吗啡预先给药对去甲肾上腺素诱导大鼠心肌细胞NF-κB活化的影响

Effects of morphine pretreatment on norepinephrine-induced activation of NF-κB in cultured rat cardiomyocytes

目的 观察吗啡预先给药对去甲肾上腺素（NE）诱导大鼠心肌细胞NF-κB活化的影响，探讨吗啡在细胞水平对缺血心肌的保护作用机制。方法 出生1-3d的SD大鼠，分离、培养心肌细胞，将培养4～5d呈亚融合状态下的细胞随机分为5组（n：4）：对照组（C组）、10^-6mol·L^-1NE组（NE1组）、10^-7mol·L^-1 NE组（NE2组）、10^-8mol·L^-1 NE组（NB组）、10^-5mol·L^-1吗啡＋10^-8mol·L^-1 NE组（M＋NE组）。NE1组、NE2组、NB组细胞培养基内分别加入相应浓度的NE，M＋NE组在加入NE前30min加入10^-5mol·L^-1吗啡。加入NE后24h，进行下述指标的测定，采用流式细胞术测定心肌细胞胞浆NF-κB表达水平；采用免疫细胞化学染色技术测定NF-κB、TNF-α表达，采用酶免疫试验测定TNF-α含量，采用反转录PCR技术测定TNF-αmRNA表达。结果 与C组相比，NE1组、NE2组、NE3组细胞浆NF-κB水平降低，其中NB组NF-κB表达水平最低，NR组、NE3组TNF-α含量及TNF-αmRNA表达升高，陋组NF-κB、TNF-α表达升高（P〈0．05）；与NE3组比较，M＋NE组细胞浆NF-κB水平升高，TNF-α含量及TNF-αmRNA表达降低（P〈0．05）。结论 10^-5mol·L^-1吗啡预先给药可在一定程度上抑制NE诱导大鼠心肌细胞NF-κB的活化，从而减少了TNF-α的释放。

Objective To investigate the effects of morphine pretreatment on norepinephrine （NE）-induced activation of NF-κB in cultured neonatal rat cardiomyocytes. Methods The cultured rat cardiomyocytes were obtained from 1-3 day old SD rats and cultured for 4-5 days. The cells were randomly divided into 5 groups ： group A control; group B NE 10^-6mol·L^-1（NE1）; group C NE 10^-7 mol·L^-1（NF2）; group D NE 10^-8 mol·L^-1 （NE3 ） and group E morphine 10-5 mol·L^-1 ＋ NE 10^-8mol·L^-1（M ＋ NE）. In group E morphine was added to the culture media 30min before NE. The following measurements were made 24 h after NE had been added： （ 1 ） percentage of NF-κB expression in the cytoplasm of cardiomyocytes indicating the degree of activation of NF-κB （flow cytometry ） ; （ 2 ） expression of NF-κB and TNF-α （ immuno-histochemistry ） ; （ 3 ） TNF-α concentration （enzyme immunometric assay EIA） and TNF-α mRNA expression （RT-PCR）.Results The expression and the degree of activation of NF-κB and the level of TNF-α were significantly increased in the 3 NE groups （ NE1. 2.3 ） dose-dependently as compared with control group and group E （M ＋ NE）. Conclusion Morphine pretreatment can inhibit NE-induced activation of NF-κB in cardiomyocytes and in turn decrease TNF-α release.