脑苷肌肽促进星形胶质细胞分泌GDNF
保护AAPH诱导神经元损伤的实验研究

中国卒中杂志 ›› 2015, Vol. 10 ›› Issue (04): 291-297.

脑苷肌肽促进星形胶质细胞分泌GDNF
保护AAPH诱导神经元损伤的实验研究

郭安臣1，2，3，4，赵一龙2，4，5，苏芳2，4，5，李巍巍2，4，5，王拥军2，3，4，5，王群2，3，4，5

1100050 北京
首都医科大学附属北京
天坛医院临床医学研究
实验室
2脑血管病转化医学北京
市重点实验室
3北京脑重大疾病研究院
4国家神经系统疾病临床
研究中心
5首都医科大学附属北京
天坛医院神经病学中心

收稿日期:2014-12-04 出版日期:2015-04-20 发布日期:2015-04-20
通讯作者: 王群 wangq@ccmu.edu.cn
基金资助:
国家自然科学基金资助项
目（81171097; 81271312）

Cattle Encephalon Glycoside and Ignotin Ameliorates the Injury of Neurons Through
Increasing the Level of GDNF of Astrocytes

*Laboratory for Clinical Medical Research,
Beijing Tiantan Hospital, Capital Medical University, Beijing 100050, China

Received:2014-12-04 Online:2015-04-20 Published:2015-04-20

摘要/Abstract

摘要：

【摘要】目的研究脑苷肌肽对缺血性卒中患者发挥神经保护作用的可能机制，探讨星形胶质细胞在脑保护中的作用。方法取10只孕16～18 d SD大鼠的胚胎、及10只24 h SD乳鼠的脑组织分别用于原代神经元及原代星形胶质细胞培养，经免疫组化染色证实培养的细胞分别为神经元和星形胶质细胞后，给予1 mmol/L、 5 mmol/L、10 mmol/L、20 mmol/L和40 mmol/L 2，2-偶氮二（2-甲基丙基咪）二盐酸盐｛2，2’-Azobis ［（2-methylpropionamidine）dihydrochloride，AAPH］｝模拟缺血性卒中诱导神经元和星形胶质细胞损伤，并通过利用分光光度计分析细胞活力，观察AAPH对细胞活力的影响；观察0.025 μg/ml、0.05 μg/ml 和0.1 μg/ml脑苷肌肽对星形胶质细胞的保护作用，同时制备脑苷肌肽－星形胶质细胞条件培养基，比较条件培养基对AAPH诱导的神经元损伤的保护作用。结果经分光光度计分析40 mmol/L AAPH为合适诱导损伤浓度，在AAPH损伤4 h后，分别给予不同浓度的脑苷肌肽共同孵育24 h。结果显示0.025 μg/ml、0.05 μg/ml和0.1 μg/ml浓度的脑苷肌肽均能够减轻AAPH造成的星形胶质细胞损伤，但0.05 μg/ml和0.1 μg/ml的脑苷肌肽的保护作最为显著。 0.1 μg/ml脑苷肌肽－星形胶质细胞条件培养基能够通过促进胶质细胞分泌胶质源性神经生长因子，进而保护AAPH诱导的神经元损伤（P＜0.05）。而将0.1 μg/ml的脑苷肌肽作用24 h后的细胞上清液作为条件培养基，在40 mmol/L AAPH诱导神经元损伤后加入脑苷肌肽-星形胶质细胞条件培养基作用 24 h。检测神经元细胞活力，发现脑苷肌肽-星形胶质细胞条件培养基能够逆转AAPH造成的神经元损伤，差异具有显著性（P＜0.05）。结论脑苷肌肽作为临床常用的神经保护剂，其可能的作用机制之一是促进星形胶质细胞分泌胶质源性神经生长因子，发挥神经保护作用。

文章导读： 本实验通过体外研究发现星形胶质细胞是卒中后神经保护的重要靶点，对神经保护新药的研发有
重要的理论指导意义。

关键词: 脑苷肌肽；星形胶质细胞；神经元；脑源性神经营养因子；2; 2-偶氮二（2-甲基丙基
咪）二盐酸盐

Abstract:

【Abstract】 Objective To study the possible mechanism of cattle encephalon glycoside and ignotin (CEGI) to protect the neuron from damage. At the same time, the neuroprotective function of the astrocytes in brain injury was detected. Methods Ten pregnant Sprague Dawley (SD) rats and 10 neonatal SD rats were used to culture primarily neurons and astrocytes in the present study. To mimic the ischemic stroke, we added 40 mmol/L 2, 2'-Azobis (2-methylpropionamidine)dihydrochloride (AAPH) into the culture system to induce the cell injury. And the cell viabilities were detected with spectrophotometer. The protective functions of 0.025 μg/ml, 0.05 μg/ml and 0.1 μg/ml of CEGI were tested in the astrocyte culture system. At the same time, 0.1 μg/ml of CEGI-astrocyte conditional media were made to protect the neurons from AAPH induced injury. And the protective mechanism of CEGI was also detected. Results 40 mmol/L AAPH can be used to induce astrocyte injury. CEGI can ameliorate the AAPH induced astrocyte injury. 0.1 μg/ml of CEGI-astrocyte conditional media can increase the cell viability of neurons, and also CEGI-astrocyte conditional media can inhibit the apoptosis of neurons induced by AAPH. Western blot experiments demonstrated that CEGI-astrocyte conditional media could increase the level of glial cell-derived neurotrophic factor (GDNF) released from astrocytes to protect against neuronal damage induced by AAPH. Conclusion CEGI protects the neuron from injury by stimulating astrocyte secreting GDNF. And astrocytes are the target for neuroprotective drug screening.

Key words: Cattle Encephalon Glycoside and Ignotin; Reactive astrocyte; Neuron; Glial cellderived
neurotrophic factor; 2, 2'-Azobis (2-methylpropionamidine) dihydrochloride

郭安臣1，2，3，4，赵一龙2，4，5，苏芳2，4，5，李巍巍2，4，5，王拥军2，3，4，5，王群2，3，4，5. 脑苷肌肽促进星形胶质细胞分泌GDNF
保护AAPH诱导神经元损伤的实验研究[J]. 中国卒中杂志, 2015, 10(04): 291-297.

GUO An-Chen*, ZHAO Yi-Long, SU Fang,LI Wei-Wei, WANG Yong-Jun, WANG Qun.. Cattle Encephalon Glycoside and Ignotin Ameliorates the Injury of Neurons Through
Increasing the Level of GDNF of Astrocytes[J]. Chinese Journal of Stroke, 2015, 10(04): 291-297.

参考文献

1 Flynn RW, MacWalter RS, Doney AS. The cost of

cerebral ischaemia[J]. Neuropharmacology, 2008,

55:250-256.

2 Sutherland BA, Minner up J, Balami JS, et al.

Neuroprotection for ischaemic stroke:translation from

the bench to the bedside[J]. Int J Stroke, 2012, 7:407-

418.

3 Hines DJ, Haydon PG. Inhibition of a SNARE

sensitive pathway in astrocytes attenuates damage

following stroke[J]. J Neurosci, 2013, 33:4234-4240.

4 陈慧, 崔庆宏, 张拥波, 等. 以星形胶质细胞为靶点治

疗缺血性脑卒中的机制探讨[J]. 神经损伤与功能重

建, 2012, 7:50-53.

5 Iade cola C, An r athe r J. St roke re sea rch at a

crossroad:asking the brain for directions[J]. Nature

Neurosci, 2011, 14:1363-8.

6 Hacke W, Kaste M, Bluhmki E, et a1. For the ECASS

investigators. Thrombolysis with altheplase 3 to 4. 5

hours after acute ischemic stroke[J]. N Engl J Med,

2008, 359:1317-1329.

7 Kikuchi K, Uchikado H, Morioka M, et al. Clinical

neuroprotective drugs for treatment and prevention of

stroke[J]. Int J Mol Sci, 2012, 13:7739-7761.

8 Shualb A, Hussin MS. The past and future of

neuroprotection in cerebral ischaemic stroke[J]. Euro

Neurol, 2008, 59:4-14.

9 Parpura V, Heneka MT, Montana V, et al. Glial cells

in (patho) physiology[J]. J Neurochem, 2012, 121:4-27.

10 Blair R, Leavit T, Cynthia S, et al. Mature astroctes

transform into transitional radia glia within adult

mouse neocortex that supports directed migration of

transplanted immature neurons[J]. Exp Neurol, 1999,

157:43-57.

11 Louw DF, Masada T, Sutherland GR. Ischemic

neu rona l i nju r y i s amel ior at ed by a s t rocy t e

activation[J]. Can J Neurol Sci, 1998, 25:102-107.

12 Ba r r e t o G, Wh i t e R E , Ou y a n g YB, e t a l .

Astrocytes:Targets for neuroprotection in stroke[J].

Cent Nerv Syst Agents Med Chem, 2011, 11:164-173.

13 Kang MC, Cha SH, Wijesinghe W, et al. Protective

effect of marine algae phlorotannins against AAPHinduced

oxidative stress in zebrafish embryo[J]. Food

Chem, 2013, 138:950-955.

14 Z h a n g Y, Mi l a t o v i c D, As c h n e r M, e t a l .

Neuroprotection by tamoxifen in focal cerebral

ischemia is not mediated by an agonist action at

estrogen receptors but is associated with antioxidant

activity[J]. Exp Neurol, 2007, 204:819-827.

15 Kim EA, Lee SH, Ko CI, et al. Protective effect of

fucoidan against AAPH-induced oxidative stress in

zebrafish model[J]. Carbohydr Polym, 2014, 102:185-

191.

16 杨永升, 王津津. 脑苷肌肽对rd小鼠视网膜细胞保护

作用的研究[J]. 眼科新进展, 2012, 22:1131-1133.

17 Lin LF, Doherty D, Lile, et al. GDNF, a glial cell line-der ived neurot rophic factor for midbrain

dopaminergic neurons[J]. Science, 1993, 260:1130-

1132.

18 Miyazaki H, Okumay Y, Fujii Y, et al. Glial cell linederived

neurotrophic factor protects against delayed

neuronal death after transient forebrain ischemia in

rats[J]. NeuroScience, 1999, 89:643-647.

19 Kitagawa H, Hayashi T, Mitsumoto Y, et al. Reduction

of ischemic brain injury by topical application of glial

cell line-derived neurotrophic factor after permanent

middle cerebral artery occlusion in rats[J]. Stroke,

1998, 29:1417-1422.