Abstract

Introduction

An increasing imbalance between the number of donor organs and potential liver transplant recipients has led to the development of novel strategies to increase the pool of organ donors (1). The declaration of brain death (BD) as a point of no return has been accepted by most societies, and organs derived from BD donors currently represent the main source of organs used in transplantation (1,2). Since the first BD donor organ transplantation in the 1960s, clinical transplant results of donor organs have significantly improved (3). To date, the grafts obtained from BD donors have yielded positive outcomes. However, BD is a dynamic and rather unphysiological course of events that influences a number of physiological processes in the human body, whereby potential grafts are damaged before liver transplantation (4). Several experiments demonstrated that systemic and hormonal changes occur immediately under BD conditions, followed by oxidative stress, an inflammatory response, and tissue ischemia reperfusion, all of which result in liver damage (5–8). BD itself has a complicated influence on the liver, thus limiting the number of suitable organs for liver transplantation (6–9). The mechanism underlying the deteriorating effect of BD on organs has not been fully established. Therefore, it is necessary to investigate the characterization of liver injury under conditions of BD to improve the outcome of liver transplantation.

As the resident macrophages in the liver, Kupffer cells (KCs) express key renin angiotensin system (RAS) components, and RAS activity potentially participates in pathology and physiology (10). The activation of donor KCs is closely correlated with intense phagocytosis, a high expression of membranous molecules, antigen presentation, and the secretion of numerous cytokines, which all participate in the immune and pro-inflammatory or anti-inflammatory reactions (11). However, the exact role of KCs in liver injury under conditions of BD remains unknown. The purpose of this study was to explore the role of KCs in inflammation and apoptosis in liver injury under conditions of BD in a rat model.

Materials and methods

Results

KC depletion aggravates liver injury under conditions of BD

Histopathological changes in the liver

Liver histological sections were stained with H&E, and liver injury was graded according to the degree of necro-inflammation (HAI-NI) using the Knodell score system (16,17). We observed that the zones of necrosis were located around pericentral areas in the rats, with the exception of rats undergoing sham operation. The hepatocytes in the GdCl₃ group exhibited aggravated vacuolar degeneration and edema, and a greater number of inflammatory cells infiltrated the portal area compared with the B and S groups. However, there were no differences in injury between the saline group and the BD group. Saline had no effect on hepatocyte injury under conditions of BD (Fig. 1). The pathological Knodell scores of the livers exposed to GdCl₃ treatment were significantly increased compared with those of the B and S groups (P<0.05). However, no significant difference was noted between the B and S groups (P>0.05).

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Figure 1.

Liver injury following BD. Pretreatment of rats with GdCl₃ aggravates liver injury following BD. Liver pathological microsections from each model rat were scored according to hepatocyte necrosis, inflammatory cell infiltration in lobules, vascular endothelial injury and thrombus formation (arrow). Scale bar=50 µM. Knodell scores are expressed as the mean ± standard deviation. ^#and *P<0.05 vs. the BD group and the Saline group. ^ΔP>0.05 vs. the BD group. BD, brain dead; GdCl₃, gadolinium chloride.

Plasma liver function markers

After 6 h of BD, ALT levels increased 4-fold compared with those in the sham operation group. AST levels followed the same trend as ALT levels. Six h after BD, pretreatment with GdCl₃ caused increases in ALT and AST levels compared with those in the saline group (P<0.05). However, pretreatment with saline had no effect on ALT and AST levels. Statistically significant differences in serum ALT and AST levels were noted between the saline and BD groups. GdCl₃ treatment prior to BD increased AST/ALT ratio without significant statistical difference (P>0.05; Fig. 2). These results reveal that GdCl₃ treatment significantly aggravated hepatocyte injury as assessed by ALT and AST levels and necrotic cell death without significant statistical difference of AAR.

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Figure 2.

(A) ALT and AST levels in each group. GdCl₃ treatment prior to BD increased hepatocellular damage. The data are expressed as the means ± standard deviation (SD). ^#and *P<0.05 vs. the BD group, saline group and sham group. (B) AST/ALT ratios in each group. GdCl₃ treatment prior to BD increased AST/ALT ratio without significant statistical difference (P>0.05). (C) Inflammatory cytokine expression in liver. The results are presented as the fold change after normalization to baseline of the sham operation group, and the data are expressed as the means ± SD. TNF-α, IL-1β and IL-10 levels were upregulated in each liver group after BD. TNF and IL-1β levels were upregulated in GdCl₃-treated animals vs. diluent-treated controls and the BD group, whereas IL-10 levels were significantly downregulated in GdCl₃-treated animals vs. diluent-treated controls and the BD group. No significant differences were noted between the diluent-treated controls and the BD group. ALT, alanine aminotransferase; AST, aspartate aminotransferase; IL, interleukin; BD, brain dead; GdCl3, gadolinium chloride; TNF-α, tumor necrosis factor-α.

Effect of KC depletion on liver cell apoptosis under BD conditions

Liver apoptosis

Apoptotic cells were quantified in each field. In situ labeling of cell nuclei using the TUNEL assay revealed that BD significantly increased hepatic cell apoptosis in the saline and BD groups 6 h after BD compared with that in the sham operation group. The AI increased 6 h after BD. However, hepatic cell apoptosis was dramatically aggravated 6 h after GdCl₃ treatment (P<0.05; Fig. 3).

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Figure 3.

Liver AI. The AI is expressed as the percentage of apoptotic cells among the total number of cells. The level of apoptosis was increased in GdCl₃-treated animals compared with those in the diluent-treated controls and the BD group. No significant differences were noted between the diluent-treated controls and the BD group. ^#and *P<0.05 vs. the BD group, saline group and sham group. AI, apoptotic index; BD, brain dead; GdCl3, gadolinium chloride.

Western blot analysis of apoptosis-related proteins in liver tissue

To determine the importance of hepatic cell apoptosis in KC-mediated protective mechanisms and BD-induced hepatic injury, western blot analysis of apoptosis-related proteins was performed on total hepatic protein. Caspase-3, cleaved caspase-3 and Bcl-2 expression levels were examined by western blot analysis. Caspase-3 and cleaved caspase-3 expression levels were significantly increased, whereas Bcl-2 expression was significantly decreased 2 and 6 h after BD compared with those in the sham operation group (P<0.05). After GdCl₃ treatment, caspase-3 and cleaved caspase-3 expression levels were significantly decreased, whereas Bcl-2 expression was significantly increased compared with those in the saline and BD groups (P<0.05; Fig. 4).

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Figure 4.

Western blot analysis of apoptosis-related proteins in liver tissue. Each protein band was quantified and normalized to β-actin, and the results of every group are presented as the fold change after normalization to the baseline of the sham operation group. ^#P<0.05 vs. the BD and saline groups. *P>0.05 vs. the BD group. BD, brain dead.

Discussion

As resident macrophages of the liver, KCs excrete significant amounts of pro-inflammatory and anti-inflammatory cytokines and play a central role in the detrimental effects of liver transplantation. Studies have demonstrated that inhibition of KC activation through pharmacological mechanisms improved the outcomes of liver transplantation. Although different categories of hepatic macrophages were suppressed by liposomal clodronate to varying degrees, KCs were most susceptible to suppression (12,18). Therefore, GdCl₃ was used to inhibit KC function in our study. Several scholars reported that KCs were protective in liver tissues subjected to total ischemia/reperfusion (I/R) in transplantation (19). KCs are directly involved in inducing liver transplantation tolerance and hepatic I/R injury. Furthermore, KCs are activated during the onset of BD induction in animals (6). Upon GdCl₃ treatment to deplete KCs, we demonstrated that extensive KC elimination aggravated liver injury after BD in rats. The levels of the pro-inflammatory cytokines TNF-α and IL-1β increased, whereas expression of the anti-inflammatory cytokine IL-10 in the liver decreased. In addition, liver cell apoptosis was accelerated by suppression of KCs. Our finding suggested that KCs play a protective role in liver injury after BD potentially based on IL-10 expression and liver apoptosis suppression.

Secondary to BD, inflammation is driven by both the innate and adaptive immune systems in addition to numerous cytokines (19). Studies have demonstrated that BD is associated with significant upregulation of inflammatory cytokines through several mechanisms, thus promoting severe injury after liver transplantation (20,21). Upon methylprednisolone treatment, soluble ILs and TNF-α were significantly decreased, which significantly ameliorated liver injury post-transplantation (22).

IL-1β is an important mediator of the pro-inflammatory response and is produced by activated macrophages as a proprotein (6,23). Research has demonstrated that BD promotes the induction of IL-1β and TNF-α based on the activation of non-parenchymal cells in the liver (6,24). IL-10 suppresses the production of pro-inflammatory cytokines and upregulates inhibitors of IL-1β and TNF-α, leading to impairment or reversal of the effects of pro-inflammatory mediators (25). In the liver under BD conditions, IL-10 was primarily produced by KCs, as reported previously (26). We observed that IL-1β and IL-10 were significantly increased in the liver tissues of BD rats. IL-1β and TNF-α expression in the liver increases 6 h after BD, whereas GdCL₃ treatment increased IL-1β and TNF-α levels. However, IL-10 was significantly decreased upon treatment with GdCL₃. The data indicate that in the absence of KCs, the production of liver cytokines was significantly imbalanced. We suggest that the absence of KCs and their production of IL-10 lead to a pro-inflammatory response under BD conditions, which aggravates liver injury after BD in rats.

Previous studies have demonstrated that BD induces hepatocellular apoptosis and liver dysfunction (9,12,27). Therefore, we examined the effect of KCs on liver apoptosis upon treatment with GdCl₃ under BD conditions. Caspase-3 is a major executioner caspase that is cleaved at an aspartate residue to become activated. Cleaved caspase-3 degrades multiple cellular proteins and is responsible for morphological changes in cells during apoptosis. Previous research has demonstrated that apoptosis was obviously regulated by cleaved caspase-3 and Bcl-2 protein expression. Further studies revealed that the administration of the pan-caspase inhibitor IDN-6556 during liver transplantation offers local therapeutic protection against hepatocellular apoptosis and liver injury (28). Our present supporting evidence demonstrates that KC function is inhibited by GdCl₃ treatment, shifting the balance of pro-apoptotic and anti-apoptotic molecules to favor cell death. We observed that BD induces hepatocellular apoptosis, upregulation of cleaved caspase-3 expression and suppression of Bcl-2 expression. Under conditions of GdCl₃ pretreatment, increased liver apoptosis was noted with increased cleaved caspase-3 and caspase-3 expression and decreased Bcl-2 expression. KC elimination by GdCl₃ aggravated liver injury in a manner dependent on the regulation of apoptosis under BD conditions in rats by shifting the balance of cleaved caspase-3 and Bcl-2 molecules to favor cell death. Therefore, KCs may protect against BD by inhibiting hepatocyte apoptosis. Similarly, caspase-3 and cleaved caspase-3 expression increased, and Bcl-2 expression decreased. Given that caspase-3 promotes apoptosis and Bcl-2 impedes apoptosis, KCs may regulate cleaved caspase-3 and Bcl-2 to fight against apoptosis in BD. In summary, these results provide strong evidence that KCs protect liver function under BD conditions through the regulation of a pro-inflammatory state and apoptosis.

Murine KCs are protective in total hepatic I/R injury (19,29), and these results were similar to those presented in our research. KCs are exceptionally plastic cells that can polarize to specific activation states and perform various functions in different microenvironments. It has been reported that alternatively activated M2 KCs can promote caspase-3-dependent apoptosis of classically activated M1 KCs, thus providing a protective mechanism. The M2-mediated apoptosis of M1 KCs was shown to be arginase dependent by way of IL-10 (30). Another study demonstrated that GdCl₃ played an important protective role in early I/R injury and suppressed bile duct cell apoptosis during liver transplantation (31). During BD, liver-specific parameters of plasma endotoxin levels and the endotoxin-neutralizing capacity were compromised independent of hemodynamic status (32). Despite varying inflammatory profiles in organs after BD, BD does not accelerate I/R injury in transplantation (33). This discrepant finding is likely due to the predominant inflammatory component resulting from plasma endotoxin levels in our BD model, which differs from I/R injury. In this study, we demonstrated that KCs appear to play a protective role in liver subjected to BD. No single method is available to inhibit apoptosis and apoptosis genes to verify the role of KCs in liver injury protection in the context of BD. Therefore, the protective effects of KCs in liver injury observed in this study may only represent one aspect of the overall protective mechanism. In addition, it may be concluded that the effect of KCs in liver injury under conditions of BD involves a combined action requiring the participation of numerous mechanisms. Further studies may also be necessary to understand the exact mechanism.

In summary, our study indicates that KCs play a protective role in livers subjected to BD, which appears to be due to KC secretion of the potent anti-inflammatory cytokine IL-10. We also demonstrated that GdCl₃ efficiently inhibits the activity of KCs, which participate in the onset of liver injury through its effect on pro-inflammatory and anti-inflammatory activation. All of these results suggest that KCs are a potential target of GdCl₃ for the prevention and treatment of liver injury under BD conditions in rats.

Acknowledgements

Glossary

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