烟熏对大鼠肺泡巨噬细胞Dectin-1受体表达及对烟曲霉感染应答的影响  

Effects of cigarette smoke on rat alveolar macrophages Dectin-1 receptor expression and anti-Aspergillus defense

作　　者：邵宏涛 陈玉宝[2] 孙禾 施毅 Shao Hongtao;Chen Yubao;Sun He;Shi Yi(Department of Geriatric,Integrated Traditional Chinese and Western Medicine Hospital affiliated to Nanjing University of Chinese Medicine,Jiangsu Province Academy of Traditional Chinese Medicine,Nanjing 201128,China;Department of Respiratory and Critical Care Medicine,Nanjing First Hospital,Nanjing Medical University,Nanjing 210006,China;Department of Respiratory and Critical Care Medicine,Shanghai Eastern Hospital,Shanghai Tongji University,Shanghai 200080,China;Department of Respiratory and Critical Care Medicine,Nanjing Jinling Hospital,Medical School of Nanjing University,Nanjing 210002,China)

机构地区：[1]南京中医药大学附属中西医结合医院老年科,江苏省中医药研究院,南京201128 [2]南京医科大学附属南京医院(南京市第一医院)呼吸与危重症医学科,南京210006 [3]上海同济大学附属上海东方医院呼吸与危重症医学科,上海200080 [4]南京大学医学院附属金陵医院呼吸与危重症医学科,南京210002

出　　处：《国际呼吸杂志》2022年第18期1382-1391,共10页International Journal of Respiration

基　　金：国家自然科学基金(81200004);南京市医学科技发展重点项目(ZKX13014)。

摘　　要：目的探讨香烟烟雾提取物(CSE)对肺泡巨噬细胞(AMs)Dectin-1受体表达及对烟曲霉感染应答能力的影响。方法实验研究。大鼠肺泡巨噬细胞株NR8383加入CSE后再使用烟曲霉静止期孢子(RC)刺激以建立烟曲霉体外感染细胞模型,流式细胞仪、激光共聚焦显微镜、蛋白质印迹检测Dectin-1表达;siRNA沉默和慢病毒载体过表达Dectin-1后通过吞噬实验检测AMs对RC吞噬能力,定量实时聚合酶链反应、酶联免疫吸附试验检测相关细胞因子表达。结果激光共聚焦显微镜(15.29±2.88比24.56±4.01,t=11.75,P<0.05)及流式细胞仪(17.73±4.95比25.94±7.12,t=17.27,P<0.05)均显示CSE降低AMs表面Dectin-1受体的表达。RC体外刺激后Dectin-1 mRNA的表达出现时间依赖性增加,对照组在所有时间点都较CSE组显著增加(P值均<0.05);蛋白表达结果类似:对照组明显高于CSE组(18 h:0.755±0.035比0.360±0.047;24 h:0.968±0.035比0.552±0.049,P值均<0.05)。CSE降低AMs对RC吞噬能力(吞噬率:53.33%±9.95%比75.17%±8.66%;吞噬指数:2.17±0.58比7.67±1.53,P值均<0.05)。siRNA下调Dectin-1表达联合CSE可以进一步降低NR8383细胞对RC的吞噬力(P<0.05),但单纯下调Dectin-1表达对RC的吞噬能力差异无统计学意义(P>0.05)。Dectin-1介导细胞因子肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)和IL-4的产生:TNF-α、IL-1β在对照组增加尤为明显,感染6 h后达峰值,每个时间点比较差异均有统计学意义(P值均<0.05)。与之相反,IL-4在CSE组升高更为显著,每个时间点比较差异均有统计学意义(P值均<0.05)。IL-10表达也增加,但2组间比较差异无统计学意义(P>0.05)。使用siRNA抑制/慢病毒过表达Dectin-1对4种细胞因子mRNA和蛋白产生同向作用。结论CSE可引起AMs Dectin-1受体低表达和功能障碍,导致烟曲霉感染后吞噬能力降低和细胞因子异常表达。CSE引起的AMs抗曲霉防御能力下降部分是通过Dectin-1介导的。Objective To investigate the effect of cigarette smoke extract(CSE)on the expression of Dectin-1 receptor in alveolar macrophages(AMs)and the ability of responding to Aspergillus fumigatus infection.Methods It is an experimental research.After smoking the rat alveolar macrophage cell line NR8383,an infected cell model of Aspergillus fumigatus was established in vitro.Flow cytometry,laser confocal microscopy,and Western Blot were used to detect Dectin-1 expression;phagocytic ability to resting conidia(RC)was detected by phagocytosis experiment following siRNA silencing and lentiviral vector over-expressing Dectin-1,and the expression of related cytokines was detected by qRT-PC and ELISA.Results We found that CSE decreased the Dectin-1 receptor expression on AMs surfaces both by laser confocal microscopy(15.29±2.88 vs 24.56±4.01)and by flow cytometry(17.73±4.95 vs 25.94±7.12)(P<0.01).Dectin-1 mRNA expression increased in a time-dependent manner after RC stimulation in vitro.Compared with the CSE group,expression of Dectin-1 in the control group was much higher at all time points(P<0.05).Western blot analysis showed the similar results(18 h:0.755±0.035 vs 0.360±0.047;24 h:0.968±0.035 vs 0.552±0.049;P<0.01).CSE decreased the phagocytic ability of AMs to RC(phagocytosis rate:53.33%±9.95%vs 75.17%±8.66%;phagocytic index:2.17±0.58 vs 7.67±1.53,P<0.01).The cigarette smoke-exposed NR8383 cells that had been transfected with the siDectin-1 were more impaired in the phagocytosis of RC than the control group(P<0.05).However,there showed no statistical significance only by knockdown experiment(P>0.05).We found that TNF-α,IL-1β,IL-10,and IL-4 were activated in a time-dependent manner after RC simulation both at transcriptional and translational levels.Increase in TNF-αand IL-1βexpressions were much greater in the control group than in the CSE group(all P<0.05),while opposite result was noted for IL-4(P<0.05).No difference between the two groups was noted for IL-10(P>0.05).Using siRNA to suppress and lentivirus

关 键 词：巨噬细胞 肺泡 DECTIN-1 烟曲霉 吸烟 

分 类 号：R519[医药卫生—内科学]