Protective effect of midazolam against injury to PC12 cells induced by N-methyl-D-aspartate / 中华麻醉学杂志

ABSTRACT

Objective To evaluate the protective effect of midazolam (MID) on PC 12 cells against injury induced by N-methyl-D-aspartate (NMDA) -Methods The differentiated PC12 cell strain was isolated and cultured in DMEM full nutrient liquid medium and incubated in CO2 incubator at 37℃ and 5 % CO2 for 3-4 days. The experiment consisted of 3 groups (1) control group; (2) NMDA group and (3) MID treatment group. In NMDA group NMDA 300 ?mol?L-1 was added to DMEM liquid medium. MID group was further divided into five subgroups according to different concentrations of midazolam (MID) added to DMEM liquid medium in addition to NMDA 300 ?mol?L-1MID Ⅰ -Ⅴ subgroups (midazolam 0.33, 1, 3, 10, 30?mol?L-1 ). The PC 12 cells were then cultured for another few hours. Cellular viability was assessed by lactic dehydrogenase (LDH) assay and MTT assay. Meanwhile the [Ca2+ ] was measured by Fura-2/AM fluorescence and nitric oxide synthase (NOS) activity was measured with ultraviolet spectrophotometer. Results Exposure to NMDA 300 ?mol?L-1 for 4 h resulted in increase in release of LDH from PC 12 cells and decrease in optical density (OD570nm) absorbed by living cells, indicating that NMDA induced injury to PC12 cells. The presence of midazolam 0.33, 1, 3, 10 ?mol?L-1 ( MID subgroup I -IV ) decreased LDH release and increased OD570nm value. Exposure to NMDA 300 ?mol?L-1 for 4h also resulted in increase in intracellular Ca2+ concentration ([Ca2+ ];) and NOS activity in PC 12 cells. Midazolam 3 and 30?mol?L-1 significantly decreased [Ca2+ ]; and NOS activity as compared with NMDA group.Conclusion Midazolam can attenuate the NMDA-induced injury to PC12 cells, decrease the Ca2+ overloading and NOS activity in PC 12 cells. The inhibitory effects of midazolam on [Ca2+ ]; overloading and NOS activity may be involved in the mechanism of its protective action.