刺五加皂甙诱导PC12细胞对缺血的耐受及相关机制探讨

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

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、Western 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 PC12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 ASE 24 h 预处理则显著抑制9 h 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9 h,可明显减轻缺血9 h 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 PC12细胞 HIF-1α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。

Objective To study the tolerance to ischemia induced by Acanthopannx Senticosns 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 MTr assay and analysis of cellular morphology. The expression of hypoxia-inducing factor （HIF）-la, 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 electmphoretic 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（500μg/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 μg/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-1α 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-1α the overexpression of its downstream target EPO may be the molecular mechanism in tolerance of PC12 cells to ischemia induced by ASE pretreatment.