尼古丁通过增强自噬减轻脂多糖诱导的急性肺损伤炎症反应  

Nicotine alleviates LPS-induced inflammatory response in acute lung injury mice by enhancing autophagy

作　　者：袁关利 刘银凤[2] 韩卓笑 孟爱宏[1] Yuan Guanli;Liu Yinfeng;Han Zhuoxiao;Meng Aihong(Department of Respiratory and Critical Care Medicine,the Second Hospital of Hebei Medical University,Shijiazhuang 050000,China;Department of Breast Surgery,the First Hospital of Qinhuangdao,Qinhuangdao 066000,China;Department of Respiratory and Critical Care Medicine,the First Hospital of Qinhuangdao,Qinhuangdao 066000,China)

机构地区：[1]河北医科大学第二医院呼吸与危重症医学科,石家庄050000 [2]秦皇岛市第一医院乳腺外科,秦皇岛066000 [3]秦皇岛市第一医院呼吸与危重症医学科

出　　处：《国际呼吸杂志》2022年第13期989-996,共8页International Journal of Respiration

基　　金：河北省自然科学基金资助项目(H2019206263);河北省重点研发计划项目(19277760D);河北省应用基础研究计划(15967753D)。

摘　　要：目的探讨尼古丁在脂多糖(LPS)诱导的急性肺损伤炎症反应中的作用及机制。方法本研究为实验研究。动物实验部分,应用LPS构建急性肺损伤小鼠模型,将C57BL/6J小鼠24只,按体质量排序,采用蛇形分组法分为4组,每组6只,对照组、LPS组、LPS+尼古丁组、LPS+尼古丁+3-MA组。留取肺组织行HE染色,肺泡灌洗液行酶联免疫吸附试验(ELISA)检测白细胞介素1β(IL-1β)水平。细胞实验部分,采用MH-S细胞系,分为4组,对照组、LPS组、LPS+尼古丁组、LPS+尼古丁+3-MA组,取细胞上清液行ELISA检测IL-1β水平,采用细胞裂解液提取总蛋白行蛋白免疫印迹法检测NLRP3、Pro-caspase1、Cleaved-caspase1、LC3BⅡ、LC3BⅠ、P62蛋白水平,免疫荧光检测LC3B表达,透射电镜观测自噬小体数量。结果LPS组较对照组,肺组织出现明显炎性细胞浸润。支气管肺泡灌洗液和MH-S细胞上清液中IL-1β水平升高;LPS+尼古丁组较LPS组,肺组织炎性细胞浸润减轻,支气管肺泡灌洗液和细胞上清液中IL-1β水平降低;LPS+尼古丁+3-MA组较LPS+尼古丁组,肺组织炎性细胞浸润加重,支气管肺泡灌洗液和MH-S细胞上清液中IL-1β水平升高,差异均有统计学意义(F值分别为446.40、656.40,P值均<0.001)。蛋白免疫印迹法检测各组MH-S细胞裂解液,LPS组较对照组NLRP3、Pro-caspase1、Cleaved-caspase1表达水平升高,LPS+尼古丁组较LPS组NLRP3、Pro-caspase1、Cleaved-caspase 1表达水平下降,LPS+尼古丁+3-MA组较LPS+尼古丁组NLRP3、Pro-caspase1、Cleaved-caspase1表达水平升高,差异均有统计学意义(F值分别为180.00、301.70、105.80,P值均<0.001)。使用透射电镜观察4组MH-S细胞自噬小体,采用蛋白免疫印迹法检测4组MH-S细胞中P62及LC3BⅡ/LC3BⅠ表达显示,与对照组比较,LPS组自噬小体数量增多,P62表达水平降低,LC3BⅡ/LC3BⅠ升高;LPS+尼古丁组较LPS组自噬小体数量增多,P62表达水平降低,LC3BⅡ/LC3BⅠ升高,LPS+尼古丁+3-MA组较LPSObjective To explore the effects and mechanism of nicotine on LPS-induced acute lung injury in mice.Methods This is an experimental study.Mice models for acute lung injury were established by LPS in the present study.The 24 C57BL/6J mice were sorted by body weight and divided into 4 groups based on the Serpentine grouping including the control group,LPS group,LPS+nicotine group,and LPS+nicotine+3-MA group with 6 mice in each group.Lung tissue was stained with H&E,and 1interleukin-11β(IL-1β)were detected by the enzyme-linked immunosorbent assay(ELISA)in bronchoalveolar lavage fluid.MH-S cells were divided into 4 groups including the control group,LPS group,LPS+nicotine group,LPS+nicotine+3-MA group,the supernatant of cells was detected for IL-1βlevel by ELISA,and the total protein extracted from cell lysate was detected by Western blot for confirming NLRP3,Pro-caspase1,Cleaved-caspase1,LC3BI,LC3BII,P62 protein levels,immunofluorescence was applied to detect the expression of LC3B,and transmission electron microscopy was applied to observe the number of autophagosomes.Results Compared with the control group,the evident inflammatory cell infiltration in the lung tissues and the increased IL-1βin bronchoalveolar lavage fluid and cell supernata were found in the LPS group.Compared with the LPS group,the less inflammatornt cell infiltration in the lung tissues and the decreased IL-1βin bronchoalveolar lavage fluid and cell supernatant were detected in the LPS+nicotine group.Compared with the LPS+nicotine group,the evident inflammatory cell infiltration in the lung tissues and the increased IL-1βin bronchoalveolar lavage fluid and cell supernata were found in the LPS+nicotine+3-MA group.and the differences were statistically significant(The F values were 446.40 and 656.40,respectively;all P<0.001).Western blotting was applied to detect MH-S cell lysates in all groups,the expression levels of NLRP3,Pro-caspase1,and Cleaved-caspase 1 were elevated in the LPS group than the control group,which statistically reduced i

关 键 词：急性肺损伤 自噬 NLRP3炎症小体 尼古丁 模型 动物 

分 类 号：R563[医药卫生—呼吸系统]