尼古丁诱导人肠道上皮细胞自噬水平的研究

  目的  探讨尼古丁对人肠道上皮细胞自噬水平的影响和可能的分子机制。  方法  正常培养人肠道上皮细胞, 采用不同浓度(0、2.5、5、10μM)尼古丁处理细胞24h。倒置显微镜观察细胞形态变化; 通过CCK8法检测肠道上皮细胞的增殖情况; 通过Western blot检测自噬标志物LC3、beclin1和p62变化; 通过透射电子显微镜(TEM)检测自噬小体形成情况; 通过GFPLC3转染后激光共聚焦显微镜检测自噬斑点形成情况。同时, 也检测了内质网应和mTOR/AMPK/Akt信号通路变化。  结果  细胞增殖随着尼古丁浓度的增加受到明显抑制, 2.5μM尼古丁对细胞形态和增殖无显著影响。尼古丁能够上调LC3B-II和beclin1蛋白表达水平, 同时下调P62的表达, 且成浓度依赖性。TEM和激光共聚焦显示尼古丁作用后, 肠道上皮细胞自噬体数量显著增加。  结论  低浓度的尼古丁能够诱导肠道上皮细胞的自噬, 且对细胞增殖无影响, 机制与内质网应激和mTOR/Akt信号通路激活相关。 

Study on nicotine-induced autophagy in human intestinal epithelial cells

  Objective  To explore the effect of nicotine on autophagy level of human intestinal epithelial cells (hIEC) and related molecular mechanism.  Methods  Human intestinal epithelial cells were cultured normally and then treated with different concentrations (2.5, 5, 10 μM) of nicotine for 24h.Cell morphology was observed by inverted microscope.The proliferation of intestinal epithelial cells was detected by CCK8.Western blot analysis was performed to assess expression of autophagy markers, including LC3, beclin1 and p62.Transmission electron microscope (TEM) was used to detect the formation of autophagosomes.GFP-LC3 transfection assay observation was carried out to detect the formation of autophagy spots.Meanwhile, endoplasmic reticulum stress (ERS) and mTOR/AMPK/Akt signaling pathway change were also examined using immunoblot analysis.  Results  Cell proliferation was significantly inhibited with the increase of nicotine concentration, except 2.5μM nicotine treatment.Nicotine could increase the levels of LC3B-II and beclin1 protein expression, and decrease the expression levels of p62 in dose-dependent manner.TEM and laser confocal observations showed that nicotine activated the autophagy of hIEC by increasing the number of autophagosomes.  Conclusion  Low concentration of nicotine can induce autophagy in hIEC, which is related to activation of ERS and mTOR/Akt signaling pathway.