刺五加皂甙诱导PC12细胞对缺血的耐受及相关机制探讨

目的研究刺五加皂甙（ASE）预处理诱导PC12细胞对缺血的耐受及相关机制。方法采用MTT比色分析、细胞形态学观察、Westem Blot法以及凝胶电泳迁移实验（EMSA）等方法比较ASE预处理对PC12细胞缺血模型（氧葡萄糖剥夺模型）的影响。结果在PCI2细胞缺血模型中,9h氧葡萄糖剥夺可明显降低PC12细胞活力,细胞存活率为（49．12±3．22）％。然而ASE24h预处理则显著抑制9h氧葡萄糖剥夺对细胞的损伤,细胞存活率显著提高（67．97±2．92）％,实验组与对照组间相比较差异有显著统计学意义（F=473．67,P〈0．01）;细胞形态学显示,刺五加皂甙（终浓度50μg／ml）预处理再缺血处理9h,可明显减轻缺血9h所致的细胞损害,较多细胞保留突起,突起网络依然存在,胞体完好。Western Blot结果表明ASE预处理能明显增加缺氧诱导因子-1α（HIF-1α蛋白）表达,差异有显著统计学意义（F=167．18,P〈0．01）;ASE预处理诱导表达的HIF-1具有生物学活性,它能与DNA结合,并激活下游基因促红细胞生成素（EPO蛋白）的表达,差异亦有显著统计学意义（F=128．37,P〈0．01）。结论ASE预处理可以诱导PC12细胞对缺血的耐受,其诱导耐受的作用可能与ASE预处理诱导PCI2细胞HIF—let稳定表达、提高HIF-1与DNA结合活性以及增加其下游基因EPO蛋白表达有关。

Acanthopanax Senticosus Saponins induced tolerance to ischemia and its possible molecular mechanism in PC12 cells

OBJECTIVE: To study the tolerance to ischemia induced by Acanthopannx Senticosus Saponins (ASE) in PC12 cells and the involved mechanism. METHODS: An ischemic model was developed in PC12 cell line by treatment with oxygen-glucose deprivation. The effects of ASE pretreatment on tolerance of PC12 cells to ischemia were evaluated by MTT assay and analysis of cellular morphology. The expression of hypoxia-inducing factor (HIF)-1alpha, erythropoietin (EPO) after the pretreatment with ASE was detected by Western blotting. The DNA binding activities of HIF-1 in PC12 cells with the pretreatment of ASE were demonstrated by using electrophoretic mobility shift assay (EMSA). RESULTS: In ischemia model, the viability of PC12 cells was decreased to (49.12 +/- 3.22)% after oxygen-glucose deprivation for 9 hours. However, ASE (50 microg/ml) pretreatment could remarkably increase the viability of PC12 cells by (67.97 +/- 2.92)%. There were significant differences between the experimental group and control group (F = 473.67, P < 0.01). The cellular morphology showed that PC12 cells exposed for 7 days to nerve growth factor (NGF) exhibited round, smooth cell bodies with normal processes and that processes formed extensive network. At 9 hour after ischemia, cell bodies of many PC12 cells were found shrinken, the processes were disrupted and network disappeared. However, pretreatment with ASE (50 microg/ml) could largely prevent the morphological damage to PC12 cells that would have caused by subsequent exposure to 9 h ischemic insult, many cellular bodies were intact and many processes and network of PC12 cells still existed. The expression of HIF-1alpha increased after pretreatment with ASE shown by Western blot. There were significant differences between the experimental group and control group (F = 167.18, P < 0.01). The DNA binding activities of HIF-1 in PC12 cells after pretreatment with ASE was significantly increased, and it could activate the expression of EPO (F = 128.37, P < 0.01). CONCLUSIONS: The pretreatment with ASE could induce tolerance against ischemia in PC12 cells. The elevated expression and increased DNA binding activity of HIF-1alpha, the overexpression of its downstream target EPO may be the molecular mechanism in tolerance of PC12 cells to ischemia induced by ASE pretreatment.