p38 MAPK抑制剂通过抑制NLRP3途径介导的细胞焦亡对小鼠慢性阻塞性肺疾病损伤的改善作用  

Improvement effect of p38 MAPK inhibitor on chronic obstructive pulmonary disease injury in mice through inhibiting cell pyrotosis mediated by NLRP3 pathway

作　　者：李明 王秋婷[1] 陈山[1] 石慧芳[1] LI Ming;WANG Qiuting;CHEN Shan;SHI Huifang(Department of Respiratory Medicine,Second Affiliated Hospital,Hainan Medical College,Haikou 570311,China)

机构地区：[1]海南医学院第二附属医院呼吸内科,海南海口570311

出　　处：《吉林大学学报（医学版）》2022年第3期744-754,共11页Journal of Jilin University：Medicine Edition

基　　金：海南省卫健委卫生健康行业科研项目(20A200056)。

摘　　要：目的:探讨p38丝裂原活化蛋白激酶(p38 MAPK)抑制剂SB303580(SB)对小鼠慢性阻塞性肺疾病(COPD)进展的影响,并阐明其可能的作用机制。方法:将48只C57BL/6小鼠分为对照组、SB组、COPD组和COPD+SB组,每组12只。COPD组和COPD+SB组小鼠给予香烟烟雾和脂多糖(LPS)建立COPD模型。吸入乙酰甲胆碱(Mch)后观察各组小鼠气道阻力以评价各组小鼠肺功能,HE染色观察各组小鼠肺组织病理形态表现,细胞计数法和ELISA法检测各组小鼠肺泡灌洗液(BALF)中白细胞总数、中性粒细胞、巨噬细胞和淋巴细胞数及肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、白细胞介素1β(IL-1β)和白细胞介素18(IL-18)水平,体外应用烟草提取物(CSE)刺激小鼠巨噬细胞系RAW264.7细胞,将细胞分为对照组、SB组、CSE组和SB+CSE组。细胞免疫荧光染色观察各组巨噬细胞中NOD样受体蛋白3(NLRP3)及裂解型半胱氨酸蛋白酶3(cleaved caspase-3)的表达,流式细胞术检测各组细胞焦亡率和早期凋亡率,Western blotting法检测各组小鼠肺组织和巨噬细胞中磷酸化p38(p-38)、焦亡相关蛋白和核因子κB(NF-κB)信号通路相关蛋白表达水平。结果:成功构建COPD小鼠模型。与对照组比较,COPD组和COPD+SB组小鼠气道阻力,BALF中白细胞总数、中性粒细胞、巨噬细胞和淋巴细胞数量及TNF-α、IL-6、IL-1β和IL-18水平以及肺组织中p-p-38、NLRP3、半胱氨酸蛋白酶1(caspase-1)、凋亡相关斑点样蛋白(ASC)、NF-κB p65及Toll样受体4(TLR4)蛋白表达水平均明显升高(P<0.05或P<0.01);肺组织结构明显受损,气道上皮细胞脱落,部分相邻肺泡融合至肺大泡等病理学改变。与对照组比较,SB组小鼠气道阻力,BALF中白细胞总数、中性粒细胞、巨噬细胞和淋巴细胞数及TNF-α、IL-6、IL-1β、IL-18水平和肺组织中NLRP3、caspase-1、ASC、NF-κB p65及TLR4蛋白表达水平均无明显改变(P>0.05);肺组织结构正常,但肺组织中p-p-38蛋白表Objective:To investigate the effect of p38 mitogen-activated protein kinase(p38 MAPK)inhibitor SB303580(SB)on the progression of chronic obstructive pulmonary disease(COPD)in the mice,and to elucidate its possible mechanism.Methods:A total of 48 C57BL/6 mice were divided into control group,SB group,COPD group,and COPD+SB group,and there were 12 mice in each group.The mice in COPD group and COPD+SB group were given cigarette smoke and lipopolysaccharide(LPS)to establish the COPD models.The airway resistance after inhalation of methacholine(Mch)was observed to evaluate the lung function of the mice in various groups,and the pathomophology of lung tissue of the mice in various groups was observed by HE staining.The total number of white blood cells,neutrophils,macrophages and lymphocytes,the levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),interleukin-1β(IL-1β),and interleukin-18(IL-18)in pulmonary alveolar lavage fluid(BALF)of the mice in various groups were detected by cell counting and ELISA method.The mouse macrophage RAW264.7 cells were stimulated by cigaritte smoke extract(CSE)in vitro,and the cells were divided into control group,SB group,CSE group and SB+CSE group.Cell immunofluorescence staining was used to observe the expressions of NOD-like receptor protein 3(NLRP3)and cleaved caspase-3 in the macrophages in various groups,and flow cytometry was used to detect the pyroptotic rates and early apoptotic rates of the cells in various groups,and Western blotting method was used to detect the expression levels of phosphorylated p-38(p-p-38),pyroptosis-related and nuclear factorκB(NF-κB)signaling pathway related poteins in the macrophages in various groups.Results:The COPD mouse models were successfully established.Compared with control group,the airway resistance of the mice in COPD group and COPD+SB group,the total number of white blood cells,neutrophils,macrophages and lymphocytes in BALF,the levels of TNF-α,IL-6,IL-1β,IL-18 in BALF,and the expression levels of p-p-38,NLRP3,cysteine protea

关 键 词：慢性阻塞性肺疾病 p38丝裂原活化蛋白激酶抑制剂SB303580 细胞焦亡 细胞凋亡 巨噬细胞 

分 类 号：R563.13[医药卫生—呼吸系统]