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

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

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0.025μg/ml、0.05μg/ml和0.1μg/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0.025μg/ml、0.05μg/ml和0.1μg/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0.05μg/ml和0.1μg/ml的脑苷肌肽的保护作最为显著。0.1μg/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0.1μg/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。

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.