目的 探讨脑源性神经生长因子(brain-derived neurotrophic factor,BDNF)对缺氧(oxygen deprivation,OD)神经元的保护作用是否与激活自噬有关,其信号通路是否通过PI3K/Akt/mTOR途径。方法 1)建立胚鼠神经元OD损伤模型;CCK-8法观察不同浓度BDNF对OD神经元细胞活性的影响;2)检测OD神经元1,3,5 h自噬微管相关蛋白轻链3(LC3)的表达,情况观察BDNF对OD神经元的保护作用是否与自噬有关;3)检测p-Akt,p-mTOR,p-p70S6K以及LC3II的表达,对比BDNF与自噬激活剂雷帕霉素(mammalian target of rapamycin,Rapamycin)对Akt/mTOR/p70S6K的影响,以及自噬被3-MA抑制后,BDNF对Akt/mTOR/p70S6K的影响。结果 1)50 μg/L BDNF可对OD神经元起保护作用(P<0.05),100 μg/L BDNF效应次之;2)BDNF和/或Rapamycin在下调p-Akt/p-mTOR/p-p70S6K表达的同时可上调LC3II的表达;当3-MA抑制自噬后,BDNF对LC3II的上调作用被抑制。结论 BDNF通过PI3K/Akt/mTOR/p70S6K信号途径激活自噬,发挥对OD神经元的保护作用。
Abstract
Objective Based on growing evidence linking autophagy to ischemic preconditioning,it was hypothesized that autophagy was necessary for neuroprotection conferred by hypoxic preconditioning and further aimed to test whether brain-derived neurotrophic factor(BDNF) protects against hypoxic neuronal damage by autophagy activation and the possible signaling pathway. Methods Primary cultured cortical neurons of pregnant rats were treated with oxygen deprivation (OD) for 0.5~6 h to mimic hypoxic injury in vivo.Neuronal autophagy was measured with expression of microtubule associated protein light chain 3(LC3),and the LC3II,p-Akt,p-mTOR,and p-p70S6K protein were detected by western-blot after treated with autophagy promotor rapamycin or autophagy inhibitor 3-methyladenine(3-MA). Results 1)Compared with the control group,cells treated with 50 μg/L BDNF had the highest cell viability.2)BDNF decreased the expression of P-Akt,p-mTOR,and p-p70S6K,and increased the expression of LC3II.Similar to the roles of rapamycin to the signals,BDNF-induced up-regulation of LC3II was inhibited by 3-MA. Conclusion BDNF protects cortical neurons against oxygen deprivation damage by autophagy via PI3K/Akt/mTOR/p70S6K signaling pathway.
关键词
脑源性神经生长因子 /
自噬 /
神经元 /
哺乳动物雷帕霉素靶蛋白 /
自噬微管相关蛋白轻链3
Key words
brain-derived neurotrophic factor /
autophagy /
cortical neurons /
mammalian target of rapamycin /
light chain 3
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] 邹峥,刘小惠,邹大卫.缺氧缺血性脑损伤[J].实用儿科临床杂志,2011,26(18):1446-1449.
[2] Chen AI,Xiong L,Tong YU,et al.The neuroprotective roles of BDNF in hypoxic ischemic brain injury[J].Biomedical Reports,2013,1:167-176.
[3] 马骄,俞丹,毛萌,等.神经丝氨酸蛋白酶抑制剂对新生大鼠神经元损伤的保护作用[J].实用儿科临床杂志,2010,27(24):1859-1861.
[4] Mizushima N,Yoshimori T,Levine B.Methods in mammalian autophagy research[J].Cell,2010,140(3):313-326.
[5] Yang Z,Klionsky DJ.An overview of the molecular mechanism of autophagy[J].Curr Top Microbiol Immunol,2009,335:1-32.
[6] Sun X,Zhou H,Luo X,et al.Neuroprotection of brain-derived neurotrophic factor against hypoxic injury invitro requires activation of extracellular signal-regulated kinase andphosphatidylinositol 3-kinase[J].Int J Dev Neurosci,2008,26(3-4):363-370.
[7] Zhu W,Nelson CM.PI3K signaling in the regulation of branching morphogenesis[J].Biosystems,2012,109(3):403-411.
[8] Geering B,Cutillas PR,Vanhaesebroeck B.Regulation of class IA PI3Ks:is there a role for monomeric PI3K subunits?[J].Biochem Soc Trans,2007,35(Pt 2):199-203.
[9] LoPiccolo J,Blumenthal GM,Bernstein WB,et al.Targeting the PI3K/Akt/mTOR pathway:effective combinations and clinical considerations[J].Drug Resist Updat,2008,11(1-2):32-50.
[10] 袁莉,姚亚民,吕丹丹,等.Rapamycin保护大鼠缺血性脑损伤和特定脑区神经新生有关[J].神经解剖学杂志,2011,27(3):263-270.
[11] 沈亦雯,汤奇胜.雷帕霉素对胶质瘤干细胞的特异性凋亡作用[J].中华临床医师杂志:电子版,2010,4(5):578-582.
[12] Wu YT,Tan HL,Huang Q,et al.Activation of the PI3K-Akt-mTOR signaling pathway promotes necrotic cell death via suppression of autophagy[J].Autophagy,2009,5(6):824-834.
[13] Chen M,Du Y,Qui M,et al.Ophiopogonin B-induced autophagy in non-small cell lung cancer cells via inhibition of the PI3K/Akt signaling pathway[J].Oncol Rep,2013,29(2):430-436.
[14] Jaber N,Dou Z,Chen JS,et al.Class III PI3K Vps34 plays an essential role in autophagy and in heart and liver function[J].Proc Natl Acad Sci USA,2012,109(6):2003-2008.
[15] Wu YT,Tan HL,Shui G,et al.Dual role of 3-methyladenine in modulation of autophagy via different temporal patterns of inhibition on class I and III phosphoinositide 3-kinase[J].J Biol Chem,2010,285(14):10850-10861.
基金
国家自然科学基金(30973215);四川省科技支撑计划(2012SZ0010);教育部创新团队和新世纪人才计划(IRT 0935)
PDF(549 KB)
