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Original Articles: Experimental Transplantation

Curcumin Has Potent Liver Preservation Properties in an Isolated Perfusion Model

Chen, Changguo; Johnston, Thomas D.; Wu, Guanghan; Ranjan, Dinesh

Author Information
doi: 10.1097/01.tp.0000232674.52951.ff

Abstract

Liver transplantation is the accepted therapy for end-stage liver diseases. Despite recent improvements in organ preservation with University of Wisconsin solution (UW), limitations in cold ischemic time, and its effect on the quality of preserved organs compromises organ transplantation. The pathophysiologic mechanism of tissue injury after and during cold preservation remains a subject of controversy (1, 2). It is reported that cold hypoxic storage and reoxygenation during liver preservation leads to oxidative stress from generated reactive oxygen radicals (ROS) (3, 4) and leads to inflammation (5). Many strategies and pharmacological agents have been used to protect the preserved organ from this injury. Curcumin, an aromatic polyphenol component of the spice turmeric, has been used as a wound-healing agent and has known anti-inflammatory, antitumor, and antioxidant properties (6–9). Curcumin is known as a potent inhibitor of protein kinase C, protein tyrosine kinase, and inhibits transcription factor NFκB activation (10). In a rat kidney ischemia-reperfusion (I/R) model, curcumin significantly decreased serum creatine (a marker for I/R injury) and decreased chemokine MCP-1 and RANTES (markers for inflammation) expression after I/R injury (11). In this study, we report the preliminary findings of our experiments using curcumin as an additive to phosphate buffered saline (PBS) and to other known clinical preservative solutions Euro-Collins (EC) and University of Wisconsin solution (UW) for hypothermic preservation of rat liver.

MATERIALS AND METHODS

Nonfasting adult Sprague-Dawley rats weighing 250–300 grams each were used in all experiments.

Euro-Collins Solution

The EC solution (Baxter Healthcare, Deerfield, Ill.) was obtained from the Transplant Center, University of Kentucky. The composition of EC is: Na+, 10 (mmol/L); K+, 115 (mmol/L); Cl, 15 (mmol/L); bicarbonate, 10 (mmol/L); phosphate 58 (mmol/L); and glucose 35 (g/l) (12).

UW Solution

The UW solution (Du Pont Pharmaceuticals, Wilmington, Del.) was obtained from the Transplant Center, University of Kentucky. The composition of UW is: K+, 125 (mmol/L); Na+, 30 (mmol/L); Mg2+, 5 (mmol/L); phosphate, 25 (mmol/L); SO42−, 5 (mmol/L); lactobionate, 100 (mmol/L); raffinose, 30 (mmol/L); allopurinol, 1 (mmol/L); adenosine, 5 (mmol/L); glutathione, 3 (mmol/L); pentastarch, 5 (%) (13).

Krebs-Henseleit Solution

The composition of Krebs-Henseleit solution prepared with double deionized water was as follows (in mM): NaCl, 118; KCl, 4.7; CaCl2, 2.55; MgSO4, 1.18; KH2PO4, 1.18; NaHCO3, 24.88; and glucose, 11.1. The pH of the solution was 7.3–7.4 (14).

Curcumin Solution Preparation

Curcumin solutions were freshly prepared before each experiment by directly adding curcumin (in DMSO) to PBS, EC, or UW to final curcumin concentrations of 25–200 μM and final vehicle DMSO concentration of 0.1%, and then stirred for 30 min before use. The same concentration of DMSO (0.1%) was added to PBS, EC and UW as vehicle control. No effect of DMSO (0.1%) alone in PBS, EC, or UW on rat liver perfusion was detected in the beginning of our experiments (data not shown).

Experimental Design

The animals were divided into three groups of 10 each. The rats were anesthetized with sodium pentobarbital (40–50 mg/kg body weight) by intraperitoneal injection. The abdomen was entered through a midline incision, and the liver was exposed and freed from the surrounding tissue. The common duct was cut and cannulated proximally with 1- to 2-mm plastic tube. Heparin sodium (100 units) was injected intravenously, and the portal vein was isolated and cannulated with a 2-mm catheter. Flush solution as outlined below was infused at a pressure of 18 cm H2O. The infrarenal vena cava was transected and the liver was removed and transferred to the perfusion apparatus for functional studies (15). The livers in the normal (NL) group were flushed with 37°C PBS, EC or UW solution (20 ml each) according to the respective corresponding group of experiment and then immediately perfused on the perfusion apparatus with oxygenated Krebs-Henseleit bicarbonate buffer solution. The control PBS, EC or UW group livers were flushed with 4°C PBS, EC or UW solution (20 ml each) and stored at 4°C for 24–48 hr as required by the experimental protocol. The study (PBS+C), (EC+C) or (UW+C) group livers were flushed with 4°C PBS, EC or UW solution with 25–200 μM curcumin (in EC) (20 ml each), 100 μM curcumin in PBS and UW (20 ml each), and stored at 4°C of the same solution for 24–48 hr. After 24–48 hr storage, the PBS, EC, UW, PBS+C, EC+C, and UW+C livers were transferred to the perfusion apparatus and perfused.

Perfusion of Isolated Rat Livers

The set-up for isolated rat liver function studies was according to published methods with minor modifications (Figure 1) (1, 16). Oxygenated Krebs-Henseleit bicarbonate buffer solution was used for all perfusions. The liver was connected to the end of perfusion column (H) via portal vein. The perfusion solution, which was heated in a container (A), bubbled with a gas mixture (95% O2, 5% CO2), and maintained at 37°C, was pumped by a roller pump. The fluid was passed through a filter and then through a temperature-stabilization chamber (B). The temperature-stabilized perfusion solution then went into the perfusion line from which the portal vein was perfused. The remainder went into the fluid column (H), which was also temperature-controlled, returning then to the container via the overflow tubing. The perfusion pressure was controlled by the height of the perfusion column (H). In our study, the pressure was adjusted to 18 cm H2O. The fluid returning from the liver was collected in a container (D) placed below the liver and was then returned to the fluid container by other Masterflex pump for recirculation. The temperature of the system, including the fluid container, the heating chamber, the perfusion column, and the humidified temperature-controlled chamber, was maintained at approximately 37°C during the perfusion period by using a heat pump and circulating water. Perfusion flow was monitored continuously by a Transonic flow-meter. Liver function was studied for 120 min. In each liver preparation, a volume of 500 ml was required for perfusion.

FIGURE 1.
FIGURE 1.:
The perfusion apparatus monitors flow rate into the rat liver portal vein, which is perfused with oxygenated Krebs-Hensleit solution (pH 7.4, 37°C) at a constant pressure of 18 cm H2O. The recirculated effluent can be sampled for analysis and bile output measured gravimetrically.

Liver Function Studies During Perfusion

During perfusion, portal flow and pressure were monitored continuously, from which portal vein perfusion flow rate was recorded every 10 min. All perfusion fluid was collected at the end of the experiment and liver enzymes (alanine aminotransferase [ALT], aspartate aminotransferase [AST] and lactate dehydrogenase [LDH]) were measured by CIBA-Corning Express-Plus Analyzer using reagents ordered from Chiron/Diagnostics and protocols from CIBA-Corning. Bile secretion was collected during the perfusion period, and bile production rates were calculated according to published method (16).

At the end of the experiment, tissue samples were taken from the liver and fixed in 10% buffered formalin for histological studies. Additional tissue samples were placed in an oven at 80°C for three days to determine the dry weight.

Statistical Analysis

Two-way analysis of variance (ANOVA) was used for repeated measurements. If significance was established, the Student-Newman-Keuls test was used to analyze the difference between individual groups. A value of P<0.05 was considered significant. All results were expressed as means± SEM.

RESULTS

Kinetic Study of Curcumin Concentration on Rat Liver Preservation in EC

As shown in Table 1, curcumin at 100 μM significantly increased portal vein perfusion flow rate from EC control of 19.3±0.5 (cc/gm-min) at 30 min and 22.0±0.4 at 120 min to 27.3±0.5 and 28.6±0.8, respectively. Curcumin at 100 μM significantly increased bile output from EC control of 17±2.5 to 43.0±3.0. Curcumin at 100 μM reduced liver enzyme AST and ALT release from EC control of 11.4±1.8 and 13.2±2.2 to 5.7±1.0 and 6.2±1.4, respectively. Curcumin at 25 μM and 200 μM did not as significantly enhance liver preservation as at 100 μM in EC. It is interesting to note that curcumin at 100 μM with 24 hr preservation time enhanced EC+C preservation equivalent to UW and close to the liver quality of normal group (NL) with zero preservation time.

TABLE 1
TABLE 1:
Kinetic study of curcumin on rat liver preservation

In agreement with the kinetic pattern in Table 1, another batch of experiments in portal vein perfusion flow rate confirmed that curcumin at 100 μM is the optimal concentration to enhance EC in liver preservation (Figure 2) up to the level of NL and UW after 24 hr cold preservation and perfusion for up to 120 min. However, curcumin at 25, 50, and 150 μM, induced lower flow rate than EC. Because curcumin at 100 μM was found to be the optimum concentration in our system, we decided to use this concentration in the other experiments.

FIGURE 2.
FIGURE 2.:
Curcumin concentration kinetics in rat liver preservation. The quality of EC+C preserved livers was measured by portal vein perfusion flow rate and compared with EC, UW and NL. Curcumin at 100 μM enhanced EC preservation to NL level.

Curcumin Enhanced Rat Liver Preservation in PBS

Rat livers preserved with 100 μM curcumin+PBS for 24 hr had portal vein flow rate higher than that preserved in PBS alone during the 120 min perfusion period. However, curcumin did not enhance PBS to the UW or NL level. The average flow rates (between 0–120 min) were: PBS, 33.29±3.3; PBS+100 μM Cur, 51.4±2.9; NL, 78.14±2.66, UW, 69.81±1.79. PBS+Cur(100 μM) was significantly different from PBS (P<0.01, n=13) (figure not shown).

Curcumin (100 μM) significantly increased bile production (μl/h/g-dry weight) from PBS control of 21.11±2.1 to 33.1±1.7 (P<0.05, n=10) in PBS+C. Curcumin(100 μM) significantly decreased liver enzyme release (units/g-dry weight) from PBS control of 15.19±2.5 (ALT), 14.32±1.7 (AST) and 49.18±6.01 (LDH) to 7.58±0.95 (ALT), 7.38±0.44 (AST) and 33.41±3.87 (LDH) (P<0.05, n=10) in PBS+C (figure not shown).

Curcumin Enhanced EC in Ex-vivo Model

In an ex-vivo liver model, curcumin at 100 μM enhanced portal vein flow rate from control EC up to the NL level during the 0–120min perfusion period. The average flow rate (between 0–120 min) were: EC, 60.37±1.6; EC+C, 80.33±2.18; NL, 78.14±2.66. EC+C was significantly different from EC (P<0.01, n=13) (Figure 3).

FIGURE 3.
FIGURE 3.:
Curcumin (100 μM) enhanced rat liver preservation in EC as measured by portal vein perfusion flow rate and compared with EC and NL.

Curcumin Enhanced EC Preservation Resulting in Reduced Liver Enzyme Release

During the 120-minute perfusion period, total ALT production determined in the perfusate was (units/g/dry weight): 3.32±0.67 in NL group, 6.21±1.4 in EC+C (100 μM) group, 13.2±2.24 in EC group. NL, EC+C groups vs. EC group P<0.05. AST was 5.21±0.8 in NL group, 5.68±1.0 in EC+C group, 11.34±1.8 in EC group. LDH was 10.96±2.35 in NL group, 12.34±4.6 in EC+C group, 22.8±5.8 in EC group (Figure 4). All the NL and EC+C groups' enzymes were lower than EC group (P<0.05, n=10).

FIGURE 4.
FIGURE 4.:
Comparison of liver enzymes ALT, AST, and LDH released into the perfusate among the three preservation techniques (EC, EC+C [100 μM], and NL). *P<0.05, EC+C, NL vs. EC, n=10.

Curcumin Enhanced EC Preservation Resulting in Increased Bile Production

During the 120 min perfusion period, in the EC preserved livers the bile production (μl/h/g dry weight) was 17.8±2.4; in the EC+C (100 μM) group, the bile production was 43.0±3; in the NL group, the bile production was 46.2±4.4. EC+C was significantly different from EC (P<0.05, n=10) (figure not shown).

Curcumin Enhanced Rat Liver Preservation in UW

Based on the results that curcumin enhanced rat liver preservation in EC and makes the preserved liver with quality equivalent to that of UW (the portal vein flow rate [120′] in EC+C [100 μM] was 28.6±0.8 [cc/gm-min] versus UW 26.6±1.8; Bile production was 43±3.0 [μl/gm-min] versus UW 46±4.2; AST secretion was 5.7±1.0 [Units/gm dry-weight] versus UW 5.5±0.8 [Table 1]) preserved livers and comparable to the quality of no hypothermic storage (NL) group (Table 1 and Figure 2), we asked whether this occurs because curcumin substitutes for a critical component in UW that may be missing in EC. Alternatively, curcumin might have an entirely novel effect and might be capable of enhancing UW preservation. Our results in Table 2 indicate that as the duration of hypothermic storage increased, a significant enhancement of UW's preservation of flow rate and bile production was seen with curcumin (UW×36 hr and UW×48 hr). After 36 hr of hypothermic storage, the flow rate at 30 min increased from control of 7.9±0.5 (cc/g-min) to 17.3±0.5 and the flow rate at 120 min increased from control of 11.1±1.0 to 19.3±0.7 (P<0.05, n=10). Bile output increased from control of 16.5±1.3 to 21.0±2.0 (μl/g-min) (P<0.05, n=10). After 48 hr of hypothermic storage, the flow rate at 30 min increased from control of 6.2±0.5 (cc/g-min) to 15.1±0.7 and the flow rate at 120 min increased from control of 11.0±0.9 to 16.4±0.6 (P<0.05, n=10). Bile output increased from control of 10.2±2.3 to 17.1±1.3 (μl/g-min) (P<0.05, n=10). However, we did not observe significant improvement in AST and ALT, markers of hepatic-cellular injury, by addition of curcumin to UW when livers were preserved for longer than 24 hr (Table 2).

TABLE 2
TABLE 2:
Curcumin (Cur) enhances rat liver preservation in University of Wisconsin solution

DISCUSSION

Liver preservation between donor operation and recipient operation is an important step in liver transplantation. Organ injury after ischemia and reperfusion remains one of the most formidable limiting factors in the field of transplantation. The precise mechanisms of the liver injury following cold preservation are unclear (17). There are many factors that induce ischemia/reperfusion injuries in the liver: 1) energy depletion and metabolism interruption: loss of mitochondrial respiration leading to adenosine triphosphate (ATP) depletion and deterioration of energy dependent metabolic pathways and transport (18); 2) sinusoidal endothelial cell damage, apoptosis and necrosis (19); 3) Kupffer cell induced oxidative stress (20); and 4) inflammatory damage from recruited neutrophils (21). Among these factors, inflammation is now considered to be one of the most important factor underlying ischemia/reperfusion injury (22). New organ preservation solutions are formulated to prevent or reduce the aforementioned factors. For example, the use of calcium and potassium channel blockers to prevent K+ efflux and Ca++ influx, thus to reduce the Ca++ induced ATP depletion in the cells and to prevent cell edema; the use of metallo-proteinase (MMP) inhibitors to prevent MMP, especially MMP-2 induced tissue inflammation (23); the use of complement inhibitors to prevent multimolecular assembly of C5b-9 that is able to damage cell membrane, thus induces cell lysis and organ damage (24, 25); the use of heme oxygenases, such as heme oxygenase-1 (HO-1), to down- regulate inflammation (26, 27) and the use of N-acetyl cysteine (NAC) to reduce oxidative stress (28).

Curcumin is an anti-inflammatory, antioxidant, antitumor, and antithrombotic natural product. It can cause inhibition of bioactivating enzymes and induces scavenging of reactive intermediates or free radicals and antiproliferative activity (6, 7, 29, 30). Curcumin also was demonstrated to have hepatoprotective effect in animals (31). Recent study has shown that curcumin prevented pro-inflammatory agents, that were generated during donor liver cold preservation, inducing adhesion molecule ICAM-1 and E-selectin expression in cultured endothelial cells (5). Curcumin is reportedly a potent stimulator of stress-induced expression of stress proteins, such as heat shock protein Hsp27, Hsp70, and alpha B crystalline (32). Curcumin is also known as a novel HO-1 inducer in different cell types and protects cells from oxidative stress (33, 34). A most recent study reported that pretreatment with curcumin in human hepatocytes induced HO-1 expression and protected hepatocyte from injury in a simulated cold preservation and warm reperfusion in vitro model (35). In rat kidney ischemia-reperfusion model, curcumin treatment significantly increased anti-oxidant enzyme Mn-SOD expression (36). Due to its low bioavailability and bio-stability when given enterally or even intravenously, the clinical application of curcumin so far has been limited. There are several approaches to increase blood level of curcumin, such as by oral administration of pipereine, an inhibitor of β-glucoronidation, increased curcumin blood level 154% in rat and 2000% in human (37, 38). Transformation of curcumin to a more water soluble product, and more bioavailable formulation while still maintaining its pharmacological activity is another measure, such as acetylation of curcumin (39, 40). One advantage of applying curcumin to organ preservation is that: organ preservation with curcumin based preservative solutions bypasses the hurdles of bioavailability as the solution is delivered directly to the organ, rather than having to go through the process of absorption and delivery as would be necessary for pharmacological use.

In our studies, the EC+C group livers while preserved for 24 hr, were still indistinguishable from NL group livers that had zero preservation time and had preservation characteristics significantly better than EC group in both functional (portal flow, bile production) and biochemical (liver enzymes release indicating liver injury) studies. The volume of bile secreted during perfusion has been employed as an index of hepatic viability (41, 42). Bile flow may represent the single most reliable criterion and it is immediately available and simple to measure. In our studies bile production was significantly increased in EC+C as compared to EC group, and curcumin enhanced bile production in the EC group to the NL group level. At 100 μM concentration, curcumin enhanced the preservation characteristics the most and therefore was deemed to be the optimum concentration for preservation (Table 1 and in Figure 2). We speculate that at lower concentrations (25–50 μM), curcumin may not be strong enough to antagonize oxidants and to induce anti-inflammation mechanisms while at higher concentration curcumin (200 μM) may induce side effects such as apoptosis (43). Curcumin enhanced liver preservation in UW after 36 hr and 48 hr of hypothermic storage may have the application value for better graft outcome for donated livers not transplanted within 24 hr. It is of note that curcumin even enhanced PBS as indicated by significantly increased portal vein flow rates and bile production and significantly decreased liver enzymes release into the perfusate. This indicates that curcumin has its specific effects in PBS and in UW and the putative mechanisms will need to be investigated in the future.

However, the exact mechanism of curcumin's action remains unclear. This will require further investigation as curcumin possesses several properties, such as anti- inflammatory and antioxidant, that may be playing a role in the organ preservation effects. We plan to further investigate the effects of curcumin on liver preservation in in vitro study with clinical relevant models. We also plan to do histology and immuno-histology in the preserved organs and to do in vivo transplantation of EC+C or UW+C preserved rat livers to rat recipients in comparison with EC or UW alone for survival rate and other biological markers. It will be of interest to further investigate if curcumin protects liver sinusoidal endothelial cells and whether curcumin upregulates heat shock proteins (Hsp27, Hsp70, Hsp72) and HO-1 expression during cold preservation and ischemia/reperfusion, as these proteins are known to protect rat livers from ischemia-reperfusion injuries (32, 44–47).

In conclusion, our results show that curcumin enhances liver preservation in isolated perfusion model. Additional studies, such as histology and immunohistology, are required to further define the specific mechanisms and its in vivo function after liver preservation and transplantation. This study may lead to future clinical utility.

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Keywords:

Curcumin; Liver preservation; Euro-Collins solution; University of Wisconsin solution

© 2006 Lippincott Williams & Wilkins, Inc.