一氧化碳对肢体缺血再灌注患者血清所致中性粒细胞凋亡的影响及核因子κB 的作用

Effects of carbon monoxide on polymorphonuclear leukocyte apoptosis stimulated by limb ischemiareperfusion patient serum and the role of nuclear factor-kB

目的观察外源性一氧化碳(CO)对肢体缺血再灌注(IR)患者血清所致中性粒细胞(PMN)凋亡的影响及核因子(NF)kB 的作用。方法分离培养人外周血 PMN,置于24-孔培养板,分为4组(n=16):对照组,对照+CO,IR 和 IR+CO 组。IR 和 IR+CO 组细胞应用肢体 IR 患者血清代替培养基孵育,其余两组应用健康志愿者血清培养。IR+CO 组和对照+CO 组细胞置含有0.025%CO 和5% CO_2的空气中孵育。对照组和 IR 组细胞始终在含5%CO_2空气中孵育。应用流式细胞学技术检测 PMN 凋亡率,进行 PMN DNA 电泳观察梯状带情况,应用电泳迁移率(EMSA)检测 PMN 核内 NF-kB 活性。结果与对照组相比,IR 组 PMN 凋亡百分率显著降低、NF-kB 活性明显增强;与 IR组相比,IR+CO 组 PMN 凋亡百分率显著增高、NF-kB 活性明显降低,出现典型 DNA 梯状带。结论外源性 CO 可改善肢体 IR 患者血清所致的 PMN 凋亡抑制,其机制与下调 NF-kB 活性有关。

Objective To explore the role of exogenous carbon monoxide （CO） in apoptosis of polymorphonuclear leukocyte （PMN） stimulated by ischemia-reperfusion （IR）. Methods PMNs isolated from the venous blood of a healthy volunteer, incubated, put in 24-well plates, and randomly divided into 4 groups of 8 wells： control group （exposed to 5% CO2）, control ＋ CO group （exposed to 0. 025% CO and 5% CO2 for I hour and then serum of healthy person was used to replace the culture fluid ）, IR group （exposed to 5% CO2 and then IR serum was used to replace the culture fluid ）, and IR ＋ CO group （exposed to 0. 025% CO and 5% CO2 for I hour and then serum of healthy person was used to replace the culture fluid）. The IR serum was obtained from 8 male patients with osteoarthritis of knee undergoing knee replacement. After 24-hour incubation the PMNs underwent flow cytometry and electrophoresis to examine the apoptosis of PMNs. Electrophoretic mobility shift assay （EMSA） was used to detect the NF-κB binding activity. Results The PMN apoptotic rate of the IR ＋ CO group was 9. 38% ± 1.58%, significantly higher than that of the control group （4. 18% ± 1.02%, P 〈0. 05）. The PMN apoptotic rate of the IR group was 2. 15% ± 1.02%, significantly lower than that of the control group （ P 〈 0. 05 ）. However, the PMN apoptotic rate of the control ＋ CO group was 4. 16% ± 1.12% , not significantly different from that of the control group （P 〉 0. 05）. Electrophoresis showed that PMN apoptosis DNA ladder was seen in the control, control ＋ CO, and IR ＋ CO groups, but not in the IR group. EMSA showed that after co-incubation of PMN nuclear extract and isotope- labeled NF-KB probe in term of the strength of radiation self-development band the result the IR group was significantly greater than that of the control group, and the result of the IR ＋ CO group was significantly lower than that of the IR group, however, there was no significant difference between the control and control ＋ CO groups. Conclusion Exogenous CO improves the inhibitory effect of IR blood on the PMN apoptosis with a mechanism of suppressing the NF-κB binding activity.