NKCC1、KCC2及mTOR相关蛋白4E-BP1在外伤性癫痫中的表达及意义
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摘要
目的探讨主要表达于神经元细胞膜上的NA~+-K~+-2CL-转运体(NKCC1)、K~+-CL-转运体(KCC2)及表达于神经元细胞质中m TOR通路信号蛋白4E结合蛋白1(4E-BP1)在外伤性癫痫(PTE)癫痫灶中的表达及临床意义。方法收集2010年1月至2015年12月福建医科大学附属第一医院神经外科外伤性癫痫患者术后脑组织标本14例作为实验组;选取脑外伤患者行减压或清创手术获取的和各种病变患者手术入路不可避免要切除的正常脑组织8例作为对照组。用Western blot和实时定量荧光PCR(RT-PCR)检测14例PTE癫痫脑组织、8例对照组"正常脑组织"NKCC1、KCC2、4E-BP1的表达。结果 Western blot显示PTE病灶脑组织中4E-BP1、NKCC1相对灰度值(0.61±0.12、0.92±0.19)高于正常脑组织(0.27±0.05、0.67±0.66),差异有统计学意义(P<0.05)。PTE病灶脑组织中KCC2相对灰度值(0.58±0.99)低于正常脑组织(0.72±0.06),差异有统计学意义(P<0.05)。RT-PCR结果显示,PTE病灶脑组织中4E-BP1、NKCC1相对灰度值(30.84±1.32、27.81±1.92)高于正常脑组织(26.94±1.24、23.52±0.74),差异有统计学意义(P<0.05)。PTE病灶脑组织中KCC2相对灰度值(21.55±1.01)低于正常脑组织(24.59±1.02),差异有统计学意义(P<0.05)。PTE组中NKCC1/KCC2比值(1.29±0.11)高于对照组(0.96±0.26),差异有统计学意义(P<0.05)。结论NKCC1、KCC2和m TOR通路信号蛋白4E-BP1的异常改变,可能是外伤后脑组织组织学改变及反复癫痫发作的重要分子机制。
Objective To investigagte the expression and significance of NA~+-K~+-2 CLco-transporter(NKCC1), K~+-CL-co-transporter(KCC2), which are located on the neuronal membrane,and mammalian target of rapamycin(m TOR) associated protein 4 E blinding protein 1(4 E-BP1), which is located in cytoplasm of neuron, in post-traumatic epilepsy(PTE). Methods To collect 14 postoperative brain tissue specimens of post-traumatic epilepsy patients admitted to our hospital from 2010 to 2015 as the experiment group; 8 cases of normal brain tissue as the control group. And detect the expression of NKCC1, KCC2 and 4 E-BP1 both in 14 cases of PTE tissue and 8 cases of normal brain tissue by Western blot and RT-PCR. Results The content of NKCC1 and 4 E-BP1 in PTE was higher than that in normal brain tissue(P<0.05) by Western blot and RT-PCR; while the content of KCC2 was opposite(P <0.05). Besides, in PTE the ratio of NKCC1 to KCC2 was higher(1.29 ±0.11), compared to normal brain tissue(0.96±0.26)(P<0.05). Conclusion Abnormal expression of NKCC1, KCC2 and m TOR associated protein 4 E-BP1 may be an important molecular mechanism of the histological changes of brain, after traumatic brain injured, and repeated seizures.
Objective To investigagte the expression and significance of NA~+-K~+-2 CLco-transporter(NKCC1), K~+-CL-co-transporter(KCC2), which are located on the neuronal membrane,and mammalian target of rapamycin(m TOR) associated protein 4 E blinding protein 1(4 E-BP1), which is located in cytoplasm of neuron, in post-traumatic epilepsy(PTE). Methods To collect 14 postoperative brain tissue specimens of post-traumatic epilepsy patients admitted to our hospital from 2010 to 2015 as the experiment group; 8 cases of normal brain tissue as the control group. And detect the expression of NKCC1, KCC2 and 4 E-BP1 both in 14 cases of PTE tissue and 8 cases of normal brain tissue by Western blot and RT-PCR. Results The content of NKCC1 and 4 E-BP1 in PTE was higher than that in normal brain tissue(P<0.05) by Western blot and RT-PCR; while the content of KCC2 was opposite(P <0.05). Besides, in PTE the ratio of NKCC1 to KCC2 was higher(1.29 ±0.11), compared to normal brain tissue(0.96±0.26)(P<0.05). Conclusion Abnormal expression of NKCC1, KCC2 and m TOR associated protein 4 E-BP1 may be an important molecular mechanism of the histological changes of brain, after traumatic brain injured, and repeated seizures.
引文
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[17]Wu H,Shao A,Zhao M,et al.Melatonin attenuates neuronal apoptosis through up-regulation of K(+)-Cl(-)cotransporter KCC2 expression following traumatic brain injury in rats[J].J Pineal Res,2016,61(2):241-250.
[18]Butler CR,Boychuk JA,Smith BN.Differential effects of rapamycin treatment on tonic and phasic GABAergic inhibition in dentate granule cells after focal brain injury in mice[J].Exp Neurol,2016,280:30-40.
[19]Zeng LH,Rensing NR,Wong M.The mammalian target of rapamycin(m TOR)signaling pathway mediates epileptogenesis in a model of temporal lobe epilepsy[J].J Neurosci,2009,29(21):6964-6972.
[2]Rao VR,Parko KL.Clinical approach to posttraumatic epilepsy[J].Semin Neurol,2015,35(1):57-63.
[3]Irimia A,Van Horn JD.Epileptogenic focus localization in treatment-resistant post-traumatic epilepsy[J].J Clin Neurosci,2015,22(4):627-631.
[4]Talos DM,Sun H,Kosaras B,et al.Altered inhibition in tuberous sclerosis and type IIb cortical dysplasia[J].Ann Neurol,2012,71(4):539-551.
[5]Kim JY,Liu CY,Zhang F,et al.Interplay between DISC1 and GABA signaling regulates neurogenesis in mice and risk for schizophrenia[J].Cell,2012,148(5):1051-1064.
[6]Hunt RF,Boychuk JA,Smith BN.Neural circuit mechanisms of post-traumatic epilepsy[J].Front Cell Neurosci,2013,7:89.
[7]Berdichevsky Y,Dryer AM,Saponjian Y,et al.PI3K-Akt signaling activates m TOR-mediated epileptogenesis in organotypic hippocampal culture model of post-traumatic epilepsy[J].J Neurosci,2013,33(21):9056-9067.
[8]Orlova KA,Tsai V,Baybis M,et al.Early progenitor cell marker expression distinguishes type II from type I focal cortical dysplasias[J].J Neuropathol Exp Neurol,2010,69(8):850-863.
[9]Wu C,Sun D.GABA receptors in brain development,function,and injury[J].Metab Brain Dis,2015,30(2):367-379.
[10]Rice A,Rafiq A,Shapiro SM,et al.Long-lasting reduction of inhibitory function and GABA A subunit m RNA expression in a model of temporal lobe epilepsy[J].Proc Natl Acad Sci USA,1996,93(18):9665-9669.
[11]Lakhani R,Vogel K R,Till A,et al.Defects in GABA metabolism affect selective autophagy pathways and are alleviated by m TOR inhibition[J].EMBO Mol Med,2014,6(4):551-566.
[12]Kalkman HO.Alterations in the expression of neuronal chloride transporters may contribute to schizophrenia[J].Prog Neuropsychopharmacol Biol Psychiatry,2011,35(2):410-414
[13]Hyde TM,Lipska BK,Ali T,et al.Expression of GABA signaling molecules KCC2,NKCC1,and GAD1 in cortical development and schizophrenia[J].J Neurosci,2011,31(30):11088-11095.
[14]Loscher W,Puskarjov M,Kaila K.Cation-chloride cotransporters NKCC1 and KCC2 as potential targets for novel antiepileptic and antiepileptogenic treatments[J].Neuropharmacology,2013,69:62-74.
[15]Wang F,Wang X,Shapiro LA,et al.NKCC1 up-regulation contributes to early post-traumatic seizures and increased posttraumatic seizure susceptibility[J].Brain Struct Funct,2016,222(3):1543-1556.
[16]Schwarzbach E,Bonislawski DP,Xiong G,et al.Mechanisms underlying the inability to induce area CA1 LTP in the mouse after traumatic brain injury[J].Hippocampus,2006,16(6):541-550.
[17]Wu H,Shao A,Zhao M,et al.Melatonin attenuates neuronal apoptosis through up-regulation of K(+)-Cl(-)cotransporter KCC2 expression following traumatic brain injury in rats[J].J Pineal Res,2016,61(2):241-250.
[18]Butler CR,Boychuk JA,Smith BN.Differential effects of rapamycin treatment on tonic and phasic GABAergic inhibition in dentate granule cells after focal brain injury in mice[J].Exp Neurol,2016,280:30-40.
[19]Zeng LH,Rensing NR,Wong M.The mammalian target of rapamycin(m TOR)signaling pathway mediates epileptogenesis in a model of temporal lobe epilepsy[J].J Neurosci,2009,29(21):6964-6972.