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白介素--6保护小脑颗粒神经元抵抗NMDA的神经毒性作用

王晓春, 邱一华, 彭聿平

南通大学医学院生理学教研室与江苏省神经再生重点实验室.江苏,南通 226001

摘要

近来研究发现,细胞因子白介素--6(interleukin--6,IL--6)不仅是重要的免疫调节因子,而且也是重要的神经调节因子。中枢神经系统中IL--6的作用是复杂的,尤其是对脑损伤时IL--6所发挥的作用及其作用机制尚不十分清楚。为此,作者利用N--甲基--D--天门冬氨酸(N--methyl--D--aspartate,NMDA)处理小脑颗粒神经元引起急性损伤的细胞模型,探讨IL--6对神经元损伤的保护及其作用机制。取出生后8 d幼鼠的小脑,进行小脑颗粒神经元培养。将IL--6(5或10 ng/mL)加入到小脑颗粒神经元的培养液中孵育8 d,然后用NMDA(100#mu#mol/L)刺激小脑颗粒神经元30 min以造成神经元损伤。用噻唑兰(methyl--thiazole--tetrazolium,MTT)比色法检测神经元的活性;用末端脱氧核苷酸转移酶介导的原位缺口末端标记(terminal deoxynucleotidyl transferase--mediated dUTP nick endlabeling,TUNEL)法检测神经元的凋亡;用激光扫描共聚焦显微镜(confocal laser scanning microscope,CLSM)观察神经元内游离Ca~(2+)浓度的动态变化。用抗gp130单克隆抗体(75 ng/mL)抑制IL--6的活性,然后观察IL--6抗NMDA神经毒性作用的变化;并利用Western blot法检测IL--6对小脑颗粒神经元表达IL--6的细胞内信号转导蛋白-----信号转导子和转录激活子3(signal transducer and activator of transcription 3,STAT3)和细胞外信号调节激酶(extracellular signal regulated kinase 1/2,ERK1/2)磷酸化水平的影响。NMDA刺激未经IL--6处理的小脑颗粒神经元,导致神经元活性降低、神经元凋亡增加和神经元内Ca~(2+)超载。NMDA刺激经IL--6慢性预处理的小脑颗粒神经元,其损伤程度与未经IL--6处理的神经元相比明显减轻,包括神经元活性明显增强、神经元凋亡显著减少和神经元内Ca~(2+)超载减轻.抗gp130抗体可阻断IL--6减轻NMDA激发的神经元内Ca~(2+)超载的作用;经IL--6慢性处理的小脑颗粒神经元,其细胞内STAT3和ERK1/2的磷酸化水平显著增加。结果表明,IL--6能保护神经元抵抗由NMDA诱导的兴奋性神经毒性,此神经保护机制与IL--6减轻神经元内Ca~(2+)超载密切相关,而且可能通过激活神经元内IL--6的信号转导路径调节细胞内的基因表达而实现。

关键词： 小脑颗粒神经元; 白介素-6; N-甲基-D-天冬氨酸; 神经毒性; 神经保护; 凋亡; Ca~(2+)超载; gP130; 信号转导子和转录激活子3; 细胞外信号调节激酶1/2

Interleukin--6 protects cerebellar granule neurons from NMDA--induced neurotoxicity

Wang Xiaochun, Qiu Yihua, Peng Yuping

Department of Physiology, Faculty of Medicine, and Key Laboratory of Neuroregeneration of Jiangsu Province, Nantong University.Nantong 226001,Jiangsu

Abstract

Interleukin-6 (IL-6) is an important cytokine that participates in inflammation reaction and cell growth and differentiation in the immune and nervous systems. However, the neuroprotection of IL-6 against N-methyl-D-aspartate (NMDA)-induced neurotoxicity and the related underlying mechanisms are still not identified. In the present study, the cultured cerebellar granule neurons (CGNs) from postnatal (8-day) infant rats were chronically exposed to IL-6 for 8 d, and then NMDA (100 μmol/L) was applied to the cultured CGNs for 30 min. Methyl-thiazole-tetrazolium (MTT) assay, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method and confocal laser scanning microscope (CLSM) were used to detect neuronal vitality, apoptosis and dynamic changes of intracellular Ca~(2+) levels in the neurons, respectively. Anti-gp130 monoclonal antibody (75 ng/mL) was employed to the cultured CGNs with IL-6 to inhibit IL-6 activity so as to evaluate the role of gp130 (a 130 kDa glucoprotein transducing IL-6 signal) in mediating IL-6 neuroprotection. Western blot was used to measure the expressions of phospho-signal transducer and activator of transcription 3 (STAT3) and phospho-extracellular signal regulated kinase 1/2 (ERK1/2) in the cultured CGNs. The NMDA stimulation of the cultured CGNs without IL-6 pretreatment resulted in a significant reduction of the neuronal vitality, notable enhancement of the neuronal apoptosis and intracellular Ca~(2+) overload in the neurons. The NMDA stimulation of the CGNs chronically pretreated with IL-6 caused a remarkable increase in the neuronal vitality, marked suppression of neuronal apoptosis and intracellular Ca~(2+) overload in the neurons, compared with that in the control neurons without IL-6 pretreatment. Furthermore, anti-gp130 antibody blocked the inhibitory effect of IL-6 on NMDA-induced intracellular Ca~(2+) overload in the neurons. The levels of phospho-STAT3 and phospho-ERK1/2 were significantly higher in IL-6-pretreated CGNs than those in IL-6-untreated neurons. The results suggest that chronic IL-6 pretreatment of CGNs protects the neurons against NMDA-induced neurotoxicity. The neuroprotective effect of IL-6 is closely related to its suppression of NMDA-induced intracellular Ca~(2+) overload and is possibly mediated by gp130/JAK-STAT3 and gp130/RASERK1/2 transduction pathways.

Key words: Cerebellar granule neurons;interleukin-6;N-methyl-D-aspartate;Neurotoxicity;neuroprotection;Apoptosis;Ca~(2+) overload;gp130;signal transducer and activator of transcription 3;extracellular signal regulated kinase 1/2

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引用本文：

王晓春, 邱一华, 彭聿平. 白介素--6保护小脑颗粒神经元抵抗NMDA的神经毒性作用[J]. 生理学报 2007; 59 (2): 150-156.

Wang Xiaochun, Qiu Yihua, Peng Yuping. Interleukin--6 protects cerebellar granule neurons from NMDA--induced neurotoxicity. Acta Physiol Sin 2007; 59 (2): 150-156 (in Chinese with English abstract).