inflammasome and Muc5ac in airway of asthmatic mice

目的 观察NLRP3炎症小体拮抗剂MCC950干预对哮喘小鼠气道黏蛋白Muc5ac表达水平的影响,探讨NLRP3炎症小体在哮喘气道黏液高分泌中的作用及其机制。方法 无特定病原体级的6~8周龄的雌性BALB/c小鼠50只,随机分为正常对照组(NS组)、哮喘组(AS组)、MCC950低剂量干预组(ML组)、MCC950高剂量干预组(MH组)及地塞米松组(Dex组),每组10只。各组小鼠支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)分别行总细胞计数、瑞士–吉姆萨染色行白细胞分类计数、酶联免疫吸附试验检测白细胞介素(interleukin,IL)-18、IL-1β浓度;各组小鼠肺组织病理石蜡切片分别行伊红染色、阿尔新蓝–碘酸雪夫染色图像分析,免疫组织化学及实时荧光定量聚合酶链反应检测肺组织Muc5ac、NLRP3及Caspase-1蛋白表达水平及相对mRNA表达量。结果 与NS组比较,AS组小鼠BALF细胞总数、嗜酸粒细胞百分比、IL-18及IL-1β浓度升高(P<0.05),肺组织气道周围炎症细胞浸润评分、气道黏液物质阳性相对着色面积增加(P<0.05),肺组织Muc5ac、NLRP3及Caspase-1蛋白表达水平及相对mRNA表达量升高(P<0.05);与AS组比较,MCC950干预组小鼠上述指标均减低(P<0.05);与Dex组比较,MCC950干预组小鼠上述指标均升高(P<0.05);ML组与MH组比较,上述指标差异无统计学意义。结论 MCC950可能通过抑制NLRP3、Caspase-1、IL-18及IL-1β表达,下调气道黏蛋白Muc5ac表达,减轻气道黏液高分泌。

Objective To observe the effect of NLRP3 inflammasome inhibitor MCC950 intervention on airway Muc5ac level in asthmatic mice,and to explore the role and mechanism of NLRP3 inflammasome in asthmatic airway mucus hypersecretion.Methods A total of 50 SPF grade BALB/c female mice aged 6-8 weeks were randomly divided into normal control group(NS group),asthma model group(AS group),dexamethasone group(Dex group),MCC950 high-dose intervention group(MH group)and MCC950 low-dose intervention group(ML group),with 10 mice in each group.Furthermore,the bronchoalveolar lavage fluid(BALF)of mice in each group was counted by total cell count,associated with white blood cell different count.In addition,the concentrations of interleukin(IL)-18 and IL-1β in BALF were tested by enzyme-linked immunosorbent assay;The lung tissues were prepared into paraffin-embedded sections,which were then subject to hematoxylin-eosin(HE)staining,Alcian blue-periodic acid Schiff base staining and Masson staining to observe the pathological changes of lung tissues.Immunohistochemistry was used to detect the protein expression levels of Muc5ac,NLRP3 and caspase-1 in lung tissues.Real-time quantitative polymerase chain reaction was performed to detect the relative mRNA expressions of Muc5ac,NLRP3 and Caspase-1 in lung tissues.Results Compared with NS group,AS group showed significant increase in total cell count of BALF,the percentage of eosinophils,the infiltration score of inflammatory cells around the airway,the positive relative staining area of airway mucus and the deposition area of airway collagen fibers in mice(P<0.05),upregulated protein expression levels of Muc5ac,NLRP3 and Caspase-1 in lung tissues(P<0.05),elevated relative mRNA expressions of Muc5ac,NLRP3 and Caspase-1 in lung tissues(P<0.05),and raised concentrations of IL-18 and IL-1β in BALF(P<0.05).While compared with AS group,the above indicators were reduced in MH group and ML group(P<0.05).Moreover,in relative to Dex group,these indicators were increased in MH group ML group(P<0.05).In addition,no statistically significant difference was observed in the aforementioned indications between MH group and ML group.Conclusions MCC950 intervention can inhibit airway inflammation and airway mucus secretion in asthmatic mice.Its mechanism is speculated to be related to the suppression of NLRP3,Caspase-1,IL-18 and IL-1β expressions,downregulation of Muc5ac expression,and inhibition in airway mucus hypersecretion.