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Reperfusion Injury and Acute Rejection of Allogeneic Liver Transplant in Rats
Relationship Between Ischemia/Reperfusion Injury and Acute Rejection of Allogeneic Liver Transplant in Rats Y. Wang, J. Wu, B. Jiang, J. Wang, C. Liu, C. Peng, and B. Tian ABSTRACT Objective. This study aimed to investigate the relationship between the severity of ischemia/reperfusion (I/R) injury and the acute rejection (AR) of allogeneic liver transplants in rats. Methods. The experimental rats were divided in different groups: normal control group (sham group, group I); syngeneic liver transplant control group (similar gene group, group II); and allogeneic liver transplant groups (groups III to VI). The rats were humanely killed at 1, 3, 5, and 7 days after transplantation or sham operation to determine the severity of I/R injury, rejection classification, and hepatocyte apoptosis. Messenger RNA (mRNA) and protein expression levels of Fas, perforin, and granzyme B were assessed in the liver tissues using real-time polymerase chain reaction and immunohistochemistry, respectively. Results. The rejection scores of the transplanted liver tissues gradually increased until these scores were proportional to the severity of I/R injury in groups III, IV, and V. The maximum scores were reached at 7 days after transplantation as the duration of transplantation was extended. The mRNA and protein expression levels of Fas, perforin, and granzyme B were significantly increased at 1, 3, 3, 5, and 7 days after liver reperfusion in groups III, IV, and V compared with those in groups I, II, and VI (P < .05). Conclusion. The occurrence of AR after allogeneic liver transplantation in rats was positively correlated with the severity of I/R injury. Given that I/R injury caused serious damage to the transplanted liver, the occurrence of AR consequently decreased.
T
HE INCIDENCE rate of acute rejection (AR) after liver transplantation is approximately 40% to 80%. AR causes functional losses affecting the survival of liver transplant recipients. In China, liver transplantation mostly involves noneheart-beating donors. In addition to the innate characteristics of donor livers, the primary factors influencing the quality of donor livers are the severity of warm ischemia, cold ischemia, and ischemia/reperfusion (I/R) injury. I/R injury is an important cause of AR and chronic rejection [1,2]. This type of injury affects specific immune processes after liver transplantation and an early functional loss of the transplanted liver. New sources of donor livers are currently developed, although existing donor liver resources are maximized. New solutions have been proposed to solve the shortage of donor liver cells. For instance, the quality of donor livers should be improved to reduce the injury of donor livers in vitro or after recovery from perfusion and prevent the onset of various complications after liver transplantation, particularly the incidence of AR. 0041-1345/13/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.06.019 50
Therefore, the reduction of the severity of I/R injury after liver transplantation to prevent and control AR is clinically important. In this study, an animal model of AR was established after the liver was transplanted in rats to study the effect of different degrees of I/R injuries and hepatocyte apoptosis during AR after liver transplantation. This animal model was also used to determine the expression levels of Fas, perforin, and granzyme B. Our preliminary findings revealed the relationship between the severities of I/R injury and AR after liver transplantation. The mechanisms involved were also determined.
From the Department of Hepatobiliary Surgery, Hunan Provincial People’s Hospital, Changsha, Hunan Province, China. Address reprint requests to Jin-shu Wu, Department of Hepatobiliary Surgery, Hunan Provincial People’s Hospital, Changsha 410005, Hunan Province, China. E-mail: ygbzcn@ 126.com ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 46, 50e55 (2014)
I/R INJURY AND AR
MATERIALS AND METHODS Animals The donors were male and female Sprague-Dawley rats. The recipients were 2- to 3-week-old male Lewis rats, weighing 220 g to 280 g (Experimental Animal Department of Central South University), with clean feeding. The donors were provided with food or drink before the experiment. By contrast, the recipients were not provided with food for 6 hours prior to transplantation. The number of recipients was slightly higher than the number of donors. The experimental rats were divided into 6 groups: group I, syngeneic sham group; group II, syngeneic liver transplantation group subjected to warm ischemia and cold ischemia for 2 minutes (1.42 0.64 minutes) and 80 minutes, respectively; and groups III to VI, allogeneic liver transplantation groups. The rats in group III were subjected to warm ischemia and cold ischemia for 2 minutes (1.42 0.64 minutes) and 80 minutes, respectively. The rats in group IV were subjected to warm ischemia and cold ischemia for 2 minutes (1.42 0.64 minutes) and 10 hours, respectively. The rats in group V were subjected to warm ischemia and cold ischemia for 15 minutes (13.56 1.78 minutes) and 80 minutes, respectively. The rats in group VI were subjected to warm ischemia and cold ischemia for 15 minutes (13.56 1.78 minutes) and 10 hours, respectively. Each group was further divided into 4 subgroups and then humanely killed at 1, 3, 5, or 7 days after transplantation, respectively (n ¼ 3; Table 1). The transplanted livers were harvested on the respective days after transplantation. Blood was then withdrawn from the inferior vena cava. This study was performed in strict compliance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol on animal use was reviewed and approved by the Institutional Animal Care and Use Committee of the Hunan Provincial People’s Hospital, China.
METHODS
Liver transplantation was performed according to an improved version of Kamada’s two-cuff technique. In brief, the subhepatic inferior vena cava and the portal vein were connected based on the cuff technique. The suprahepatic vena cava was then sutured. Stenting was performed for bile ducts. The hepatic artery was reconstructed by the bracket method. The suprahepatic inferior vena cava was reconstructed with an improved continuous suture. Liver perfusion was controlled using an automatic vein infusion pump. The perfusion rate was set at 2 mL/min with a total perfusion volume between 25 and 30 mL to ensure an even perfusion. The durations of warm and cold ischemia of the donor livers in each group were consistent with those of the experimental groups. The average time of the anhepatic phase ranged from 20 minutes to 24 minutes (21.78 1.68 minutes). The experimental rats were randomly grouped. The harvested liver tissues were fixed with 10% formaldehyde, embedded in paraffin, serially sliced in 4-mm thick sections, and stained with hematoxylin and eosin. The liver tissues were observed under an ordinary optical microscope, and the expression of donor liver cell apoptosis was detected using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. Apoptosis kits (Wuhan Boster, China) were used. TdT enzyme-free reaction buffer (PBS)
51 Table 1. The Experimental Rats Were Divided Into 6 Groups, Ischemia Time (x s) Ischemia Time Group
Warm Ischemia Time
I: syngeneic sham group 0 II: syngeneic liver 1.42 min 0.64 min transplantation group III: allogeneic liver 1.42 min 0.64 min transplantation group IV: allogeneic liver 1.42 min 0.64 min transplantation group V: allogeneic liver 13.56 min 1.78 min transplantation group VI: allogeneic liver 13.56 min 1.78 min transplantation group
Cold Ischemia Time
0 80 min 80 min 10 h 80 min 10 h
was used as a negative control sample. Tissue sections treated with DNase I were used as a positive control sample. The cells with a buffy-stained nucleus viewed under an optical microscope were considered apoptotic. The morphology of an apoptotic cell should meet the following requirements: a single cell; no inflammatory reactions or apoptosis around the cell; cell membrane shrinkage; and a densely stained nucleus presenting buffy-stained particles or fragments. Five visual fields (400) were selected for each section and the apoptotic cells were counted. The apoptotic index (AI) was calculated using the following formula: AI ¼ (the number of apoptotic cells/the total number of cells) 100%. The protein expression levels of Fas, perforin, and granzyme B were detected in paraffin-embedded liver tissues using immunohistochemical staining. Immunohistochemistry was performed according to the manufacturer’s instructions. Ten visual fields (400) viewed under a light microscope were selected randomly for each section. An HPIAS-1000 automatic pathological image analysis system was used to determine the mean absorbance of the buffystained granules in the hepatic cells. The messenger RNA (mRNA) expression levels of their respective genes were detected using real-time polymerase chain reaction (PCR). Total RNA was extracted from the hepatic tissue using one-step extraction with a Trizol kit (Canada Bio Basic Inc.). Ultraviolet spectrophotometry was performed to determine the density and the purity of the total RNA. Agarose gel electrophoresis was performed to detect the integrity of the total RNA. The obtained substance was reverse transcribed to complementary DNA (cDNA) for PCR. The optical densities of the target genes (Fas mRNA, perforin mRNA, and granzyme B mRNA) and b-actin were determined using the SYNGENE gel imaging system. The relative expression level of a target gene was presented as the ratio between the optical densities of the target gene and b-actin. The Fas, perforin, and granzyme B gene sequences of the rats were obtained from http://www.ncbi.nlm.nih.gov/ nuccore/ (GenBank Accession Nos.: X66539, NM016993, BC063166). The primer sequences of the Fas, perforin,
52
WANG, WU, JIANG ET AL Table 2. Score of AR of Rats After Liver Transplantation Reperfusion (x s) Group
Time (d)
1 3 5 7
I
1.58 1.63 1.58 1.55
II
0.11 0.08 0.11 0.12
1.45 1.48 1.62 1.56
III
0.12 0.13 0.16 0.08
2.62 3.78 4.67 5.18
IV
0.16*,† 0.32*,† 0.24*,† 0.27*,†
2.67 3.98 4.96 6.03
V
0.18*,† 0.36*,† 0.28*,† 0.31*,†
2.85 4.32 5.32 7.13
VI
0.21*,† 0.33*,† 0.26*,† 0.24*,†
1.87 1.98 2.12 2.05
0.22*,† 0.24*,† 0.18*,† 0.15*,†
*Vs group I; P < .05. † Vs group II; P < .05.
granzyme B, and b-actin genes were listed as follows: P1, 50 -CAAGGGACTGATAGCATCTTTGAGG-30 ; P2, 50 -CAAGGGACTGATAGCATCTTTGAGG-30 ; P3, 50 TGCTACACTGCCACTCGGTCA-30 ; P4: 50 -GCATGCT CTGTGGAGCTGTTA-30 ; P5, 50 -GACTTTGTGCTGA CTGCTGCTCAC-30 ; P6, 50 -GACTTTGTGCTGACTGC TGCTCAC-30 ; P7, 50 -GCCATCCTGCGTCT. GGACCTG-30 ; and P8, 50 -CATTTGCGGTGCACGATGGAG-30 . The reaction conditions used were as follows: denaturation at 95 C for 5 minutes; denaturation at 95 C for 15 seconds; annealing at 60 C for 60 seconds; extension at 72 C for 15 seconds; and 42 cycles, complete extension at 60 C for 10 minutes. b-actin was used as reference. Statistical Analysis
Statistical analysis was performed in SPSS version 11.5. Experimental data were presented as mean standard deviation (x s). One-way analysis of variance (ANOVA) was used to compare the means of multiple samples. q-test was performed for pairwise comparisons between means. P < .05 was considered significant. RESULTS I/R Injury
I/R injury was observed in rat liver transplantation. The severity of I/R injury after rat liver transplantation gradually increased as the duration of warm and cold ischemia was extended. Scattered degeneration and necrosis in the liver cells were postoperatively observed at specific time points in groups II and III. Degeneration and focal necrosis in the liver tissue and the bile duct were postoperatively noted at specific time points in group IV. Sheet degeneration and necrosis, moderate and severe expansion of the sinus hepaticus, eosinophil infiltration, and central venous dermatitis
were postoperatively observed in the liver tissues at specific time points in group V. Ballooning, lobule structure damage, atrophy, and disappearing hepatic cords with oppression were postoperatively observed in the liver cells at specific time points in group VI. Rejection Score of Transplanted Liver Tissue
The rejection scores of the transplanted liver tissues in the allogeneic groups were significantly increased compared with those in group I at 3, 5, and 7 days after donor liver reperfusion (P < .05). Similar significant changes were noted in the rejection scores of groups II and VI (both at P < .05) compared with the allogeneic groups. Significant differences were also observed in the rejection scores of transplanted liver tissues among groups III, IV, and V at 3, 5, and 7 days after donor liver reperfusion (P < .05). Likewise, significant differences were found in the rejection scores across different time points (P < .05), in which the maximum rate of rejection was reached at 7 days after transplantation (Table 2). AI of Transplanted Liver Tissue
A very small number of apoptotic cells were observed in the liver tissues of group I at all time points. AIs of groups II, III, IV, and V were significantly increased at 1, 3, 5, and 7 days after donor liver reperfusion (P < .05) compared with those of groups I and VI. AIs of groups III, IV, and V were also increased at 5 and 7 days after donor liver reperfusion (P < .05) compared with group II. These results are summarized in Table 3. Fas, Perforin, and Granzyme B Expressions
The protein and mRNA expressions of Fas, perforin, and granzyme B were not observed in rat liver tissues at 1, 3, 5, and 7 days after transplantation in groups I and II, whereas
Table 3. The Result of AI in Liver Tissue After Rats Liver Graft Reperfusion (%, x s) Group Time (d)
1 3 5 7
I
2.11 2.23 2.18 2.24
*Vs group I; P < .05. † Vs group II; P < .05.
II
0.36 0.43 0.32 0.35
16.82 26.38 24.27 22.26
III
3.36* 3.85* 4.43* 3.52
17.76 28.54 33.35 37.18
IV
2.42*,† 3.63*,† 4.22*,† 2.37
19.25 30.17 37.48 40.23
V
3.42*,† 3.54*,† 4.76*,† 4.67
30.76 38.23 43.34 49.16
VI
4.55*,† 2.31*,† 4.12*,† 4.45
3.78 3.18 2.02 2.16
0.25 0.24 0.62 0.16
I/R INJURY AND AR
53
Table 4. The Result of Fas, Perforin, and Granzyme B Protein Expression in Liver Tissue After Rats Liver Graft Reperfusion (x s) Group D
I
Fas 1d 3d 5d 7d Perforin 1d 3d 5d 7d GranzymeB 1d 3d 5d 7d
0.0175 0.0208 0.0191 0.0199
II
0.0036 0.0054 0.0040 0.0032
0.0705 0.0905 0.0718 0.0778
III
0.0102 0.0133 0.0036 0.0041
IV
V
VI
0.0801 0.0991 0.1265 0.1435
0.0076*,† 0.0125*,† 0.0137*,† 0.0165*,†
0.0971 0.1186 0.1376 0.1562
0.0085*,† 0.0093*,† 0.0132*,† 0.0112*,†
0.1245 0.1487 0.1732 0.1923
0.0073*,† 0.0025*,† 0.0045*,† 0.0032*,†
0.0146 0.0177 0.0199 0.0154
0.0023 0.0028 0.0034 0.0029
0 0 0 0
0 0 0 0
0.0125 0.0575 0.0752 0.0935
0.0032*,† 0.0145*,† 0.0127*,† 0.0165*,†
0.0128 0.0786 0.1262 0.1672
0.0087*,† 0.0076*,† 0.0172*,† 0.0108*,†
0.0132 0.0978 0.1574 0.1962
0.0021*,† 0.0054*,† 0.0086*,† 0.0057*,†
0.0186 0.0154 0.0168 0.0173
0.0032 0.0043 0.0037 0.0049
0 0 0 0
0 0 0 0
0.0101 0.0391 0.0565 0.0835
0.0016*,† 0.0125*,† 0.0137*,† 0.0165*,†
0.0112 0.0485 0.0668 0.1062
0.0045*,† 0.0037*,† 0.0076*,† 0.0117*,†
0.0106 0.0608 0.0796 0.1279
0.0039*,† 0.0054*,† 0.0056*,† 0.0108*,†
0.0196 0.0184 0.0172 0.0168
0.0077 0.0048 0.0054 0.0032
† Compared with Igroup; P < .05. *Compared with IIgroup; P < .05.
AR via Ca2þ overload. Cell apoptosis can also be activated in sinusoidal and microvascular endothelial cells (SAMEC) [6e8]; this process is also an important phenomenon during I/R injury and AR of transplants. I/R injury and high amounts of cytokines can stimulate SAMEC to express various chemical factors, such as functional Fas ligand (FasL), thereby causing the apoptosis of target cells expressing Fas [9e12]. Cytotoxic T lymphocytes (CTL) can attack target cells based on 2 methods. First, FasL can be released to induce apoptosis of target cells containing Fas receptors on their surfaces. The antigen can stimulate these target cells to express Fas because CTL can activate FasL expression via antigen-presenting cells and helper T cells. The FasL on CTL then binds to the Fas on target cells to induce apoptosis. Thus, the Fas/FasL pathway is the primary
limited expression was noted in group VI. By contrast, the expression levels of these genes were significantly increased in groups III, IV, and V compared with groups I, II, and VI (P < .05). The severity of I/R injury was aggravated in groups III, IV, and V at 3, 5, and 7 days after transplantation. The expression levels of these genes gradually increased with significant differences between groups (P < .05; Tables 4 and 5; Fig 1 and Fig 2). DISCUSSION
The occurrence of I/R injury is a clinically important cause of AR and chronic rejection as well as other specific immune reactions after liver transplantation and early loss of the function of transplanted livers [3e5]. I/R injury causes
Table 5. The Result of FasmRNA, PerforinmRNA and Granzyme B mRNA Expression in Liver Tissue After Rats Liver Graft Reperfusion (x s) Group Day
FasmRNA 1d 3d 5d 7d PerforinmRNA 1d 3d 5d 7d Granzyme B mRNA 1d 3d 5d 7d
I
0.356 0.352 0.328 0.295
*Compared with Igroup, P < .05. † Compared with IIgroup, P <.05.
II
0.061 0.054 0.038 0.034
0.276 0.294 0.315 0.265
III
0.016 0.018 0.022 0.012
IV
V
VI
0.284 0.357 0.526 0.693
0.011*,† 0.048*,† 0.037*,† 0.056*,†
0.321 0.385 0.576 0.734
0.013*,† 0.015*,† 0.014*,† 0.031*,†
0.354 0.405 0.598 0.787
0.0168*,† 0.0275*,† 0.0247*,† 0.0349*,†
0.385 0.411 0.404 0.356
0.025 0.026 0.027 0.022
0 0 0 0
0 0 0 0
0.075 0.154 0.302 0.458
0.012*,† 0.026*,† 0.038*,† 0.032*,†
0.115 0.184 0.365 0.525
0.015*,† 0.018*,† 0.022*,† 0.037*,†
0.162 0.225 0.402 0.608
0.016*,† 0.027*,† 0.024*,† 0.035*,†
0.055 0.063 0.058 0.067
0.025 0.028 0.016 0.021
0 0 0 0
0 0 0 0
0.063 0.182 0.345 0.506
0.019*,† 0.031*,† 0.041*,† 0.032*,†
0.125 0.197 0.387 0.575
0.018*,† 0.023*,† 0.032*,† 0.026*,†
0.139 0.225 0.402 0.608
0.016*,† 0.027*,† 0.024*,† 0.035*,†
0.052 0.059 0.062 0.058
0.018 0.023 0.017 0.022
54
WANG, WU, JIANG ET AL
Fig 1. The positive expression of Fas in liver tissue of group V 5 days after ROLT (original magnification 400).
Fig 2. The positive expression of granzyme B in liver tissue of group V 5 days after ROLT (original magnification 200).
mechanism of cell apoptosis. Simultaneously, FasL can induce apoptosis of the surrounding Fas-positive T cells. FasL can also target B cells, neutrophils, and monocytes. Second, perforin and granzyme B expressions are increased. Only activated CTL can express these enzymes. After natural killer cells recognize their target cells, cytolytic granules are released to induce apoptosis. Perforin in these cytolytic granules can target cells by forming pores in their cell membranes. Granzyme B can enter the cells and cause DNA fragmentation, dissociation, and cell apoptosis. We established different liver transplantation models with varying severities of I/R injury based on different durations of warm and cold ischemia after the preliminary experiments were performed to determine the effect of different levels of I/R injury on the occurrence of AR after liver transplantation in rats. Significant differences were observed among groups I to VI, in which the severity of I/R injury gradually increased in the donor livers after reperfusion. Histopathologic examination showed that I/R injury occurred in the rats after liver transplantation. The pathological scope and degree were proportional to the severity of I/R injury. In group VI, fused necrotic liver cells from livers exhibiting damaged hepatic lobular architecture, highly expanded sinus hepaticus, and atrophy were observed. Furthermore, the hepatic cord occasionally disappeared with oppression. The rejection scores of the transplanted liver tissues gradually increased and became proportional to the severity of I/R injury in groups III to V. The highest rejection score was obtained at 7 days after transplantation with an extended duration. No rejection was observed at each time period in groups I, II, and VI. I/R injury levels during the allogeneic rat liver transplantation ranged from mild to severe. The transport capacity of Ca2þ in the liver cell membrane system was impaired with a generally aggravated severity of I/R injury. The energy required by Ca2þ pumps in the plasmalemma and the endoplasmic reticulum decreased until the Ca2þ pump proteins in both organelles decreased. Thereafter, Ca2þ in the liver cells
reached an overloaded condition, and the local microcirculation disorder worsened. Thus, intracellular ATP levels were gradually reduced and Naþ-Kþ-ATPase activity decreased. As a result, more Naþ/Ca2þ exchange proteins in the cell membranes were activated. During reperfusion, intracellular Naþ escaped from the cells and then extracellular Ca2þ entered the cells, thereby causing a more serious Ca2þ overload. The aggravated apoptosis can simultaneously activate SAMEC to express various chemical factors to increase mRNA and protein expressions of Fas, perforin, and granzyme B and to produce more AR based on Fas/FasL and perforin/granzyme B methods [3,13]. The occurrence of AR after allogeneic rat liver transplantation was positively correlated with the severity of I/R injury. However, the high ATP levels in liver cells, liver mesenchymal cells, and SAMEC were decreased when I/R injury caused serious damage to the transplanted liver. As a result, cell degeneration and necrosis occurred and the lobular architecture was damaged. The sinus hepaticus was highly expanded, whereas the liver cord underwent atrophy and eventually disappeared. Thus, the necessary chemical factors are not released to initiate Fas/FasL [14,15] and perforin/granzyme B [16,17] mechanisms that induce apoptosis. The occurrence of AR is also reduced [18,19]. The results indicated that the occurrence of AR after allogeneic liver transplantation in rats was positively correlated with the severity of I/R injury. However, the occurrence of AR was evidently decreased after I/R injury caused serious damage to the transplanted liver. The mRNA expression levels of Fas, perforin, and granzyme B in the liver cells were positively correlated with the severity of AR, indicating that the mRNAs of perforin and granzyme B were important markers of AR. This analysis could provide the basis of monitoring and early diagnosis of AR. In groups II to V, Fas mRNA expression increased gradually as I/R injury was aggravated after donor liver reperfusion, showing significant differences between groups (P < .05). The expressions in all of these groups were significantly higher than those in groups I and VI at all
I/R INJURY AND AR
detection time points (P < .05). Such expressions reached the peaks at 7 days after transplantation. In groups III, IV, and V, Fas mRNA expressions in the hepatic tissues were significantly increased at 5 and 7 days after transplantation (P < .05) compared with those in group II. In normal rats, the expression of intrahepatic Fas mRNA was low. By contrast, the expression of intrahepatic Fas mRNA was noticeably increased in the case of I/R injury (from slight to severe) and AR after transplantation. Furthermore, the severity of I/R injury remained consistent with that of AR and showed a positive correlation with the number of apoptotic hepatic cells. These results could be attributed to the gradual aggravation of donor liver I/R injury that also gradually exacerbated Ca2þ overload and cellular apoptosis. Such an aggravation also up-regulated the Fas mRNA, thereby exacerbating AR gradually via the Fas/FasL pathway. In addition, group VI suffered from the most severe I/R injury, in which various protein pumps on the cell membrane were deactivated and caused cell degeneration and apoptosis. As a result, Fas mRNA could not be expressed and AR could not be induced. This finding indicated that the influence of I/R injury on AR of allogeneic liver transplants was slightly correlated with the activation of the Fas/FasL pathway and SAMEC apoptosis. The mRNA expressions of perforin and granzyme B were not observed at any of the time points in groups I and II. By contrast, the mRNA expressions of perforin and granzyme B in the liver cells were gradually increased as the degrees of I/R injury and AR were aggravated in groups III, IV, and V. I/R injuries of the liver tissues from the rats in group VI were the most serious without visible rejection. Only small amounts of perforin and granzyme B mRNAs were expressed at each time point after transplantation. The anchorage-independent values and Banff histological diagnostic criterion rankings for perforin and granzyme B were highly consistent. Thus, perforin and granzyme B were potentially valuable markers of AR. Their dynamic analysis could provide the basis of monitoring and early diagnosis of AR. To improve the diagnostic accuracy of AR after liver transplantation and expedite diagnosis, perforin and granzyme B expressions could be used as routine indexes of differential diagnosis of liver-punctured biopsy specimens. REFERENCES [1] Wu C, Xia Y, Wang P, et al. Triptolide protects mice from ischemia reperfusion injury by inhibition of IL-17 production. Int Immunopharmacol 2011;11:1564. [2] Abu-Amara M, Yang SY, Quaglia A, et al. Role of endothelial nitric oxide synthase in remote ischemic preconditioning of the mouse liver. Liver Transpl 2011;17:610.
55 [3] Netto MV, Fonseca BA, Dantas M, et al. Granzyne B Fasligand and perforin expression during acute cellular rejection episodes after kidney transplantation: comparison between blood and renal aspiration. Transplant Proc 2002;34:476. [4] Surquin M, Le Moine A, Flamand V, et al. Skin graft rejection elicited by bete-2-mecroglobulin as a minor transplantion antigen involves multiple effector pathways, roles of fas-fas ligand interactions and Th2-dependent graft eosinophil infiltrates. J Inununol 2002;169:500. [5] Wang J, Li W, Min J, et al. Fas siRNA reduces apoptotic cell death of allogeneic transplanted hepatocytes in mouse spleen. Transplant Proc 2003;35:1594. [6] Du C, Guan Q, Yin Z, et al. Renal tubular epithelial cell apoptosis by self-injury can augment renal allograft injury Fas-FasLdependent. Transplant Proc 2003;35:2481. [7] Jiang H, Pan F, Erickson LM, et al. Deletion of DOCK2, a regulator of the actin cytoskeleton in lymphocytes, suppresses cardiac allograft rejection. J Exp Med 2005;202:1121. [8] Bittmann I, Muller C, Behr J, et al. Fas/FasL and perforin/ granzyme pathway in acute rejection and diffuse alveolar damage after allogeneic lung transplantation-a human biopsy study. Virchows Arch 2004;445:375. [9] Veale JL, Liang LW, Zhang Q, et al. Noninvasive diagnosis of cellular and antibody-mediated rejection by perforin and granzyme B in renal allografts. Hum Immunol 2006;67:777. [10] Graziotto R, Del Prete D, Rigotti P, et al. Perforin, Granzyme B, and fas ligand for molecular diagnosis of acute renal-allograft rejection: analyses on serial biopsies suggest methodological issues. Transplantation 2006;81:1125. [11] Corti B, Altimari A, Gabusi E, et al. Potential of real-time PCR assessment of granzyme B and perforin up-regulation for rejection monitoring in intestinal transplant recipients. Transplant Proc 2005;37:4467. [12] D’Errico A, Corti B, Pinna AD, et al. Granzyme B and perforin as predictive markers for acute rejection in human intestinal transplantation. Transplant Proc 2003;35:3061. [13] Yu YY, Deng XX, Ji J, et al. [Sensitivity and specificity of granzyme B and perforin in diagnosing acute rejection after liver transplantation]. Zhonghua Bing Li Xue Za Zhi 2005;34:198. [14] Wei N, Liu GT, Chen XG, et al. H1, a derivative of Tetrandrine, exerts anti-MDR activity by initiating intrinsic apoptosis pathway and inhibiting the activation of Erk1/2 and Akt1/2. Biochem Pharmacol 2011;82:1593. [15] Okazaki A, Hiraga N, Imamura M, et al. Severe necroinflammatory reaction caused by natural killer cell-mediated Fas/Fas ligand interaction and dendritic cells in human hepatocyte chimeric mouse. Hepatology 2012;56:555. [16] Chakraborty T, Bose A, Goswami KK, et al. Neem leaf glycoprotein suppresses regulatory T cell mediated suppression of monocyte/macrophage functions. Int Immunopharmacol 2012; 12:326. [17] Cerezo LA, Kuncová K, Mann H, et al. The metastasis promoting protein S100A4 is increased in idiopathic inflammatory myopathies. Rheumatology (Oxford) 2011;50:1766. [18] Yannaraki M, Rebibou JM, Ducloux D, et al. Urinary cytotoxic molecular markers for a noninvasive diagnosis in acute renal transplant rejection. Transpl Int 2006;19:759. [19] Galante NZ, Camara NO, Kallas EG, et al. Noninvasive immune monitoring assessed by flow cytometry and real time RTPCR in urine of renal transplantation recipients. Transpl Immunol 2006;16:73.
T
HE INCIDENCE rate of acute rejection (AR) after liver transplantation is approximately 40% to 80%. AR causes functional losses affecting the survival of liver transplant recipients. In China, liver transplantation mostly involves noneheart-beating donors. In addition to the innate characteristics of donor livers, the primary factors influencing the quality of donor livers are the severity of warm ischemia, cold ischemia, and ischemia/reperfusion (I/R) injury. I/R injury is an important cause of AR and chronic rejection [1,2]. This type of injury affects specific immune processes after liver transplantation and an early functional loss of the transplanted liver. New sources of donor livers are currently developed, although existing donor liver resources are maximized. New solutions have been proposed to solve the shortage of donor liver cells. For instance, the quality of donor livers should be improved to reduce the injury of donor livers in vitro or after recovery from perfusion and prevent the onset of various complications after liver transplantation, particularly the incidence of AR. 0041-1345/13/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.06.019 50
Therefore, the reduction of the severity of I/R injury after liver transplantation to prevent and control AR is clinically important. In this study, an animal model of AR was established after the liver was transplanted in rats to study the effect of different degrees of I/R injuries and hepatocyte apoptosis during AR after liver transplantation. This animal model was also used to determine the expression levels of Fas, perforin, and granzyme B. Our preliminary findings revealed the relationship between the severities of I/R injury and AR after liver transplantation. The mechanisms involved were also determined.
From the Department of Hepatobiliary Surgery, Hunan Provincial People’s Hospital, Changsha, Hunan Province, China. Address reprint requests to Jin-shu Wu, Department of Hepatobiliary Surgery, Hunan Provincial People’s Hospital, Changsha 410005, Hunan Province, China. E-mail: ygbzcn@ 126.com ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 46, 50e55 (2014)
I/R INJURY AND AR
MATERIALS AND METHODS Animals The donors were male and female Sprague-Dawley rats. The recipients were 2- to 3-week-old male Lewis rats, weighing 220 g to 280 g (Experimental Animal Department of Central South University), with clean feeding. The donors were provided with food or drink before the experiment. By contrast, the recipients were not provided with food for 6 hours prior to transplantation. The number of recipients was slightly higher than the number of donors. The experimental rats were divided into 6 groups: group I, syngeneic sham group; group II, syngeneic liver transplantation group subjected to warm ischemia and cold ischemia for 2 minutes (1.42 0.64 minutes) and 80 minutes, respectively; and groups III to VI, allogeneic liver transplantation groups. The rats in group III were subjected to warm ischemia and cold ischemia for 2 minutes (1.42 0.64 minutes) and 80 minutes, respectively. The rats in group IV were subjected to warm ischemia and cold ischemia for 2 minutes (1.42 0.64 minutes) and 10 hours, respectively. The rats in group V were subjected to warm ischemia and cold ischemia for 15 minutes (13.56 1.78 minutes) and 80 minutes, respectively. The rats in group VI were subjected to warm ischemia and cold ischemia for 15 minutes (13.56 1.78 minutes) and 10 hours, respectively. Each group was further divided into 4 subgroups and then humanely killed at 1, 3, 5, or 7 days after transplantation, respectively (n ¼ 3; Table 1). The transplanted livers were harvested on the respective days after transplantation. Blood was then withdrawn from the inferior vena cava. This study was performed in strict compliance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol on animal use was reviewed and approved by the Institutional Animal Care and Use Committee of the Hunan Provincial People’s Hospital, China.
METHODS
Liver transplantation was performed according to an improved version of Kamada’s two-cuff technique. In brief, the subhepatic inferior vena cava and the portal vein were connected based on the cuff technique. The suprahepatic vena cava was then sutured. Stenting was performed for bile ducts. The hepatic artery was reconstructed by the bracket method. The suprahepatic inferior vena cava was reconstructed with an improved continuous suture. Liver perfusion was controlled using an automatic vein infusion pump. The perfusion rate was set at 2 mL/min with a total perfusion volume between 25 and 30 mL to ensure an even perfusion. The durations of warm and cold ischemia of the donor livers in each group were consistent with those of the experimental groups. The average time of the anhepatic phase ranged from 20 minutes to 24 minutes (21.78 1.68 minutes). The experimental rats were randomly grouped. The harvested liver tissues were fixed with 10% formaldehyde, embedded in paraffin, serially sliced in 4-mm thick sections, and stained with hematoxylin and eosin. The liver tissues were observed under an ordinary optical microscope, and the expression of donor liver cell apoptosis was detected using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. Apoptosis kits (Wuhan Boster, China) were used. TdT enzyme-free reaction buffer (PBS)
51 Table 1. The Experimental Rats Were Divided Into 6 Groups, Ischemia Time (x s) Ischemia Time Group
Warm Ischemia Time
I: syngeneic sham group 0 II: syngeneic liver 1.42 min 0.64 min transplantation group III: allogeneic liver 1.42 min 0.64 min transplantation group IV: allogeneic liver 1.42 min 0.64 min transplantation group V: allogeneic liver 13.56 min 1.78 min transplantation group VI: allogeneic liver 13.56 min 1.78 min transplantation group
Cold Ischemia Time
0 80 min 80 min 10 h 80 min 10 h
was used as a negative control sample. Tissue sections treated with DNase I were used as a positive control sample. The cells with a buffy-stained nucleus viewed under an optical microscope were considered apoptotic. The morphology of an apoptotic cell should meet the following requirements: a single cell; no inflammatory reactions or apoptosis around the cell; cell membrane shrinkage; and a densely stained nucleus presenting buffy-stained particles or fragments. Five visual fields (400) were selected for each section and the apoptotic cells were counted. The apoptotic index (AI) was calculated using the following formula: AI ¼ (the number of apoptotic cells/the total number of cells) 100%. The protein expression levels of Fas, perforin, and granzyme B were detected in paraffin-embedded liver tissues using immunohistochemical staining. Immunohistochemistry was performed according to the manufacturer’s instructions. Ten visual fields (400) viewed under a light microscope were selected randomly for each section. An HPIAS-1000 automatic pathological image analysis system was used to determine the mean absorbance of the buffystained granules in the hepatic cells. The messenger RNA (mRNA) expression levels of their respective genes were detected using real-time polymerase chain reaction (PCR). Total RNA was extracted from the hepatic tissue using one-step extraction with a Trizol kit (Canada Bio Basic Inc.). Ultraviolet spectrophotometry was performed to determine the density and the purity of the total RNA. Agarose gel electrophoresis was performed to detect the integrity of the total RNA. The obtained substance was reverse transcribed to complementary DNA (cDNA) for PCR. The optical densities of the target genes (Fas mRNA, perforin mRNA, and granzyme B mRNA) and b-actin were determined using the SYNGENE gel imaging system. The relative expression level of a target gene was presented as the ratio between the optical densities of the target gene and b-actin. The Fas, perforin, and granzyme B gene sequences of the rats were obtained from http://www.ncbi.nlm.nih.gov/ nuccore/ (GenBank Accession Nos.: X66539, NM016993, BC063166). The primer sequences of the Fas, perforin,
52
WANG, WU, JIANG ET AL Table 2. Score of AR of Rats After Liver Transplantation Reperfusion (x s) Group
Time (d)
1 3 5 7
I
1.58 1.63 1.58 1.55
II
0.11 0.08 0.11 0.12
1.45 1.48 1.62 1.56
III
0.12 0.13 0.16 0.08
2.62 3.78 4.67 5.18
IV
0.16*,† 0.32*,† 0.24*,† 0.27*,†
2.67 3.98 4.96 6.03
V
0.18*,† 0.36*,† 0.28*,† 0.31*,†
2.85 4.32 5.32 7.13
VI
0.21*,† 0.33*,† 0.26*,† 0.24*,†
1.87 1.98 2.12 2.05
0.22*,† 0.24*,† 0.18*,† 0.15*,†
*Vs group I; P < .05. † Vs group II; P < .05.
granzyme B, and b-actin genes were listed as follows: P1, 50 -CAAGGGACTGATAGCATCTTTGAGG-30 ; P2, 50 -CAAGGGACTGATAGCATCTTTGAGG-30 ; P3, 50 TGCTACACTGCCACTCGGTCA-30 ; P4: 50 -GCATGCT CTGTGGAGCTGTTA-30 ; P5, 50 -GACTTTGTGCTGA CTGCTGCTCAC-30 ; P6, 50 -GACTTTGTGCTGACTGC TGCTCAC-30 ; P7, 50 -GCCATCCTGCGTCT. GGACCTG-30 ; and P8, 50 -CATTTGCGGTGCACGATGGAG-30 . The reaction conditions used were as follows: denaturation at 95 C for 5 minutes; denaturation at 95 C for 15 seconds; annealing at 60 C for 60 seconds; extension at 72 C for 15 seconds; and 42 cycles, complete extension at 60 C for 10 minutes. b-actin was used as reference. Statistical Analysis
Statistical analysis was performed in SPSS version 11.5. Experimental data were presented as mean standard deviation (x s). One-way analysis of variance (ANOVA) was used to compare the means of multiple samples. q-test was performed for pairwise comparisons between means. P < .05 was considered significant. RESULTS I/R Injury
I/R injury was observed in rat liver transplantation. The severity of I/R injury after rat liver transplantation gradually increased as the duration of warm and cold ischemia was extended. Scattered degeneration and necrosis in the liver cells were postoperatively observed at specific time points in groups II and III. Degeneration and focal necrosis in the liver tissue and the bile duct were postoperatively noted at specific time points in group IV. Sheet degeneration and necrosis, moderate and severe expansion of the sinus hepaticus, eosinophil infiltration, and central venous dermatitis
were postoperatively observed in the liver tissues at specific time points in group V. Ballooning, lobule structure damage, atrophy, and disappearing hepatic cords with oppression were postoperatively observed in the liver cells at specific time points in group VI. Rejection Score of Transplanted Liver Tissue
The rejection scores of the transplanted liver tissues in the allogeneic groups were significantly increased compared with those in group I at 3, 5, and 7 days after donor liver reperfusion (P < .05). Similar significant changes were noted in the rejection scores of groups II and VI (both at P < .05) compared with the allogeneic groups. Significant differences were also observed in the rejection scores of transplanted liver tissues among groups III, IV, and V at 3, 5, and 7 days after donor liver reperfusion (P < .05). Likewise, significant differences were found in the rejection scores across different time points (P < .05), in which the maximum rate of rejection was reached at 7 days after transplantation (Table 2). AI of Transplanted Liver Tissue
A very small number of apoptotic cells were observed in the liver tissues of group I at all time points. AIs of groups II, III, IV, and V were significantly increased at 1, 3, 5, and 7 days after donor liver reperfusion (P < .05) compared with those of groups I and VI. AIs of groups III, IV, and V were also increased at 5 and 7 days after donor liver reperfusion (P < .05) compared with group II. These results are summarized in Table 3. Fas, Perforin, and Granzyme B Expressions
The protein and mRNA expressions of Fas, perforin, and granzyme B were not observed in rat liver tissues at 1, 3, 5, and 7 days after transplantation in groups I and II, whereas
Table 3. The Result of AI in Liver Tissue After Rats Liver Graft Reperfusion (%, x s) Group Time (d)
1 3 5 7
I
2.11 2.23 2.18 2.24
*Vs group I; P < .05. † Vs group II; P < .05.
II
0.36 0.43 0.32 0.35
16.82 26.38 24.27 22.26
III
3.36* 3.85* 4.43* 3.52
17.76 28.54 33.35 37.18
IV
2.42*,† 3.63*,† 4.22*,† 2.37
19.25 30.17 37.48 40.23
V
3.42*,† 3.54*,† 4.76*,† 4.67
30.76 38.23 43.34 49.16
VI
4.55*,† 2.31*,† 4.12*,† 4.45
3.78 3.18 2.02 2.16
0.25 0.24 0.62 0.16
I/R INJURY AND AR
53
Table 4. The Result of Fas, Perforin, and Granzyme B Protein Expression in Liver Tissue After Rats Liver Graft Reperfusion (x s) Group D
I
Fas 1d 3d 5d 7d Perforin 1d 3d 5d 7d GranzymeB 1d 3d 5d 7d
0.0175 0.0208 0.0191 0.0199
II
0.0036 0.0054 0.0040 0.0032
0.0705 0.0905 0.0718 0.0778
III
0.0102 0.0133 0.0036 0.0041
IV
V
VI
0.0801 0.0991 0.1265 0.1435
0.0076*,† 0.0125*,† 0.0137*,† 0.0165*,†
0.0971 0.1186 0.1376 0.1562
0.0085*,† 0.0093*,† 0.0132*,† 0.0112*,†
0.1245 0.1487 0.1732 0.1923
0.0073*,† 0.0025*,† 0.0045*,† 0.0032*,†
0.0146 0.0177 0.0199 0.0154
0.0023 0.0028 0.0034 0.0029
0 0 0 0
0 0 0 0
0.0125 0.0575 0.0752 0.0935
0.0032*,† 0.0145*,† 0.0127*,† 0.0165*,†
0.0128 0.0786 0.1262 0.1672
0.0087*,† 0.0076*,† 0.0172*,† 0.0108*,†
0.0132 0.0978 0.1574 0.1962
0.0021*,† 0.0054*,† 0.0086*,† 0.0057*,†
0.0186 0.0154 0.0168 0.0173
0.0032 0.0043 0.0037 0.0049
0 0 0 0
0 0 0 0
0.0101 0.0391 0.0565 0.0835
0.0016*,† 0.0125*,† 0.0137*,† 0.0165*,†
0.0112 0.0485 0.0668 0.1062
0.0045*,† 0.0037*,† 0.0076*,† 0.0117*,†
0.0106 0.0608 0.0796 0.1279
0.0039*,† 0.0054*,† 0.0056*,† 0.0108*,†
0.0196 0.0184 0.0172 0.0168
0.0077 0.0048 0.0054 0.0032
† Compared with Igroup; P < .05. *Compared with IIgroup; P < .05.
AR via Ca2þ overload. Cell apoptosis can also be activated in sinusoidal and microvascular endothelial cells (SAMEC) [6e8]; this process is also an important phenomenon during I/R injury and AR of transplants. I/R injury and high amounts of cytokines can stimulate SAMEC to express various chemical factors, such as functional Fas ligand (FasL), thereby causing the apoptosis of target cells expressing Fas [9e12]. Cytotoxic T lymphocytes (CTL) can attack target cells based on 2 methods. First, FasL can be released to induce apoptosis of target cells containing Fas receptors on their surfaces. The antigen can stimulate these target cells to express Fas because CTL can activate FasL expression via antigen-presenting cells and helper T cells. The FasL on CTL then binds to the Fas on target cells to induce apoptosis. Thus, the Fas/FasL pathway is the primary
limited expression was noted in group VI. By contrast, the expression levels of these genes were significantly increased in groups III, IV, and V compared with groups I, II, and VI (P < .05). The severity of I/R injury was aggravated in groups III, IV, and V at 3, 5, and 7 days after transplantation. The expression levels of these genes gradually increased with significant differences between groups (P < .05; Tables 4 and 5; Fig 1 and Fig 2). DISCUSSION
The occurrence of I/R injury is a clinically important cause of AR and chronic rejection as well as other specific immune reactions after liver transplantation and early loss of the function of transplanted livers [3e5]. I/R injury causes
Table 5. The Result of FasmRNA, PerforinmRNA and Granzyme B mRNA Expression in Liver Tissue After Rats Liver Graft Reperfusion (x s) Group Day
FasmRNA 1d 3d 5d 7d PerforinmRNA 1d 3d 5d 7d Granzyme B mRNA 1d 3d 5d 7d
I
0.356 0.352 0.328 0.295
*Compared with Igroup, P < .05. † Compared with IIgroup, P <.05.
II
0.061 0.054 0.038 0.034
0.276 0.294 0.315 0.265
III
0.016 0.018 0.022 0.012
IV
V
VI
0.284 0.357 0.526 0.693
0.011*,† 0.048*,† 0.037*,† 0.056*,†
0.321 0.385 0.576 0.734
0.013*,† 0.015*,† 0.014*,† 0.031*,†
0.354 0.405 0.598 0.787
0.0168*,† 0.0275*,† 0.0247*,† 0.0349*,†
0.385 0.411 0.404 0.356
0.025 0.026 0.027 0.022
0 0 0 0
0 0 0 0
0.075 0.154 0.302 0.458
0.012*,† 0.026*,† 0.038*,† 0.032*,†
0.115 0.184 0.365 0.525
0.015*,† 0.018*,† 0.022*,† 0.037*,†
0.162 0.225 0.402 0.608
0.016*,† 0.027*,† 0.024*,† 0.035*,†
0.055 0.063 0.058 0.067
0.025 0.028 0.016 0.021
0 0 0 0
0 0 0 0
0.063 0.182 0.345 0.506
0.019*,† 0.031*,† 0.041*,† 0.032*,†
0.125 0.197 0.387 0.575
0.018*,† 0.023*,† 0.032*,† 0.026*,†
0.139 0.225 0.402 0.608
0.016*,† 0.027*,† 0.024*,† 0.035*,†
0.052 0.059 0.062 0.058
0.018 0.023 0.017 0.022
54
WANG, WU, JIANG ET AL
Fig 1. The positive expression of Fas in liver tissue of group V 5 days after ROLT (original magnification 400).
Fig 2. The positive expression of granzyme B in liver tissue of group V 5 days after ROLT (original magnification 200).
mechanism of cell apoptosis. Simultaneously, FasL can induce apoptosis of the surrounding Fas-positive T cells. FasL can also target B cells, neutrophils, and monocytes. Second, perforin and granzyme B expressions are increased. Only activated CTL can express these enzymes. After natural killer cells recognize their target cells, cytolytic granules are released to induce apoptosis. Perforin in these cytolytic granules can target cells by forming pores in their cell membranes. Granzyme B can enter the cells and cause DNA fragmentation, dissociation, and cell apoptosis. We established different liver transplantation models with varying severities of I/R injury based on different durations of warm and cold ischemia after the preliminary experiments were performed to determine the effect of different levels of I/R injury on the occurrence of AR after liver transplantation in rats. Significant differences were observed among groups I to VI, in which the severity of I/R injury gradually increased in the donor livers after reperfusion. Histopathologic examination showed that I/R injury occurred in the rats after liver transplantation. The pathological scope and degree were proportional to the severity of I/R injury. In group VI, fused necrotic liver cells from livers exhibiting damaged hepatic lobular architecture, highly expanded sinus hepaticus, and atrophy were observed. Furthermore, the hepatic cord occasionally disappeared with oppression. The rejection scores of the transplanted liver tissues gradually increased and became proportional to the severity of I/R injury in groups III to V. The highest rejection score was obtained at 7 days after transplantation with an extended duration. No rejection was observed at each time period in groups I, II, and VI. I/R injury levels during the allogeneic rat liver transplantation ranged from mild to severe. The transport capacity of Ca2þ in the liver cell membrane system was impaired with a generally aggravated severity of I/R injury. The energy required by Ca2þ pumps in the plasmalemma and the endoplasmic reticulum decreased until the Ca2þ pump proteins in both organelles decreased. Thereafter, Ca2þ in the liver cells
reached an overloaded condition, and the local microcirculation disorder worsened. Thus, intracellular ATP levels were gradually reduced and Naþ-Kþ-ATPase activity decreased. As a result, more Naþ/Ca2þ exchange proteins in the cell membranes were activated. During reperfusion, intracellular Naþ escaped from the cells and then extracellular Ca2þ entered the cells, thereby causing a more serious Ca2þ overload. The aggravated apoptosis can simultaneously activate SAMEC to express various chemical factors to increase mRNA and protein expressions of Fas, perforin, and granzyme B and to produce more AR based on Fas/FasL and perforin/granzyme B methods [3,13]. The occurrence of AR after allogeneic rat liver transplantation was positively correlated with the severity of I/R injury. However, the high ATP levels in liver cells, liver mesenchymal cells, and SAMEC were decreased when I/R injury caused serious damage to the transplanted liver. As a result, cell degeneration and necrosis occurred and the lobular architecture was damaged. The sinus hepaticus was highly expanded, whereas the liver cord underwent atrophy and eventually disappeared. Thus, the necessary chemical factors are not released to initiate Fas/FasL [14,15] and perforin/granzyme B [16,17] mechanisms that induce apoptosis. The occurrence of AR is also reduced [18,19]. The results indicated that the occurrence of AR after allogeneic liver transplantation in rats was positively correlated with the severity of I/R injury. However, the occurrence of AR was evidently decreased after I/R injury caused serious damage to the transplanted liver. The mRNA expression levels of Fas, perforin, and granzyme B in the liver cells were positively correlated with the severity of AR, indicating that the mRNAs of perforin and granzyme B were important markers of AR. This analysis could provide the basis of monitoring and early diagnosis of AR. In groups II to V, Fas mRNA expression increased gradually as I/R injury was aggravated after donor liver reperfusion, showing significant differences between groups (P < .05). The expressions in all of these groups were significantly higher than those in groups I and VI at all
I/R INJURY AND AR
detection time points (P < .05). Such expressions reached the peaks at 7 days after transplantation. In groups III, IV, and V, Fas mRNA expressions in the hepatic tissues were significantly increased at 5 and 7 days after transplantation (P < .05) compared with those in group II. In normal rats, the expression of intrahepatic Fas mRNA was low. By contrast, the expression of intrahepatic Fas mRNA was noticeably increased in the case of I/R injury (from slight to severe) and AR after transplantation. Furthermore, the severity of I/R injury remained consistent with that of AR and showed a positive correlation with the number of apoptotic hepatic cells. These results could be attributed to the gradual aggravation of donor liver I/R injury that also gradually exacerbated Ca2þ overload and cellular apoptosis. Such an aggravation also up-regulated the Fas mRNA, thereby exacerbating AR gradually via the Fas/FasL pathway. In addition, group VI suffered from the most severe I/R injury, in which various protein pumps on the cell membrane were deactivated and caused cell degeneration and apoptosis. As a result, Fas mRNA could not be expressed and AR could not be induced. This finding indicated that the influence of I/R injury on AR of allogeneic liver transplants was slightly correlated with the activation of the Fas/FasL pathway and SAMEC apoptosis. The mRNA expressions of perforin and granzyme B were not observed at any of the time points in groups I and II. By contrast, the mRNA expressions of perforin and granzyme B in the liver cells were gradually increased as the degrees of I/R injury and AR were aggravated in groups III, IV, and V. I/R injuries of the liver tissues from the rats in group VI were the most serious without visible rejection. Only small amounts of perforin and granzyme B mRNAs were expressed at each time point after transplantation. The anchorage-independent values and Banff histological diagnostic criterion rankings for perforin and granzyme B were highly consistent. Thus, perforin and granzyme B were potentially valuable markers of AR. Their dynamic analysis could provide the basis of monitoring and early diagnosis of AR. To improve the diagnostic accuracy of AR after liver transplantation and expedite diagnosis, perforin and granzyme B expressions could be used as routine indexes of differential diagnosis of liver-punctured biopsy specimens. REFERENCES [1] Wu C, Xia Y, Wang P, et al. Triptolide protects mice from ischemia reperfusion injury by inhibition of IL-17 production. Int Immunopharmacol 2011;11:1564. [2] Abu-Amara M, Yang SY, Quaglia A, et al. Role of endothelial nitric oxide synthase in remote ischemic preconditioning of the mouse liver. Liver Transpl 2011;17:610.
55 [3] Netto MV, Fonseca BA, Dantas M, et al. Granzyne B Fasligand and perforin expression during acute cellular rejection episodes after kidney transplantation: comparison between blood and renal aspiration. Transplant Proc 2002;34:476. [4] Surquin M, Le Moine A, Flamand V, et al. Skin graft rejection elicited by bete-2-mecroglobulin as a minor transplantion antigen involves multiple effector pathways, roles of fas-fas ligand interactions and Th2-dependent graft eosinophil infiltrates. J Inununol 2002;169:500. [5] Wang J, Li W, Min J, et al. Fas siRNA reduces apoptotic cell death of allogeneic transplanted hepatocytes in mouse spleen. Transplant Proc 2003;35:1594. [6] Du C, Guan Q, Yin Z, et al. Renal tubular epithelial cell apoptosis by self-injury can augment renal allograft injury Fas-FasLdependent. Transplant Proc 2003;35:2481. [7] Jiang H, Pan F, Erickson LM, et al. Deletion of DOCK2, a regulator of the actin cytoskeleton in lymphocytes, suppresses cardiac allograft rejection. J Exp Med 2005;202:1121. [8] Bittmann I, Muller C, Behr J, et al. Fas/FasL and perforin/ granzyme pathway in acute rejection and diffuse alveolar damage after allogeneic lung transplantation-a human biopsy study. Virchows Arch 2004;445:375. [9] Veale JL, Liang LW, Zhang Q, et al. Noninvasive diagnosis of cellular and antibody-mediated rejection by perforin and granzyme B in renal allografts. Hum Immunol 2006;67:777. [10] Graziotto R, Del Prete D, Rigotti P, et al. Perforin, Granzyme B, and fas ligand for molecular diagnosis of acute renal-allograft rejection: analyses on serial biopsies suggest methodological issues. Transplantation 2006;81:1125. [11] Corti B, Altimari A, Gabusi E, et al. Potential of real-time PCR assessment of granzyme B and perforin up-regulation for rejection monitoring in intestinal transplant recipients. Transplant Proc 2005;37:4467. [12] D’Errico A, Corti B, Pinna AD, et al. Granzyme B and perforin as predictive markers for acute rejection in human intestinal transplantation. Transplant Proc 2003;35:3061. [13] Yu YY, Deng XX, Ji J, et al. [Sensitivity and specificity of granzyme B and perforin in diagnosing acute rejection after liver transplantation]. Zhonghua Bing Li Xue Za Zhi 2005;34:198. [14] Wei N, Liu GT, Chen XG, et al. H1, a derivative of Tetrandrine, exerts anti-MDR activity by initiating intrinsic apoptosis pathway and inhibiting the activation of Erk1/2 and Akt1/2. Biochem Pharmacol 2011;82:1593. [15] Okazaki A, Hiraga N, Imamura M, et al. Severe necroinflammatory reaction caused by natural killer cell-mediated Fas/Fas ligand interaction and dendritic cells in human hepatocyte chimeric mouse. Hepatology 2012;56:555. [16] Chakraborty T, Bose A, Goswami KK, et al. Neem leaf glycoprotein suppresses regulatory T cell mediated suppression of monocyte/macrophage functions. Int Immunopharmacol 2012; 12:326. [17] Cerezo LA, Kuncová K, Mann H, et al. The metastasis promoting protein S100A4 is increased in idiopathic inflammatory myopathies. Rheumatology (Oxford) 2011;50:1766. [18] Yannaraki M, Rebibou JM, Ducloux D, et al. Urinary cytotoxic molecular markers for a noninvasive diagnosis in acute renal transplant rejection. Transpl Int 2006;19:759. [19] Galante NZ, Camara NO, Kallas EG, et al. Noninvasive immune monitoring assessed by flow cytometry and real time RTPCR in urine of renal transplantation recipients. Transpl Immunol 2006;16:73.