AMPK在香烟烟雾提取物诱导的支气管上皮细胞凋亡中的调节作用  

Effect of AMPK on the apoptosis in NHBE cells of COPD induced by CSE

作　　者：谢旺[1] 罗百灵[1] 张立[1] 郭现玲 

机构地区：[1]中南大学湘雅医院呼吸内科,湖南长沙410008

出　　处：《中国现代医学杂志》2014年第30期28-33,共6页China Journal of Modern Medicine

基　　金：国家自然科学基金(No:30971324);慢性阻塞性肺疾病与肺癌协同创新临床研究(No:2013BAI09B09)

摘　　要：目的内质网应激是导致慢性阻塞性肺疾病病程中细胞凋亡的重要机制之一,该研究探讨AMPK在香烟烟雾提取物诱导的支气管上皮细胞凋亡中的调节作用。方法实验分四组:空白对照组、AMPK特异性抑制剂(Compound C)组、CSE组、CSE+Compound C组,体外培育人支气管上皮细胞(NHBE)并分别给予CSE及Compound C处理。采用Western blot法检测CHOP、Caspase 4及p-AMPK蛋白表达水平,Hoechst33342染色观察细胞形态学改变及流式细胞学检测细胞凋亡率。结果与正常对照组相比,CSE干预组中p-AMPK/AMPK蛋白表达下降,差异具有显著性(P<0.05);CHOP、Caspase4蛋白表达增加,差异具有显著性(P<0.05);Compound C+CSE组与正常对照组比较,p-AMPK/AMPK显著下降,Caspase4/GAPDH、CHOP/GAPDH显著增加,差异具有显著性(P<0.05);流式细胞学检测显示:无论早期凋亡率还是总凋亡率,CSE组、Compound C+CSE组与正常对照组对比均增加,差异有显著性(P<0.05)。予以Hoechst 33342进行细胞核染色显示:与对照组相比,CSE组、Compound C+CSE组细胞均出现典型的凋亡特征,细胞核呈现浓染、颗粒块状荧光及明显的形态裂化;各实验组均出现不同程度凋亡,以早期凋亡为主,流式细胞学检测显示:空白对照组、CSE组、Compound C+CSE组流式细胞学检测的早期凋亡率依次为(1.480±0.217)%、(26.860±2.946)%、(43.620±2.536)%,细胞凋亡率依次递增(P<0.05),而Compound C组早期凋亡率(3.560±1.209)%与对照组差异无显著性(P>0.05)。结论 CSE能诱导NHBE细胞发生凋亡,并呈时间浓度依赖性;AMPK的表达对细胞抵抗CSE诱导的内质网应激可能具有一定的保护效应。【Objective】Endoplasmic reticulum stress-mediated apoptosis is an important pathogenic factor of chronic obstructive pulmonary disease. The aim of this study was to investigate the role of AMPK on CSE-induced apoptosis in human bronchial epithelial cell.【Methods】Normal human bronchial epithelial cells were cultured and treated with CSE alone or together with Compound C in vitro. The experiment was divided into four groups： Control group, CSE group, Compound C group, CSE＋Compound C group. Expressions of p-AMPK, AMPK, CHOP, caspase 4was detected by Western blot. Features of apoptosis were analyzed with using AnnexinV-PI flow cytometry and Hoechst 33342. 【Results】The expression of p-AMPK/AMPK was lower in the CSE and Compound C＋CSE treated group compared to the control group（P〈0.05）, but The expression of Caspase4 and CHOP protein were higher than those of the control group（P〈0.05）, the same result is also found in Compound C＋CSE group VS. control group and CSE group（P〈0.05）. All groups were stained with Hoechst33342, Cells in CSE group, Compound C＋CSE group haveshowed strong nuclear staining, particle block of fluorescence and obvious morphology cracking, the character of apoptosis in Compound C＋CSE group is more obvious than CSE group. AnnexinV-PI flow cytometry showed that the early and total apoptosis rates of the CSE group, Compound C＋CSE group were higher than the control group（P〈0.05）. Thesame result is also found in Compound C＋CSE group VS. CSE group（P〈0.05）. The results of apoptosis rate in control group, CSE group, Compound C＋CSE group were（1.480±0.217）%,（26.860±2.946）%,（43.620±2.536）%. Early apoptosis rate in the Compound C group（3.560±1.209）% has no significant difference compare to the control group（P〉0.05）.【Conclusions】CSE can induce apoptotic cell death with time and concentration dependent.The expression of AMPK may exert a protective effect on the apoptosis in NHBE cells of COPD induced by CSE.

关 键 词：慢性阻塞性肺疾病 腺苷酸活化蛋白激酶 内质网应激 凋亡 

分 类 号：R563[医药卫生—呼吸系统]