为评估巴豆醛对人支气管上皮细胞(BEAS-2B)的毒性效应,使用细胞计数试剂盒(CCK-8)检测BEAS-2B细胞存活率,免疫荧光法检测自噬小体的聚集和定位,Western Blot实验检测自噬标志物微管相关蛋白1轻链3β(LC3B-II)及两个自噬相关通路磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶标(mTOR)和肝脏激酶B1(LKB1)/单磷酸腺苷活化蛋白激酶(AMPK)/Unc-51-类激酶(ULK1)的变化.结果表明:①巴豆醛暴露可显著降低BEAS-2B细胞存活率.②巴豆醛处理可引起自噬小体的聚集,改变LC3B-II蛋白表达量;巴豆醛诱导细胞自噬具有明显的时间效应关系,BEAS-2B细胞自噬强度随巴豆醛暴露时间延长先升高后降低.③PI3K/Akt/mTOR通路中磷酸化的PI3K、Akt和4E结合蛋白1(4E-BP1)呈下降趋势,磷酸化的mTOR和核糖体蛋白S6激酶(p70 S6K)先下降后升高;LKB1/AMPK/ULK1通路中LKB1、AMPK和ULK1活性均呈升高趋势.因此,两个通路均参与调控巴豆醛诱导的BEAS-2B细胞自噬水平的升高.%To assess the toxic effects of crotonaldehyde on human bronchial epithelial cells BEAS-2B,cell counting kit-8(CCK-8)assay was adopted to detect the viability of BEAS-2B cells; immunofluorescence assay to the accumulation and location of autophagosomes; and Western Blot assay to autophagy-marker microtubule-associated protein 1 light chain 3 beta(LC3B-II), the variations of two pathways of phosphatidylinositol 3-kinase(PI3K)/ protein kinase B(Akt)/ mammalian target of rapamycin(mTOR)and liver kinase B1(LKB1)/adenosine monophosphate-activated protein kinase(AMPK)/ Unc-51-like kinase 1 (ULK1). The results indicated that:1)The viability of BEAS-2B cells decreased significantly after exposure to crotonaldehyde. 2)Crotonaldehyde significantly promoted the accumulation of autophagosomes and changed the expression level of LC3B-II. Induced autophagy had apparent time-effect relationship. The level of autophagy in BEAS-2B cells was up-regulated prior to down-regulation with the increase of exposure time. 3)The relative expression of phospho-(P-)PI3K,P-Akt and P-4E-binding protein1(4E-BP1)in PI3K/Akt/mTOR pathway decreased,while that of P-mTOR and P-ribosomal protein S6 kinase(p70 S6K)decreased first and then increased. The activities of LKB1,AMPK,and ULK1 in LKB1/AMPK/ULK1 pathway increased. Collectively, the two pathways mediated the up-regulation of autophagy in BEAS-2B cells exposed to crotonaldehyde.
展开▼
