尼古丁在诱导的肺泡巨噬细胞自噬及肺炎中的作用
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摘要
目的:探讨尼古丁在诱导的小鼠肺泡巨噬细胞自噬及肺炎发生中的作用。方法:通过碘化丙啶(PI)/Hochest33258染色法检测尼古丁诱导巨噬细胞MH-S死亡;通过Western印迹和电子透射显微镜检测尼古丁诱导MH-S细胞自噬的发生;采用中性红摄取实验检测尼古丁处理后巨噬细胞的吞噬能力;通过攻毒实验检测尼古丁诱导的肺炎。结果:不同浓度尼古丁作用下PI染色的死细胞呈上升趋势;LC3BⅡ蛋白表达具有尼古丁剂量依赖性,并且1μmol/L的尼古丁能够增强LPS预刺激的MH-S细胞自噬发生。电子透射显微镜结果显示在1μmol/L尼古丁的作用下,细胞内自噬体数量增加;MH-S的中性红摄取能力与尼古丁浓度剂量呈负相关性;尼古丁能够诱导小鼠发生肺炎,并降低小鼠体重。结论:尼古丁能够增强肺泡巨噬细胞的自噬水平,并且可以诱导肺炎的发生,有助于进一步研究吸烟与肺炎的关系。
Objective: To evaluate the function of nicotine in induced alveolar macrophages autophagy and pneumonia. Methods: PI/Hochest33258 staining was carried out for nicotine induced cell death assay. Western blotting and image of transmission electron microscopy(TEM) were employed for evaluation of cell autophagy. Neutral red uptake was used for macrophage phagocytosis assay, Pneumonia induced by intranasally infected with nicotine was identified by H&E and continuous weight monitoring. Results: PI stained death cells were shown in a nicotine dose-dependent manner. LC3 B Ⅱ was induced in nicotine pretreated MH-S cell in a dose-dependent increasing manner according to Western blotting assay. Obviously, nicotine enhance autophagy in LPS pre-treated cell. Multiply autophagosome could be identified through image of TEM. Neutral red uptake capacity of macrophage MH-S was negatively correlated with nicotine concentration dose. Mice lung H&E staining and lose of weight show nicotine induced pneumonia in vivo. Conclusion: Nicotine in cigarette smoke extract obviously increased alveolar macrophages autophagy and pneumonia in vivo are generated in this study and laid the foundation for the further research of relationship between smoking and pneumonia.
Objective: To evaluate the function of nicotine in induced alveolar macrophages autophagy and pneumonia. Methods: PI/Hochest33258 staining was carried out for nicotine induced cell death assay. Western blotting and image of transmission electron microscopy(TEM) were employed for evaluation of cell autophagy. Neutral red uptake was used for macrophage phagocytosis assay, Pneumonia induced by intranasally infected with nicotine was identified by H&E and continuous weight monitoring. Results: PI stained death cells were shown in a nicotine dose-dependent manner. LC3 B Ⅱ was induced in nicotine pretreated MH-S cell in a dose-dependent increasing manner according to Western blotting assay. Obviously, nicotine enhance autophagy in LPS pre-treated cell. Multiply autophagosome could be identified through image of TEM. Neutral red uptake capacity of macrophage MH-S was negatively correlated with nicotine concentration dose. Mice lung H&E staining and lose of weight show nicotine induced pneumonia in vivo. Conclusion: Nicotine in cigarette smoke extract obviously increased alveolar macrophages autophagy and pneumonia in vivo are generated in this study and laid the foundation for the further research of relationship between smoking and pneumonia.
引文
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[3]Benowitz N L,Burbank A D.Cardiovascular toxicity of nicotine:implications for electronic cigarette use[J].Trends Cardiovasc Med,2016,26(6):515-523.
[4]Herman A I,DeVito E E,Jensen K P,et al.Pharmacogenetics of nicotine addiction:role of dopamine[J].Pharmacogenomics,2014,15(2):221-234.
[5]Demiralay R,Gursan N,Erdem H.Regulation of nicotine-induced apoptosis of pulmonary artery endothelial cells by treatment of N-acetylcysteine and vitamin E[J].Hum Exp Toxicol,2007,26(7):595-602.
[6]Wu X,Zhang H,Qi W,et al.Nicotine promotes atherosclerosis via ROS-NLRP3-mediated endothelial cell pyroptosis[J].2018,9(2):171.
[7]Kim S Y,Kang K L,Lee J C,et al.Nicotinic acetylcholine receptor alpha7 and beta4 subunits contribute nicotine-induced apoptosis in periodontal ligament stem cells[J].Mol Cells,2012,33(4):343-350.
[8]Hosseinzadeh A,Thompson P R,Segal B H,et al.Nicotine induces neutrophil extracellular traps[J].JLeukoc Biol,2016,100(5):1105-1112.
[9]Lee J,Luria A,Rhodes C,et al.Nicotine drives neutrophil extracellular traps formation and accelerates collagen-induced arthritis[J].Rheumatology(Oxford),2017,56(4):644-653.
[10]Ohsumi Y.Historical landmarks of autophagy research[J].Cell Res,2014,24(1):9-23.
[11]Levy J M M,Towers C G,Thorburn A.Targeting autophagy in cancer[J].Nat Rev Cancer,2017,17(9):528-542.
[12]Kimmey J M,Stallings C L.Bacterial pathogens versus autophagy:implications for therapeutic interventions[J].Trends Mol Med,2016,22(12):1060-1076.
[13]Deretic V,Saitoh T,Akira S.Autophagy in infection,inflammation and immunity[J].Nat Rev Immunol,2013,13(10):722-737.
[14]Budani M C,Tiboni G M.Ovotoxicity of cigarette smoke:a systematic review of the literature[J].Reprod Toxicol,2017,72:164-181.
[15]Liu C,Qi J,Shan B,et al.Pretreatment with cathelicidin-BF ameliorates Pseudomonas aeruginosa pneumonia in mice by enhancing NETosis and the autophagy of recruited neutrophils and macrophages[J].Int Immunopharmacol,2018,65:382-391.
[16]Shan M,Qin J,Jin F,et al.Autophagy suppresses isoprenaline-induced M2 macrophage polarization via the ROS/ERK and mTOR signaling pathway[J].Free Radic Biol Med,2017,110:432-443.
[17]Garcia-Arcos I,Geraghty P,Baumlin N,et al.Chronic electronic cigarette exposure in mice induces features of COPD in a nicotine-dependent manner[J].Thorax,2016,71(12):1119-1129.