A total of 96 healthy male Sprague-Dawley rats, weighing 250-300 g, were used in this study. Hind limb
ischemia was made on 40 rats through clamping the infrarenal aorta for 2 hours with a microvascular
clip, then limb reperfusion for 0, 4, 8, 16 and 24 hours (n=8 in each time point) was performed, respectively. Other 8 rats undergoing full surgical operation including isolation of the infrarenal aorta without occlusion were taken as the
sham operation group. Lung tissues were obtained from the 48 animals and Northern blotting and Western blotting were employed to measure the changes of HO-1
mRNA and
protein expression, respectively. Immunohistochemistry technique was used to determine the cell types responsible for HO-1 expression after limb
ischemia/reperfusion. Then hind limb
ischemia was made on other 12 rats through clamping the infrarenal aorta for 2 hours with a microvascular
clip, among whom, 6 rats were given
zinc protoporphyrin (ZnPP), an inhibitor of HO. Then limb reperfusion for 16 hours was performed on all the 12 rats. And other 12 rats underwent full surgical operation including isolation of the infrarenal aorta without occlusion, among whom, 6 rats were then given ZnPP. Then lung tissues were obtained from the 24 animals and
lung injury markers, lung histology, polymorphonuclear leukocyte (PMN) count and
malondialdehyde (MDA) content were detected, respectively. HO activity was determined through measuring the
carboxyhemoglobin (COHb) level in artery blood with a CO-oximeter after limb
ischemia/reperfusion. And the animal mortality was observed on the other 24 rats.
RESULTS: Northern blotting analysis showed that HO-1
mRNA increased significantly at 4 hours after reperfusion, peaked at 16 hours, and began to decrease at 24 hours. In contrast, no positive signal was observed in the
sham and simple
ischemia animals. Increased HO-1
mRNA levels were accompanied by similar increases in HO-1
protein. Lung PMNs and MDA content increased significantly at 4, 8, 16 and 24 hours after reperfusion, compared with the
sham controls (P<0.001), while they decreased in rats with reperfusion for 16 hours when compared with rats with reperfusion for 4 hours (P<0.001). Immunohistochemical studies showed that HO-1 was expressed in a variety of cell types, including the airway epithelia, alveolar macrophages and vascular smooth muscular cells. The blood COHb level and animal mortality increased significantly after limb
ischemia/reperfusion compared with the
sham controls (P<0.001). ZnPP administrated to the
ischemia/reperfusion animals led to a decrease in the COHb level and an increase in lung PMN number, MDA content and animal mortality (P<0.001 compared with
ischemia/reperfusion group), and the
lung injury was aggravated.
CONCLUSIONS: