钙通道阻滞剂维拉帕米对甲基苯丙胺所致大鼠小脑神经细胞凋亡的抑制作用(英文)

Inhibition of methamphetamine-induced apoptosis by the calcium channel blocker verapamil in rat cerebellar neurons

目的 :研究维拉帕米对甲基苯丙胺 (MA)导致神经细胞损伤的影响 ,探讨维拉帕米的保护机制。方法 :采用MTT法 ,乳酸脱氢酶 (LDH)法 ,观察MA的神经毒性作用。运用DNA电泳法、流式细胞仪法 ,观察MA致神经细胞凋亡的作用 ,并研究了维拉帕米的保护作用。结果 :(1 )MA(0 .2 5～ 3 0 0mmol/L)作用 4 8h和 6 0h ,能增加大鼠小脑神经细胞系R2细胞LDH的漏出率 ,分别为 4 8.2 %～ 89.3%和 6 2 .5 %～ 97.3% ,与对照组相比 ,差异有显著性 (P <0 .0 1 ) ,活细胞明显减少 ;应用流式细胞仪检测 ,在G1峰前出现亚二倍体峰 ,MA所致R2细胞凋亡呈一定的时间依赖性和剂量依赖性。DNA电泳图谱则表现出典型的梯状条带。 (2 )维拉帕米 (0 .5～ 5 0 μmol/L)减少MA导致的R2细胞LDH的漏出率 ,改善细胞形态 ,对MA诱导R2细胞凋亡有明显的抑制作用。结论 :维拉帕米对MA诱导的神经细胞凋亡具有保护作用 ,MA神经毒性作用可能与L型电压依赖性钙通道有关。

Objective: To study protective effects of verapamil on neurotoxicity induced by methamphetamine (MA) in rat cerebellar neuronal cells (R2 cells) and explore the possible mechanisms. Methods: MA neurotoxicity to R2 cells was examined by MTT reduction and LDH leakage assays. Using flow cytometer and agarose gel electrophoresis, apoptosis induced by MA in R2 cells was observed. The effects of verapamil on neurotoxicity and apoptosis induced by MA in R2 cells were determined. Results: (1) Incubation of the R2 cells with MA (0.25-3.00 mmol/L) for 48 h or 60 h concentration dependently increased the LDH content in the extra cellular bathing medium by 48.2%-89.3% and 62.5%-97.3%, respectively. Exposure to MA elicited the apoptotic DNA peak of the R2 cells (i.e. sub G1 peak) in a concentration and time dependent manner by the use of flow cytometer. Agarose gel electrophoresis showed a typical DNA laddering pattern. (2) Verapamil (0.5-5.0 μmol/L) reduced the number of dead cells and the LDH content in the extra cellular bathing medium, ameliorated morphological changes, and inhibited apoptosis induced by MA in R2 cells. Conclusion: These results suggest that verapamil has a neuroprotective effect on MA induced apoptosis, and that L type voltage dependent calcium channel may be involved in the neurotoxitic effect of MA.