尼古丁诱导人肠道上皮细胞自噬水平的研究
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摘要
【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m TOR/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m TOR/Akt信号通路激活相关。
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, 10μM) of nicotine for 24 h. 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 LC3 B-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.
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, 10μM) of nicotine for 24 h. 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 LC3 B-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.
引文
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[2]YE L,CAO Q,CHENG J.Review of inflammatory bowel disease in China[J].Thescientificworldjournal,2013,2013(2013):296470.
[3]MCKINNEY D L,VANSICKEL A R.Nicotine chemistry,pharmacology,and pharmacokinetics[M].Neuropathology of Drug Addictions and Substance Misuse.Elsevier.2016:93-103.
[4]LUNNEY P C,LEONG R W.Review article:Ulcerative colitis,smoking and nicotine therapy[J].Alimentary Pharmacology&Therapeutics,2012,36(11-12):997-1008.
[5]BERKOWITZ L,SCHULTZ B M,SALAZAR G A,et al.Impact of Cigarette Smoking on the Gastrointestinal Tract Inflammation:Opposing Effects in Crohn’s Disease and Ulcerative Colitis[J].Frontiers in Immunology,2018,9:74
[6]王海杰,谭玉珍.细胞自噬机制开启疾病治疗新途径[J].解剖学报,2017,48(1):103-105.WANG Haijie,TAN Yuzhen.Mechanism of autophagy opens a new way for treatment of diseases[J].ACTA ANATOMICA SINICA,2017,48(1):103-105.
[7]TOMOYA,IIDA,ONODERA,et al.Role of autophagy in the pathogenesis of inflammatory bowel disease[J].World Journal of Gastroenterology,2017,23(11):1944-1953.
[8]LUKE J,STEPHAN R,WEERSMA R K,et al.Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease[J].Nature,2012,491(7422):119-124.
[9]田玲玲,刘丽娜.溃疡性结肠炎治疗的现代观点[J].世界华人消化杂志,2016,7:1054-1063.TIAN Lingling,LIU Lina.Current approaches to management of ulcerative colitis[J].World Chinese Journal of Digestology,2016,24(7):1054-63.
[10]PELISSIER-ROTA M A,PELOSI L,MERESSE P,et al.Nicotineinduced cellular stresses and autophagy in human cancer colon cells:A supportive effect on cell homeostasis via up-regulation of Cox-2 and PGE 2 production[J].International Journal of Biochemistry&Cell Biology,2015,65:239-256.
[11]WECHMAN S L,PRADHAN A K,DESALLE R,et al.New Insights Into Beclin-1:Evolution and Pan-Malignancy Inhibitor Activity[M].Advances in cancer research.Elsevier.2018:77-114.
[12]JIANG P,MIZUSHIMA N.LC3-and p62-based biochemical methods for the analysis of autophagy progression in mammalian cells[J].Methods,2015,75:13-18.
[13]COSNES J,GOWER-ROUSSEAU C,SEKSIK P,et al.Epidemiology and Natural History of Inflammatory Bowel Diseases[J].Gastroenterology,2011,140(6):1785-1794.e1784.
[14]MAHID S S,MINOR K S,SOTO R E,et al.Smoking and inflammatory bowel disease:a meta-analysis[J].Mayo Clinic Proceedings,2006,81(11):1462-1471.
[15]COSNES J.What is the link between the use of tobacco and IBD?[J].Inflammatory Bowel Diseases,2010,14(S2):S14-S15.
[16]MILLER A.IARC monographs on the evaluation of the carcinogenic risk of chemicals to humans.Vol.38.Tobacco smoking:International Agency for Research on Cancer,Lyon,1986.pp.421(available through Oxford University Press).ISBN92-832-1238-X[M].Pergamon.1987.
[17]YEGANEH B,J GER R,GORMAN A M,et al.Induction of autophagy:Role of endoplasmic reticulum stress and unfolded protein response[M].Autophagy:Cancer,Other Pathologies,Inflammation,Immunity,Infection,and Aging.Elsevier.2015:91-101.
[18]HOSOMI S,KASER A,BLUMBERG R S.Role of endoplasmic reticulum stress and autophagy as interlinking pathways in the pathogenesis of inflammatory bowel disease[J].Current opinion in gastroenterology,2015,31(1):81.
[19]WONG M K,HOLLOWAY A C,HARDY D B.Nicotine Directly Induces Endoplasmic Reticulum Stress Response in Rat Placental Trophoblast Giant Cells[J].Toxicological Sciences An Official Journal of the Society of Toxicology,2016,151(1):23.
[20]GUAN Y,ZHANG L,LI X,et al.Repression of mammalian target of rapamycin complex 1 inhibits intestinal regeneration in acute inflammatory bowel disease models[J].The Journal of Immunology,2015,1303356.
[21]OKAMOTO R,WATANABE M.Role of epithelial cells in the pathogenesis and treatment of inflammatory bowel disease[J].Journal of gastroenterology,2016,51(1):11-21.
[22]EL-KHIDER F,MCDONALD C.Links of autophagy dysfunction to inflammatory bowel disease onset[J].Digestive Diseases,2016,34(1-2):27-34.
[23]KIM J,KUNDU M,VIOLLET B,et al.AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1[J].Nature cell biology,2011,13(2):132.
[24]李欣,杨作成.mTOR信号通路与细胞凋亡[J].国际免疫学杂志,2013,36(5):337-340.LI Xin,YANG Zuocheng.mTOR signaling pathway and apoptosis[J].International Journal of Immunology,2013,36(5):337-40.