Protective Effect Of Adenosine A_2A Receptor Activation In Small-for-Size Liver Transplantation In Rats

PartⅠA Model of Small-for-Size Liver Transplantation in Rats andObservation of the Main Indexes of Ischemia/Reperfusion InjuryObjectiveTo establish a method of practical and stable experimental animalmodel in rats for the study of small-for-size liver transplantation. Inaddition, the main indexes of ischemia/reperfusion injury insmall-for-size liver transplantation were observed and possible injurymechanism was evaluated.MethodMale Sprague Dawley(SD) rats were randomized into 2 groups, donorgroup and recipient group. Right middle lobe, right superior and inferiorlateral lobe, which was accounted for 42～48% of the recipient liverweight, was procured as graft. Portal veins and infrahepatic venae cava ofdonors were anastomosed end-to-end to those of recipients by usingmodified two-cuff technique. The survival rate and death cause wereobserved; The change of serum aspartate aminotransferase (AST)within 24 hours after small-for-size liver transplantation was evaluated by AST kit and the content of malondialdehyde (MDA) in liver graft after 6hours was detected. Additionally, morphological changes wereinvestigated by light microscope and electron-microscope, and apoptosiswas studied by TUNEL method.Results1. The survival rate of small-for-size liver transplantation in rats was66.67% (8/12) after one day and 33.3% (4/12) after one week.2. Compared with controls, the serum AST levels of recipients weremarkedly elevated after transplantation, peaking to 8561±1663 U/Land 9925±1738 U/L at 6 hours and 12 hours after reperfusionrespectively.3. The content of MDA after 6 hours was increased significantly insmall-for-size liver graft (1.34±0.38 nmol/mg prot) compared withnormal (0.60±0.14 nmol/mg prot, P＜0.01).4. HE staining showed severe disruption of lobular architecture,significant periportal edema, congestion and necrosis in small-for-sizeliver graft; Similarly, electron microscopy also showed severemitochondria and endoplasmic reticulum swelling of hepatocytesaccompanied by loss of microvilli compared with normal group.5. TUNEL results showed that the count of apoptosic hepatocytes wasincreased significantly in small-for-size liver graft (19.1±4.9)compared with 1.2±0.7 in normal group (P＜0.01). ConclusionThe method of modified small-for-size liver transplantation in ratsprovides practical and stable experimental animal model for the study ofsmall-for-size liver transplantation. Small-for-size liver graft in rats wassubjected severe ischemia/reperfusion injury.PartⅡAdenosine A_2A Receptor Activation Decreased Oxidative Stress inSmall-for-Size Liver Transplantation in RatsObjectiveTo study whether the activation of Adenosine A_2A Receptor (A_2AR)with CGS21680 (a selective A_2AR agonist) could alleviate the oxidativestress induced by ischemia-reperfusion injury in small-for-size livertransplantation in rats, and examine the mechanism underlying.MethodsSmall-for-size liver transplantation was performed using liver that washarvested from Sprague-Dawley rat and stored for 80 min at 4℃in coldsaline before being transplanted into recipient. Each recipient rat wasfitted with a catheter in the right external jugular vein for intravenousinfusion of reagents via a peristaltic pump. In each group, immediatelyafter reperfusion, recipients were infused with saline (control group),CGS 21680 (0.5μg/kg/min, CGS group), or CGS 21680 (0.5μg/kg/min) +ZM 241385 (0.5μg/kg/min, a specific A_2AR antagonist) (CGS+ZMgroup) continuously for 3 hours under the anesthesia. For each group, 9rats were used for the 7-day survival rate, and 6 rats were killed at 6 hoursafter reperfusion for the detection of total glutathione (GSH), tocopherol(TOC), ascorbic acid (AA), glutathione peroxidase (GSH-Px), catalase(CAT), superoxide dismutase (SOD) and malondialdehyd (MDA).Results6. The seven day survival rate of small-for-size liver transplantation inrats was 66.67% (6/9), compared with 33.33% (3/9) in control and33.33% (3/9) in CGS+ZM group.7. The serum AST was much lower in CGS group (3578±597 U/L) thanthat in control (8110±699 U/L, P＜0.01) and CGS+ZM group(7690±814 U/L, P＜0.01).8. The GSH level in CGS group (36.9±3.5 ng/mg prot) was significantlyhigher than that in control (28.4±2.2 ng/mg prot, P＜0.01) andCGS+ZM group (30.6±2.2 ng/mg prot, P＜0.05). There was nosignificant difference between control and CGS+ZM group.9. The TOC level in CGS group (0.32±0.05 ug/mg prot) wassignificantly higher than that in control (0.24±0.04 ug/mg prot,P＜0.01) and CGS+ZM group (0.25±0.05 ug/mg prot, P＜0.01). Therewas no significant difference between control and CGS+ZM group.10. The AA level in CGS group (3.06±0.65 ug/mg prot) was significantly higher than that in control (1.89±0.45 ug/mg prot, P＜0.01) andCGS+ZM group (1.67±0.43 ug/mg prot, P＜0.01). There was nosignificant difference between control and CGS+ZM group.11. The activity of GSH-Px in CGS group (56.5±7.4U/mg prot) wassignificantly higher than that in control (38.1±5.3U/mg prot, P＜0.01)and CGS+ZM group (40.7±7.0U/mg prot, P＜0.01). There was nosignificant difference between control and CGS+ZM group.12. There was no significant difference in activity of CAT between the 3groups.13. The activity of SOD in CGS group (601±59 U/mg prot) wassignificantly higher than that in control (450±64 U/mg prot, P＜0.01)and CGS+ZM group (408±54 U/mg prot, P＜0.01). There was nosignificant difference between control and CGS+ZM group.14. The content of MDA in CGS group (0.83±0.15 nmol/mg prot) wassignificantly lower than that in control (1.34±0.38 nmol/mg prot,P＜0.01) and CGS+ZM group (1.21±0.24 nmol/mg prot, P＜0.05).There was no significant difference between control and CGS+ZMgroup.ConclusionAt the time of reperfusion, excessive blood inflow (in relation to thegraft size) carries more oxygen to the cells in small-for-size grafts,subsequently generating more reactive oxygen species (ROS) and leading to oxidative injury. Activation of A_2AR attenuatesischemia-reperfusion injury in rat small-for-size grafts by enhancing theanti-oxidant enzyme activity, facilitating the generation of anti-oxidant,and alleviating lipid peroxidation.PartⅢAdenosine A_2A Receptor Activation Reduced Inflammatory Responsein Small-for-Size Liver Transplantation in RatsObjectiveTo study whether the activation of A_2AR with CGS21680 couldalleviate the inflammatory response induced by ischemia-reperfusioninjury in small-for-size liver transplantation in rats, and examine themechanism underlying.MethodsIn all groups, liver grafts were storage at 4℃for 80min ischemic timein cold saline. Each recipient rat was fitted with a catheter in the rightexternal jugular vein for intravenous infusion via a peristaltic pump.Immediately after reperfusion, recipients were infused with saline(control group), CGS21680 (at the dose of 0.5μg/kg/min, CGS group), orCGS21680 (0.5μg/kg/min)+ZM 241385 (0.5μg/kg/min) continuouslyfor 3 hours under the anesthesia.Six rats in each group were killed at 6 hours after reperfusion and the liver was removed. RT-PCR and ELISA were used to detect theexpression of TNF-α, IL-1β, IL-6 and IL-10, respectively. MPO activity,HE staining and electron microscopic analysis were also performed ongraft samples.Results1. The TNF-αmRNA level in CGS group was significantly lower thanthat in control and CGS+ZM group. The protein level of TNF-α(140±27 pg/g) was also decreased than that in control (626±108 pg/g,P＜0.01) and CGS+ZM group (723±133 pg/g, P＜0.01). There was nosignificant difference between control and CGS+ZM group.2. The IL-1βmRNA level in CGS group was significantly lower thanthat in control and CGS+ZM group. The protein level of IL-1β(73±17pg/g) was also decreased than that in control (273±57 pg/g, P＜0.01)and CGS+ZM group (253±53 pg/g, P＜0.01). There was no significantdifference between control and CGS+ZM group.3. The IL-6 mRNA level in CGS group was significantly lower than thatin control and CGS+ZM group. The protein level of IL-6 (2.27±0.41ng/g was also decreased than that in control (7.16±0.94 ng/g, P＜0.01)and CGS+ZM group (6.28±1.36 ng/g, P＜0.01). There was nosignificant difference between control and CGS+ZM group.4. There was no significant difference between the three groups for bothmRNA and protein level of IL-10. 5. The activity of MPO in CGS group (1.48±0.56 U/g prot) wassignificantly lower than that in control (6.32±0.83 U/g prot, P＜0.01)and CGS+ZM group (5.70±0.81 U/g prot, P＜0.01). There was nosignificant difference between control and CGS+ZM group.6. In control and CGS+ZM groups, H-E staining showed severedisruption of lobular architecture, significant periportal edema,congestion and necrosis, while in CGS group there was minimaldegeneration.7. In control and CGS+ZM groups, electron microscopy showed severemitochondria and endoplasmic reticulum swelling of hepatocytesaccompanied by loss of microvilli; while in CGS group, both the cellnucleus and cellular organelles had no significant breakdown.ConclutionActivation of A_2AR attenuates ischemia-reperfusion injury in ratsmall-for-size grafts by inhibiting pro-inflammatory cytokine productionand reducing neutrophil accumulation, and preserving the hepaticarchitecture.PartⅣAdenosine_2A Receptor Activation Inhibited Apoptosis inSmall-for-Size Liver Transplantation in RatsObjective To study whether the activation of A_2AR with CGS21680 could inhibitapoptosis induced by ischemia-reperfusion injury in small-for-size livertransplantation in rats, and examine the mechanism underlying.Methods1. In all groups, liver grafts were storage at 4℃for 80min ischemic timein cold saline. Each recipient rat was fitted with a catheter in the rightexternal jugular vein for intravenous infusion via a peristaltic pump.Immediately after reperfusion, recipients were infused with saline(control group), CGS21680 (at the dose of 0.5μg/kg/min, CGS group),or CGS21680 (0.5μg/kg/min)+ZM 241385 (0.5μg/kg/min)continuously for 3 hours under the anesthesia.2. Six rats in each group were killed at 6 hours after reperfusion andthe liver was removed. The anti-apoptotic Bcl-2 and the pro-apoptoticBax, Caspase-3 were determined by western blot. The apoptosis wasalso detected by TUNEL.Results1. The expression of Bcl-2 in CGS group was significantly higher thanthat in control and CGS+ZM group. There was no significantdifference between control and CGS+ZM group.2. The pro-apoptotic molecule Bax in CGS group was significantly lowerthan that in control and CGS+ZM group. There was no significantdifference between control and CGS+ZM group. 3. The apoptotic molecule Caspase-3 in CGS group was significantlylower than that in control and CGS+ZM group. There was nosignificant difference between control and CGS+ZM group.4. Detection of apoptosis by TUNEL assay showed that the frequency ofTUNEL+cells was diminished in CGS group; the apoptotic index,calculated as the percentage of TUNEL-nuclei divided by the counterstained nuclei, was significantly diminished in CGS group (7.6±2.8) ascompared with control (20.0±4.8, P＜0.01) and CGS+ZM group(18.2±4.0, P＜0.01).ConclutionA_2AR activation inhibited apoptosis in CGS21680-treated liver grafts inrats. The cellular and physiological mechanisms by which A_2AR exertedcytoprotective functions against IRI at the graft site involve enhancedantiapoptotic protein expression and diminished pro-apoptotic moleculeactivation.