[1]张志鑫,崔应麟,李彦杰,等.SYK调控缺氧/缺糖损伤诱导的大脑皮质神经元凋亡及其机制[J].第三军医大学学报,2017,39(13):1381-1386.
 ZHANG Zhixin,CUI Yinglin,LI Yanjie,et al.Spleen tyrosine kinase regulates anoxia/hypoglycemia-induced apoptosis in cultured cerebral cortical neurons[J].J Third Mil Med Univ,2017,39(13):1381-1386.
点击复制

SYK调控缺氧/缺糖损伤诱导的大脑皮质神经元凋亡及其机制(/HTML )
分享到:

《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
39卷
期数:
2017年第13期
页码:
1381-1386
栏目:
基础医学
出版日期:
2017-07-15

文章信息/Info

Title:
Spleen tyrosine kinase regulates anoxia/hypoglycemia-induced apoptosis in cultured cerebral cortical neurons
作者:
张志鑫崔应麟李彦杰任锟邢若星
河南省中医院康复科
Author(s):
ZHANG Zhixin CUI Yinglin LI Yanjie REN Kun XING Ruoxing

Department of Rehabilitation Medicine, Henan Provincial Hospital of Traditional Chinese Medicine, Zhengzhou, Henan Province, 450002, China

关键词:
SYKFra-1缺氧/缺糖损伤大脑皮质神经元凋亡
Keywords:
spleen tyrosine kinase Fra-1 anoxia/hypoglycemia injury cerebral cortical neurons apoptosis
分类号:
R345; R363.2; R743.302
文献标志码:
A
摘要:

目的         探讨抑制脾酪氨酸激酶(spleen tyrosine kinase, SYK)对缺氧/缺糖损伤诱导的大脑皮质神经元的凋亡的影响及相关机制。方法       分离培养10只SD怀孕大鼠(190~230 g, 胎龄16 d)胎鼠的大脑皮质神经元细胞并用MAP2免疫荧光法进行鉴定。建立缺氧/缺糖损伤的体外模型,caspase3荧光检测试剂盒检测caspase-3活性,Annexin-V FITC/PI法检测细胞凋亡,qRT-PCR检测SYK和凋亡相关的原癌基因Fra-1 mRNA的表达,Western Blot检测SYK、Bcl-2和Fra-1蛋白的表达。Control组:无转染处理的缺氧/缺糖损伤的神经元;SYK siRNA组:转染SYK siRNA的缺氧/缺糖损伤的神经元;SYK-Fra-1 siRNA组:同时转染SYK siRNA和Fra-1 siRNA的缺氧/缺糖损伤的神经元;non-specific siRNA组:转染non-specific siRNA的氧/缺糖损伤的神经元。结果        MAP2免疫荧光鉴定为神经元细胞。缺氧/缺糖损伤诱导神经元SYK表达(P<0.01),RNA干扰抑制SYK表达后caspase3活性(P<0.05)和细胞凋亡(P<0.01)均显著降低,但是Fra-1 mRNA (P<0.01)和蛋白(P<0.05)表达量明显上升。用RNA干扰技术同时抑制SYK和Fra-1后,caspase-3活性(P<0.01)显著上升但Bcl2蛋白表达(P<0.01)显著降低。结论      Fra-1介导了SYK对缺氧/缺糖损伤诱导的神经元凋亡的调节,为脑卒中所致的神经元缺血性损伤的治疗提供了新的靶点。

Abstract:

Objective        To investigate the effect of inhibiting spleen tyrosine kinase (SYK) on apoptosis in cerebral cortical neuron induced by anoxia/hypoglycemia (A/H) injury and explore the related mechanism. Methods        The fetal rat cerebral cortical neurons were isolated from 10 pregnant SD rats (weighting 190~230 g, gestational age of 16 d), cultured and then identified by MAP2 immunofluorescence assay. The cerebral cortical neuron model with A/H injury was established in vitro. Then the cells were divided into A/H injured neurons (control group), A/H injured neurons with SYK siRNA transfection (SYK siRNA group), A/H injured neurons with co-transfection of SYK siRNA and Fra-1 siRNA (SYK-Fra-1 siRNA group), and A/H injured neurons with non-specific siRNA transfection (non-specific siRNA group). Caspase-3 activity was detected by the caspase3 kit. Cell apoptosis was determined by Annexin-V FITC/PI assay. The mRNA expressions levels of SYK and apoptosis-related proto-oncogene Fra-1 were measured by qRT-PCR. The protein levels of SYK, Bcl-2 and Fra-1 were detected by using Western blotting. Results        The neurons were identified by MAP2 immunofluorescence assay. Anoxia/hypoglycemia injury induced increased SYK expression in the neurons (P<0.01). SYK silencing by RNA interference significantly reduced caspase-3 activity (P<0.05) and cell apoptosis (P<0.01), while the mRNA (P<0.01) and protein (P<0.05) levels of Fra-1 were obviously up-regulated. Caspase-3 activity was significantly increased (P<0.01) and Bcl-2 protein expression was markedly decreased (P<0.01) in the cells after the co-silencing of SYK and Fra-1. Conclusion       Fra-1 mediates SYK regulation in the apoptosis of cerebral cortical neurons induced by A/H injury, which provides the new target for A/H injury in neurons caused by cerebral stroke.

参考文献/References:


[1]ROLFS A, FAZEKAS F, GRITTNER U, et al. Acute cerebrovascular disease in the young: the Stroke in Young Fabry Patients study[J]. Stroke, 2013, 44(2): 340-9. DOI: https://doi.org/10.1161/STROKEAHA.112.663708
[2]TOYODA K, NINOMIYA T. Stroke and cerebrovascular diseases in patients with chronic kidney disease[J]. Lancet Neurol, 2014, 13(8): 823-33. DOI: 10.1016/S14744422(14)700262.
[3]CHEN K H, HSU H H, YANG H Y, et al. Inhibition of spleen tyrosine kinase (syk) suppresses renal fibrosis through antiinflammatory effects and down regulation of the MAPKp38 pathway[J]. Int J Biochem Cell Biol, 2016, 74:135-44. DOI: 10.1016/j.biocel.2016.03.001.
[4]ROY S, KARMAKAR M, PEARLMAN E. CD14 mediates Tolllike receptor 4 (TLR4) endocytosis and spleen tyrosine kinase (Syk) and interferon regulatory transcription factor 3 (IRF3) activation in epithelial cells and impairs neutrophil infiltration and Pseudomonas aeruginosa killing in vivo[J]. J Biol Chem, 2014, 289(2): 1174-82. DOI: 10.1074/jbc.M113.523167.
[5]SCHEIB J L, SULLIVAN C S, CARTER B D. Jedi-1 and MEGF10 signal engulfment of apoptotic neurons through the tyrosine kinase Syk[J]. J Neurosci, 2012, 32(38):13022-31. DOI: 13022-31. 10.1523/JNEUROSCI.6350-11.2012
[6]GALLAGHER D, GUTIERREZ H, GAVALDA N, et al. Nuclear factor-kappaB activation via tyrosine phosphorylation of inhibitor kappaBalpha is crucial for ciliary neurotrophic factorpromoted neurite growth from developing neurons[J]. J Neurosci, 2007, 27(36): 9664-9. DOI: 10.1523/JNEUROSCI.060807.2007
 [7]TSUJIMURA T, YANAGI S, INATOME R, et al. Syk protein-tyrosine kinase is involved in neuronlike differentiation of embryonal carcinoma P19 cells[J]. FEBS Lett, 2001, 489(2-3): 129-33. DOI: 10.1016/S0014-5793(01)02097-X.
[8]HOELLENRIEGEL J, COFFEY G P, SINHA U, et al. Selective, novel spleen tyrosine kinase (Syk) inhibitors suppress chronic lymphocytic leukemia Bcell activation and migration[J]. Leukemia, 2012, 26(7): 1576-1583. DOI: 10.1038/leu.2012.24
[9]GOBESSI S, LAURENTI L, LONGO P G, et al. Inhibition of constitutive and BCRinduced Syk activation downregulates Mcl-1 and induces apoptosis in chronic lymphocytic leukemia B cells[J]. Leukemia, 2009, 23(4): 686-97.
[10]COOPMAN P J, DO M T, BARTH M, et al. The Syk tyrosine kinase suppresses malignant growth of human breast cancer cells[J]. Nature, 2000, 406(6797): 742-7.DOI:10.1038/35021086.
[11]张通. 中国脑卒中康复治疗指南 (2011 完全版)[J][J]. 中国医学前沿杂志 (电子版), 2012, 4(6): 55-6. DOI: 10.3969/j.issn.1006-9771.2012.04.001
ZHANG T. Chinese stroke rehabilitation guidelines (2011 full version)[J]. Chin J Front Med Sci(Elect Ver), 2012, 4(6): 55-6. DOI: 10.3969/j.issn.1006-9771.2012.04.001
[12]MANZANERO S, SANTRO T, ARUMUGAM T V. Neuronal oxidative stress in acute ischemic stroke: sources and contribution to cell injury[J]. Neurochem Int, 2013, 62(5): 712-8. DOI: 10.1016/j.neuint.2012.11.009
[13]MUKHERJEE S, ZHU J, ZIKHERMAN J, et al. Monovalent and multivalent ligation of the B cell receptor exhibit differential dependence upon Syk and Src family kinases[J]. Sci Signal, 2013, 6(256): ra1. DOI: 10.1126/scisignal.2003220
[14]KOERBER R M, HELD S A, HEINE A, et al. Analysis of the anti-proliferative and the proapoptotic efficacy of Syk inhibition in multiple myeloma[J]. Exp Hematol Oncol, 2015, 4:21. DOI: 10.1186/s40164-015-0016-z
[15]MYERS D E, YIV S, QAZI S, et al. CD19-antigen specific nanoscale liposomal formulation of a SYK Psite inhibitor causes apoptotic destruction of human B-precursor leukemia cells[J]. Integr Biol (Camb), 2014, 6(8): 766-80. DOI: 10.1039/c4ib00095a
[16]SATOH J, TABUNOKI H, ISHIDA T, et al. Phosphorylated Syk expression is enhanced in NasuHakola disease brains[J]. Neuropathology, 2012, 32(2): 149-57. DOI: 10.1111/j.14401789.2011.01256.x
[17]ISKIT S, SCHLICKER A, WESSELS L, et al. Fra-1 is a key driver of colon cancer metastasis and a Fra-1 classifier predicts diseasefree survival[J]. Oncotarget, 2015, 6(41): 43146-61. DOI: 10.18632/oncotarget.6454
[18]VAZ M, MACHIREDDY N, IRVING A, et al. Oxidantinduced cell death and Nrf2dependent antioxidative response are controlled by Fra-1/AP-1[J]. Mol Cell Biol, 2012, 32(9): 1694-709. DOI: 10.1128/MCB.06390-11
[19]SHIRSAT N V, SHAIKH S A. Overexpression of the immediate early gene fra-1 inhibits proliferation, induces apoptosis, and reduces tumourigenicity of c6 glioma cells[J]. Exp Cell Res, 2003, 291(1): 91-100. DOI:10.1016/S0014-4827(03)00346-X
[20]LIN J H, LIN J Y, CHOU Y C, et al. EpsteinBarr virus LMP2A suppresses MHC class II expression by regulating the B-cell transcription factors E47 and PU.1[J]. Blood, 2015, 125(14): 2228-38. DOI: 10.1182/blood-2014-08-594689
[21]RAMOS-NINO M E, BLUMEN S R, PASS H, et al. Fra-1 governs cell migration via modulation of CD44 expression in human mesotheliomas[J]. Mol Cancer, 2007, 6:81. DOI: 10.1186/1476-4598-6-81
[22]KHALIL H, BERTRAND M J, VANDENABEELE P, et al. Caspase-3 and RasGAP: a stress-sensing survival/demise switch[J]. Trends Cell Biol, 2014, 24(2): 83-9. DOI: 10.1016/j.tcb.2013.08.002
[23]VOLKMANN N, MARASSI F M, NEWMEYER D D, et al. The rheostat in the membrane: BCL-2 family proteins and apoptosis[J]. Cell Death Differ, 2014, 21(2): 206-15. DOI: 10.1038/cdd.2013.153
[24]LLAMBI F, WANG Y M, VICTOR B, et al. BOK is a non-canonical BCL-2 family effector of apoptosis regulated by ER-associated degradation[J]. Cell, 2016, 165(2): 421-433. DOI: 10.1016/j.cell.2016.02.026
[25]ZHU Y, TCHKONIA T, FUHRMANN‐STROISSNIGG H, et al. Identification of a novel senolytic agent, navitoclax, targeting the Bcl‐2 family of anti‐apoptotic factors[J]. Aging Cell, 2016, 15(3): 428-435. DOI: 10.1111/acel.12445

更新日期/Last Update: 2017-07-14