三羟异黄酮对丙烯酰胺诱导大鼠小脑颗粒神经元凋亡的保护作用

目的：探讨三羟异黄酮（genistein,GEN）对丙烯酰胺（acrylamide,ACR）诱导的大鼠小脑颗粒神经元凋亡的保护作用。方法：取新生5～7 d的SD大鼠小脑皮质细胞进行培养,采用尼氏染色法鉴定神经元,将培养8 d的神经元随机分成5组：正常对照组、ACR染毒组（浓度为10 mmol/L）以及GEN不同浓度保护组（染毒前分别用浓度为10、25、50μmol/L的GEN预先处理细胞12 h,再给予ACR染毒24 h）。MTT法检测细胞存活率;相差显微镜及Hoechst33342染色观察细胞及其细胞核形态学变化;TUNEL法检测神经元凋亡率。结果：GEN25、50μmol/L保护组的细胞存活率与ACR染毒组比较差异无统计学意义（P〉0.05）;仅GEN 10μmol/L浓度保护组能明显提高神经元存活率,减少神经元胞体皱缩、细胞核固缩等特征,降低神经元凋亡率。结论：一定浓度的GEN（10μmol/L）能够保护ACR所致的大鼠小脑颗粒神经元凋亡,但GEN对神经元的保护作用并不随剂量增加而增强。

Protective effects of genistein against apoptosis induced by acrylamide in cultured rat cerebellar granule neurons

OBJECTIVE:To investigate the protective effects of genistein on apoptosis of rat cerebellar granule neurons induced by acrylamide.METHODS:Rat cerebellar granule neurons were prepared from the cerebellar cortex cells of 5-7day-old SD rats pups.The neurons were identified by Nissl staining method.The 8-day cultured cells passage were divided randomly into control group,acrylamide model group,genistein pretreatment groupⅠ,Ⅱ,Ⅲ(cerebellar granule neurons were pretreated with 10,25,50μmol/L genistein for 12 hours,the culture medium discarded and fresh DMEM/F12 solution added with the above mentioned concentration of genistein with 10 mmol/L acrylamide to cultured neurons for 24 hours).The neuronal viability was measured by MTT.Morphology of neurons and their nuclei were examined by phase-contrast and Hochest33342 staining,respectively.The ratio of apoptotic cells was detected by TUNEL. RESULTS:The cell survival rates of genistein pretreatment groupⅡandⅢwere not significantly higher than acrylamide model group.Genistein pretreatment groupⅠsignificantly prolonged the cell survival rate.The effects of diminished neuronal body,chromatin concentration and the ratio of apoptotic cells induced by acrylamide were markedly weakened. CONCLUSION:Genistein did not show a dose—dependent effect on protection.The appropriate concentration of 10μmol/L was found to protect against apoptosis induced by acrylamide in primary culture of cerebellar granule neurons.