[Effects of carbon monoxide on polymorphonuclear leukocyte apoptosis stimulated by limb ischemia-reperfusion patient serum and the role of nuclear factor-kappaB].
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-kappaB 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-kappaB 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.


CONCLUSIONS
Exogenous CO improves the inhibitory effect of IR blood on the PMN apoptosis with a mechanism of suppressing the NF-kappaB binding activity.
Zhou
Jun-lin
JL
Department of Orthopedics, Beijing Chaoyang Hospital, Capital University of Medical Sciences, Beijing, China.
Huang
Xin-li
XL
Chang
Jun-ying
JY
Liu
Qing-he
QH
Tian
Qing-xian
QX
DU
Xin-ru
XR
D000368:Aged; D017209:Apoptosis / Q000187:drug effects*; D002248:Carbon Monoxide / Q000494:pharmacology*; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D008297:Male; D008875:Middle Aged; D016328:NF-kappa B / Q000378:metabolism* / Q000502:physiology; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D020370:Osteoarthritis, Knee / Q000097:blood; D011485:Protein Binding / Q000187:drug effects; D015427:Reperfusion Injury / Q000097:blood*; D044967:Serum / Q000737:chemistry
D003470:Culture Media; D016328:NF-kappa B; D002248:Carbon Monoxide