毒胡萝卜素诱导大鼠皮层神经元内质网应激及丹红注射液的干预作用

目的：探讨毒胡萝卜紊诱导大鼠皮层神经元内质网应激凋亡的机制及丹红注射液的干预作用。方法：体外培养SD乳鼠皮层神经元，免疫组织化学、免疫荧光染色鉴定神经元纯度。流式细胞术Annexin V、PI双标检测凋亡率及活性caspase-3、caspase-8、caspase-7、caspase-9表达，Western Noting免疫印迹分析caspase-12、GRP78、Bcl-2、细胞色素C蛋白表达，Fura-2／AM法荧光分光光度计检测细胞内钙浓度（Ca^2＋]i）。结果：SD乳鼠皮层神经元可纯化体外培养。2μmol／L毒胡萝卜素作用神经元24、48h细胞凋亡率分别是17.88％、21.38％，丹红治疗组分别是6．30％、6．11％，两组比较差异有统计学意义（P〈0．05）。毒胡萝卜素诱导神经元GRP78表达上调，剪切活化caspase-3、caspase-8、caspase-9、caspase-12，使细胞色素C表达增加，Bcl-2表达减少。丹红注射液促进细胞Bcl-2表达，抑制细胞色素C释放，减少活化的caspase-3、caspase-8、caspase-9含量，稳定游离钙浓度。结论：毒胡萝卜素诱导神经元内质网应激反应性凋亡。丹红注射液能抑制体外培养神经元内质网应激所致凋亡。

Thapsigargin induces endoplasmic reticulum stress in rat cortical neurons and intervention of Danhong injection

AIM： To study the apoptosis induced by thapsigargin on rat cortical neurons and the intervention of Danhong injection. METHODS： Primary cortial neurons were cultured in vitro, and NSE-positive cells were detected by immunohistological chemistry and immunofluorcscence staining. Protein levels of GRP78,bcl-2, cytochrome c（cyt c）, active caspase-12 were assessed by immunoblotting cell extracts with specific antibodies. The percentage of apoptotic（annexin V positive and propidium iodide negative）cells was determined by flow cytometric analysis. Protein expression of active caspase-3, caspase-9, caspase-8, caspase-7 was measured by flow cytometric （FCM） analysis cytometry; Intracellular calcium concentrations （ Ca^2＋ ] i ） were measured by fluorescence spectrophotometer with calcium sensitive fluorescence indicator Fura-2/AM. RESULTS：As expected, GRP78 and caspase-12 prorein levels elevated in cortical neurons in response to thapsigargin（2 μmol/L,24 h and 48 h） but not to vehicle. The rate of apoptosis induced by thapsigargin 24 h and 48 h was 17.88% and 21.38%, and was depressed to 6.30% and 6.11% after treated by Danhong injection （ 8 mL/L ） respectively. Activated caspase-3,-9,-8 was detected at 24 h and 48 h after thapsigargin contribution and was reduced by Danhong injection. The enhancement of Ca^2＋ ]i in neurons were induced by thapsigargin and were depressed by Danhong injection. CONCLUSION： Thapsigargin activates ER-initiated apoptosis which can be inhibited by Danhong injection in in rat cortical neurons.