SOD mimetic improves the function, growth, and survival of small-size liver grafts after transplantation in rats

Abstract

Background: Small-for-size syndrome (SFSS) may occur when graft volume is less than 45% of the standard liver volume, and it manifests as retarded growth and failure of the grafts and more mortality. However, its pathogenesis is poorly understood, and few effective interventions have been attempted.

Aims: The present study aimed to delineate the critical role of oxidant stress in SFSS and protective effects of a superoxide dismutase mimetic, Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), on graft function, growth, and survival in the recipient rats.

Methods: Small size graft liver transplantation (SSGLT) was performed to determine the survival, graft injury, and growth. MnTBAP was administered in SSGLT recipients (SSGLT+MnTBAP).

Results: Serum alanine aminotransferase levels were sustained higher in SSGLT recipients, which were correlated with an increased apoptotic cell count and hepatocellular necrosis in liver sections. Malondialdehyde content, gene expression of tumor necrosis factor α and interleukin 1β, and DNA binding activity of nuclear factor-κB in the grafts were increased significantly in SSGLT recipients compared with sham-operated controls. Both phosphorylated p38 mitogen-activated protein kinase and nuclear c-Jun were increased in SSGLT. All these changes were strikingly reversed by the administration of MnTBAP, with an increase in serum superoxide dismutase activity. Moreover, in situ bromodeoxyuridine incorporation demonstrated that graft regeneration was much more profound in the SSGLT+MnTBAP group than in the SSGLT group. Finally, the survival of recipients with MnTBAP treatments was significantly improved.

Conclusions: Enhanced oxidant stress with activation of the p38/c-Jun/nuclear factor-κB signaling pathway contributes to SFSS-associated graft failure, retarded graft growth, and poor survival. MnTBAP effectively reversed the pathologic changes in SFSS-associated graft failure.

FIGURE 1

Animal survival, serum SOD activity, injury and MDA content of small-for-size hepatic graft. A. Survival of SSGLT recipient rats with MnTBAP treatment. MnTBAP was given once daily after the transplantation until sacrifice. Improved survival was seen in those with MnTBAP treatment 8 days after the treatment. * p<0.05 compared to the SSGLT group (n=15). B. Serum ALT levels at different time points after SSGLT. Data were presented as mean±SD, n=6 in each group. *, p<0.05; **, p<0.01 compared to the SSGLT group. C. Increased serum SOD activity in SSGLT recipient rats with MnTBAP treatment was shown. Serum SOD activity was determined at indicated time points after the transplantation. n=6. ** p<0.01 compared to SSGLT. D. Decreased liver MDA levels in SSGLT recipient rats with MnTBAP treatment. Liver MDA was determined at indicated time points spectrophotometrically to represent oxidant stress. n=6 in each group. ** p<0.01 compared to SSGLT alone.

FIGURE 2

Representative micrographs of liver histology after SSGLT with or without MnTBAP treatment. A–D: Hematoxylin and eosin (H–E) staining for small size liver graft sections at different time points after transplantation: A. 24 hrs after transplantation, B. 3 days after transplantation, C. 5 days after transplantation. D. 3 days after transplantation with MnTBAP treatment. E–G. Representative micrographs of in situ TUNEL staining of apoptotic cells in small size grafts. Each section was examined for 10 high-power fields, and both apoptotic and all cells were counted, and apoptotic cell count was expressed as follows: Apoptotic cell count (%) = (Apoptotic cells/total cell count) × 100. H. E. Sham-operated control. F. Small size graft at 30% volume 72 hours after transplantation. G. Small size graft at 30% volume from recipient rats with MnTBAP treatment. 400×. Average apoptotic cell count (%) in each group 3 days after transplantation. ** p<0.01 compared to SSGLT. n=6 in each group.

FIGURE 3

Up-regulated expression of TNF-α and IL-1β genes in the mediation of small size graft injury. Increased TNF-α (A) and IL-1β (B) gene expression was demonstrated at an early phase (2–3 days) after SSGLT, and MnTBAP significantly attenuated the enhanced TNF-α and IL-1β gene expression as demonstrated by real time RT-PCR analysis in the graft tissue. The GAPDH gene was used as a house-keeping control. n=6, *, p<0.05, **, p<0.01 compared to the SSGLT group. The sequences of the forward and reverse primers are: 5′-CGA TTT GCC ATT TCA TAC CAG-3′ and 5′-AGT ACT TGG GCA GGT TGAC-3′ for TNF-α (amplicon size: 180 bp); 5′-TGT GGA TCC CAA ACA ATA CCC-3′ and 5′-TAT GTC CCG ACC ATT GC-3′ for IL-1β (amplicon size: 175 bp); 5′-TGT GCA GTG CCA GCC TCG TCT-3′ and 5′-TTG CCG TGG GTA GAG TCA TAC-3′ for GAPDH (amplicon size: 190 bp).

FIGURE 4

Western blot analysis of activation of p38 MAPK and c-jun signaling pathway in small size graft tissue. A. Phosphorylated p38 MAPK and nuclear c-Jun levels in small size graft tissue at various time points were determined by Western blot analysis using GAPDH as a loading control (shown are the representative images). Please note that the activation of p38 MAPK was observed during the first 48 hours, whereas, nuclear c-jun was enhanced during the first 12 hours after the transplantation. The activation of both p38 MAPK and c-jun was attenuated by the treatment with MnTBAP. B&C. Densitometrical analysis of the Western blot images of p38 MAPK and c-jun was performed with image-pro plus software (n=6). * p < 0.05, ** p < 0.01 compared to the SSGLT group. D. Electrophoretic mobility shift assays (EMSA) of NF-κB in small size graft tissue after transplantation. The biotin 3'-end-labeled, double-stranded specific DNA probe is : 5'-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3' (the bold face indicates NF-κB-binding sites). EMSA image of NF-κB p65 subunit in Sham-operated, SSGLT and SSGLT + MnTBAP groups (n=4). Early activation of NF-κB was demonstrated in the graft tissue of SSGLT recipient rats compared to Sham-operated group, and decreased DNA binding activity was shown in SSGLT + MnTBAP group in comparison to SSGLT.

FIGURE 5

Graft regeneration assessed by in situ bromodeoxyuridine (BrdU) incorporation. The BrdU-positive cell counts were significantly increased in MnTBAP-treated rats compared to those without MnTBAP treatments after transplantation. Immunohistochemical staining for BrdU-positive cells in liver tissue from the control (A, D), SSGLT (B, E), and SSGLT with MnTBAP treatment (C, F) groups 48h after transplantation. Original magnification 400× (A, B, C) and 200× (D, E, F). The BrdU-positive cells were counted in each section for 10 high-power fields. * p < 0.05 compared to the SSGLT group at the same time point, n=5 at each time point of the two groups.