Heme oxygenase-1 alleviates ischemia/reperfusion injury in aged liver

Abstract

Aim: To investigate if ischemia/reperfusion (I/R) injury in aged liver could be alleviated by heme oxygenase-1 (HO-1).

Methods: Three groups of SD rats (16 mo old) were studied. Group 1: control donors received physiological saline 24 h before their livers were harvested; group 2: donors were pretreated with hemin 24 h before their livers were harvested; and group 3: donors received hemin 24 h before their livers were harvested and zinc protoporphyrin (ZnPP, HO-1 inhibitor) was given to recipients at reperfusion. The harvested livers were stored in University of Wisconsin solution (4 degrees) for 6 h, and then transplanted to syngeneic rats. Serum glutamic oxaloacetic transaminase (SGOT), apoptotic cells, and apoptotic gene were measured 3, 6, 12, 24, 48 h after reperfusion. We measured the apoptotic index by TUNEL, determined the expression of antiapoptotic Bcl-2 and proapoptotic (caspase-3) gene products by Western blot.

Results: After 3, 6, 12, 24, and 48 h of reperfusion, the SGOT levels (584.4+/-85.8 u/L, 999.2+/-125.2 u/L, 423.4+/-161.3 u/L, 257.8+/-95.8 u/L, and 122.4+/-26.4 u/L) in hemin group were significantly (all P<0.05) lower than those in saline group (1082.2+/-101.2 u/L, 1775.2+/-328.3 u/L, 840.4+/-137.8 u/L, 448.6+/-74.3 u/L, and 306.2+/-49.3 u/L). Liver HO-1 enzymatic activity correlated with beneficial effects of hemin and deleterious effects of adjunctive ZnPP treatment. Markedly less apoptotic (TUNEL+) liver cells 3, 6, 12, 24, and 48 h after reperfusion (5.16+/-0.73, 10.2+/-0.67, 9.28+/-0.78, 7.14+/-1.12, and 4.78+/-0.65) (P<0.05) could be detected in hemin liver grafts, as compared to controls (7.82+/-1.05, 15.94+/-1.82, 11.67+/-1.59, 8.28+/-1.09, and 6.36+/-0.67). We detected the increased levels of Bcl-2 (1.5-fold) expression and compared with saline controls. These differences were most pronounced at 12 h after transplantation. In contrast, an active form of proapoptotic caspase-3 (p20) protein was found to be 2.9-fold lower at 24 h in hemin-pretreated group, as compared to saline liver transplant controls.

Conclusion: HO-1 overexpression can provide potent protection against cold I/R injury. This effect depends, at least in part, on HO-1-mediated inhibition of antiapoptotic mechanism.

Figure 1

Comparison of serum SGOT levels in hemin group and in saline and hemin+ZnPP groups (P<0.05). there was no difference between the two groups. After a 6-h reperfusion, the serum SGOT level was the highest.

Figure 2

Enzymatic assay for HO-1 activity.

Figure 3

TUNEL-positive cells in (A) and saline-pretreated group (B) hemin-pretreated group (A) and (×200).

Figure 4

Western blot analyses of Bcl-2 and caspase-3 in orthotopic liver transplants at 12 h (lanes A and C) and 24 h (lanes B and D) after transplantation. At 12 and 24 h, higher levels of antiapoptotic Bcl-2 were found in hemin–treated livers (lanes A and B) as compared to saline controls (lanes C and D). Proapoptotic caspase-3 decreased in hemin–treated livers (lanes A and B) as compared to saline controls (lanes C and D).