FTY720 Pretreatment Reduces Warm Hepatic Ischemia Reperfusion Injury Through Inhibition of T-Lymphocyte Infiltration

Ischemia and reperfusion (IR) injury remains a significant problem in clinical liver transplantation. We investigated the effects of lymphocyte depletion with FTY720 in models of warm hepatic IR. Using 60-min partial warm hepatic IR, three groups of rats were studied: Sham--laparotomy alone; Control--water p.o. x 3 d before ischemia; Treatment--FTY720 p.o. x 3 d before ischemia. Animals were sacrificed for analysis at 6 h and 24 h post reperfusion. The effect of FTY720 pretreatment on survival was also studied using 150 min total hepatic IR with portojugular shunt. FTY720 treatment significantly reduced serum glutamic pyruvic transaminase and peripheral blood lymphocytes compared to controls at 6h and 24h (p < 0.0005). Histological grade was significantly improved in treated livers vs. controls (p < 0.05). CD3 immunocytochemical analysis revealed a significant reduction in T-cell infiltration in FTY720-treated livers (p < 0.0002). No difference in tissue myeloperoxidase levels was observed. Seven-day survival was significantly improved in treated rats vs. controls following total hepatic ischemia (p < 0.05). In conclusion, FTY720 ameliorates the biochemical and histological manifestations of hepatic IR by preventing T-lymphocyte infiltration and prolongs survival following a more severe ischemic insult. Myeloperoxidase data suggest this mechanism is independent of neutrophil activation. These results indicate that T lymphocytes are pivotal mediators in hepatic IR and may have important implications in liver transplantation.     

肝细胞凋亡在肝硬化大鼠肝缺血再灌注损伤中的意义

目的 研究肝硬化大鼠肝缺血再灌注（I／R）损伤和硬化肝比正常肝更容易损伤的机制是否与肝细胞凋亡有关？方法 建立原位肝I／R模型，将肝硬化大鼠随机分为2组：A组：缺血时间（I）＝20min;B组：I＝30min;C组：正常大鼠，I＝30min，比较灌注前后各组血清AST、ALT的变化和肝细胞凋亡的百分数。结果 肝硬化大鼠肝I／R后，AST、ALT明显升高，以灌注后6h为高峰，灌注24、72h后逐渐下降。灌注6h后，B组的血清转氨酶为3组中最高（P＜0．05），说明B组肝损伤最严重。肝细胞凋亡在I／R后明显增多，以灌注后6h为高峰，随后逐渐下降，变化与转氨酶一致。灌注后6h，B、A、C组肝细胞凋亡的百分数分别为20．9％、13．5％和10．7％，B组明显高于A、C两组（P＜0．01）。再灌注72h内未见明显肝细胞坏死。结论 肝细胞凋亡是肝硬化大鼠I／R损伤肝细胞死亡的主要形式，肝细胞凋亡与肝缺血时间密切相关，肝硬化肝细胞比正常肝细胞容易发生凋亡是硬化肝对缺血敏感的重要原因。     

Impact of endothelin-1 on microcirculatory disturbance after partial hepatectomy under ischemia/reperfusion in thioacetamide-induced cirrhotic rats

BACKGROUND: Endothelin (ET)-1 contributes to hepatic ischemia and reperfusion (HIR) injury in normal liver. This study was conducted to clarify the role of ET-1 in HIR injury in cirrhotic state. MATERIALS AND METHODS: Using thioacetamide-induced cirrhotic rats with spontaneous portosystemic shunt, we determined the changes in plasma aspartate aminotransferase (AST) levels, plasma and hepatic ET-1 values, 7-day survival rates, and hepatic oxygen saturation (SO(2)) by time-resolved spectroscopy as an indicator of hepatic microcirculation under intermittent or continuous total hepatic ischemia with subsequent partial hepatectomy. RESULTS: Hepatic ET-1 levels in cirrhotic rats were significantly higher than those in noncirrhotic rats. Plasma and hepatic ET-1 levels at 1, 3 and 6 h of reperfusion after intermittent hepatic ischemia were significantly lower than those after continuous hepatic ischemia. In cirrhotic animals subjected to intermittent hepatic ischemia, the elevation of plasma AST levels at 1, 3 and 6 h of reperfusion and the decline in hepatic SO(2) at the end of 60-min hepatic ischemia and after reperfusion were significantly suppressed when compared with those subjected to continuous hepatic ischemia. Pretreatment with a nonselective endothelin receptor antagonist in continuous hepatic ischemia significantly ameliorated plasma AST levels and hepatic SO(2) values with less hepatic sinusoidal congestion, resulting in an improvement in the 7-day survival rate. CONCLUSIONS: Continuous hepatic ischemia in the cirrhotic liver has disadvantages relating to microcirculatory derangement with more ET-1 production in partial hepatectomy. In liver surgery, pharmacological regulation of ET-1 production may lead to attenuation of reperfusion injuries for ischemically damaged cirrhotic liver.     

肝硬变大鼠肝脏缺血再灌注损伤

为比较硬化肝与正常肝在缺血再灌注损伤时的差异和意义。作者采用四氯化碳复制大鼠肝硬变模型,通过大鼠肝脏缺血再灌注损伤模型,检查不同时限大鼠门静脉血内毒素、肝静脉血一氧化氮。结果显示：肝硬变大鼠再灌注时门静脉内毒素水平更高;肝脏ＮＯ合成释放显著增加。作者认为肝硬变时对缺血再灌注损伤反应与正常大鼠不同,可能是肝硬变时对缺血再灌注损伤更敏感,更易发生肝功能衰竭的重要原因。     

The effect of methylprednisolone on warm ischemia-reperfusion injury in the liver

Background

Liver ischemia-reperfusion (I-R) injury is a well-known cause of morbidity and mortality following liver surgery and transplantation. Further investigation is warranted to identify measures that reduce the untoward sequelae of liver ischemia.

Methods

Male Sprague-Dawley rats (wild-type) and Zucker rats (with hepatic steatosis) were subjected to 75 minutes of 70% hepatic ischemia and 3 hours of reperfusion. The ischemic periods were based on protocols of either continuous clamping (CC) or ischemic preconditioning (IP). Prior to ischemia induction, rats were pretreated with intravenous methylprednisolone (MP; 2 mg/kg) or normal saline. Warm I-R injury was evaluated using serum levels of aspartate aminotransferase (AST), serum interleukin-6 (IL-6), and hematoxylin and eosin staining.

Results

Histology, serum IL-6, and AST release revealed that MP treatment provided significant protection as compared with ischemic controls (both CC and IP groups) only in the normal, not steatotic, livers. The inflammatory response was considerably reduced in MP groups with normal livers but not in steatotic livers. In general, the IP groups showed decreased I-R injury compared to the CC group. However, MP was able to further reduce I-R injury only in normal, not steatotic, livers.

Conclusions

MP attenuated the postischemic and inflammatory response in the normal, and not steatotic, livers. MP pretreatment might be effective in reducing warm I-R injury to livers without steatosis. The mechanism of I-R-related hepatocellular damage in steatotic liver is different than in normal liver.     

Increased sinusoidal pressure is associated with early liver weight gain in ischemia-reperfusion injury in isolated perfused rat liver

BACKGROUND: Hepatic ischemia-reperfusion (I/R) is accompanied by liver weight gain and ascites formation. This could be caused by an increase in sinusoidal pressure, a determinant of hepatic transvascular fluid movement. We determined the role of sinusoidal pressure, assessed by triple vascular occlusion pressure (P(to)), in the I/R injury in isolated rat livers perfused with leukocyte-free diluted blood bivascularly via the portal vein and hepatic artery. MATERIALS AND METHODS: Ischemia was induced at room temperature by occlusion of either the inflow lines of the hepatic artery and portal vein (the open outflow group, n = 10) or both the inflow and the outflow (hepatic venous) lines (the closed outflow group, n = 10) for 1 h, followed by 1-h reperfusion in a recirculating manner. RESULTS: Liver weight in both groups increased biphasically after reperfusion; the initial peak occurred at 3 min and the second peak at 60 min. Immediately after reperfusion, P(to) peaked, followed by a gradual decline. The initial weight increase in groups combined was significantly and positively correlated with an increase in P(to) (r = 0.716, P = 0.0002), but the second peak was independent of P(to). Liver injury, assessed by perfusate levels of hepatic enzymes and reduced bile flow rate, was observed at 60 min after reperfusion in both groups. CONCLUSIONS: These findings suggest that increased sinusoidal pressure contributes to only the early liver weight gain after reperfusion in isolated perfused rat livers. The late weight gain may be presumably due to liver injury.     

Effect of Nutritional Status on Oxidative Stress in an Ex Vivo Perfused Rat Liver

Background: Normothermic ischemia-reperfusion is a determinant in liver injury occurring during surgical procedures, ischemic state, and multiple organ failure. The preexisting nutritional status of the liver might contribute to the extent of tissue injury and primary nonfunction. The aim of this study was to determine the role of starvation on hepatic ischemia-reperfusion injury in normal rat livers.

Methods: Rats were randomly divided into two groups: one had free access to food, the other was fasted for 16 h. The portal vein was cannulated, and the liver was removed and perfused in a closed ex vivo system. Two modes of perfusion were applied in each series of rats, fed and fasting. In the ischemia-reperfusion mode, the experiment consisted of perfusion for 15 min, warm ischemia for 60 min, and reperfusion during 60 min. In the nonischemia mode, perfusion was maintained during the 135-min study period. Five rats were included in each experimental condition, yielding a total of 20 rats. Liver enzymes, potassium, glucose, lactate, free radicals, i.e., dienes and trienes, and cytochrome c were analyzed in perfusate samples. The proportion of glycogen in hepatocytes was determined in tissue biopsies.

Results: Transaminases, lactate dehydrogenase, potassium, and free radical concentrations were systematically higher in fasting rats in both conditions, with and without ischemia. Cytochrome c was higher after reperfusion in the fasting rats. Glucose and lactate concentrations were greater in the fed group. The glycogen content decreased in both groups during the experiment but was markedly lower in the fasting rats.     

Ischemic preconditioning improves energy state and transplantation survival in obese Zucker rat livers

Livers from obese donors often have fatty infiltrates and are more susceptible to ischemia-reperfusion injury and subsequent graft dysfunction. This often leads to the exclusion of organs from obese donors. We investigated whether ischemic preconditioning (IP, 10 min ischemia, 10 min reperfusion) preserves cellular metabolism in livers from obese Zucker rats during cold ischemia. Liver samples (-IP and +IP) were collected from obese and control lean rats at different time points of cold ischemia (CI) and analyzed by magnetic resonance spectroscopy (1H- and 31P-MRS) to assess whether IP improves hepatic cellular metabolism. IP significantly improved high energy metabolism in IP livers from obese rats when compared with obese controls during the first hours of CI. At 4 h of cold storage, obese IP livers were not different from control lean non-IP livers. The beneficial metabolic effect of IP on livers form obese rats, however, was absent at 8 h of reperfusion. In contrast, in livers from lean rats, IP resulted in improved high-energy metabolism during the entire observation period of 8 h. In a later part of the study, IP of liver grafts from obese rats before 4 h of cold storage improved recipient survival after graft transplantation. IP of liver grafts from obese rats before 4 h of CI increases 24-h survival of recipient animals from 25% to 88%.     

Evaluation of warm ischemia-reperfusion injury using heat shock protein in the rat liver

We focused on heat shock protein 70 (HSP70) as a marker of viability in hepatic warm ischemia-reperfusion. Segmental hepatic warm ischemia was produced in rats for 15, 30, 60, 90, 120, or 180 min. Liver sections were evaluated at 30, 60, and 120 min of reperfusion. Expression of HSP70 and messenger RNA (mRNA), apoptosis, and apoptosis-associated genes such as Bcl-2 and Bax were studied. Expression of HSP70 and mRNA was augmented as warm ischemia was prolonged, but was markedly suppressed in livers with more than 120 min of ischemia. The highest accumulation of HSP70 was observed in the nucleus. In livers subjected to longer duration of warm ischemia, necrosis and apoptosis were evident and Bcl-2 mRNA expression and Bcl-2/Bax protein ratio were markedly diminished. Apoptosis may be related to the process of cellular injury induced by warm ischemia-reperfusion. Expression of HSP70 and the Bcl-2 family can be effective markers of viability in hepatic warm ischemia-reperfusion.     

再灌注初期控制性降压对肝叶切除术病人肝缺血再灌注损伤的影响

目的 评价再灌注初期控制性降压对肝叶切除术病人肝缺血再灌注损伤的影响.方法 择期行肝叶切除术病人40例,性别不限,年龄30～60岁,体重40～70kg,ASA分级Ⅱ或Ⅲ级,将病人按分层随机方法分为2组(n=20),对照组(C组)开放肝门后10 min期间维持MAP 75～100mm Hg,控制性降压组(H组)于开放肝门前2 min开始静脉输注硝酸甘油3～6μg·kg-1·min-1实施控制性降压,再灌注10 min期间维持MAP 60～70 mm Hg.分别于缺血前(基础状态)、缺血15 min和再灌注25min时采集静脉血样,测定血浆内皮素(ET)、一氧化氮(NO)、TNF-α和IL-1的浓度.结果 与基础值比较,两组缺血15 min和再灌注25min时血浆ET、TNF-α和IL-1的浓度升高,血浆N0浓度降低(P＜0.05);与C组比较,H组再灌注25min时血浆ET、TNF-α和IL-1的浓度降低,血浆NO浓度升高(P＜0.05).结论 再灌注初期控制性降压10 min可减轻肝叶切除术病人肝缺血再灌注损伤,其机制与调节肝窦内皮细胞ET和NO的平衡及抑制炎性反应有关.

Abstract：

Objective To evaluate the effect of controlled hypotension at the beginning of reperfusion on ischemia-reperfusion (I/R) injury of the liver in patients undergoing hepatectomy. Methods Forty ASA Ⅱ or Ⅲ patients aged 30-60 yr weighing 40-70 kg undergoing elective partial hepatectomy for liver cancer were randomly divided into 2 groups ( n = 20 each): group C normal BP and group H controlled hypotension. Hepatic portal was occluded during operation. In group C normal BP was maintained during reperfusion while in group H controlled hypotension (MAP was maintained at 60-70 mm Hg) was performed for 10 min since the beginning of reperfusion.Venous blood samples were taken before hepatic ischemia (T0 ,baseline) and at 15 min of ischemia (T1) and 25 min of reperfnsion (T2 ) for determination of plasma endothelin (ET), nitric oxide(NO), TNF-α and IL-1 concentrations. Results I/R of the liver led to significant increase in plasma ET, TNF-α and IL-1 concentrations and decrease in plasma NO concentration at T1,2 as compared with the baseline values at T0 in both groups. Plasma ET,TNF-α and IL- 1 concentrations were significantly lower while plasma NO concentration was significantly higher at T2 in group H than in group C. Conclusion Ten minutes controlled hypotension in the initial stage of reperfusion can attenuate I/R-induced injury to the liver in patients undergoing hepatectomy by balancing ET with NO and inhibiting inflammation response.     

Effects of immunosuppressant FTY720 on renal and hepatic hemodynamics in the rat

BACKGROUND: FTY720 is a novel immunomodulator that may provide an opportunity for a reduction in calcineurin inhibitor dosage in transplant recipients with renal/hepatic side effects. However, the effects of FTY720 on renal or hepatic hemodynamics are unknown. The aim of this study was to establish the hemodynamic and renal actions of FTY720 at therapeutically relevant dosages. METHODS: The effects of acute and repeat oral administration of FTY720 on systemic, renal, and hepatic hemodynamics were investigated in the anesthetized male Lewis rat. Renal function and renal tubular parameters were examined in animals that received repeat high dosage of FTY720. RESULTS: Seven-day oral administration of FTY720 did not cause any significant changes in markers of hepatocyte injury, nor did it cause any reduction in renal function (elevated urea and creatinine). Histological examination of liver and kidney from animals treated with repeat FTY720 for 1 or 3 weeks did not reveal any sclerosis, tubular changes, infiltrates, or fibrosis. Hepatocyte, vascular, and biliary structures were normal. Compared with the vehicle (saline), oral administration of FTY720 at dosages up to 5 mg/kg/day for 1 week did not have any significant effects on systemic, hepatic, or renal hemodynamics. Five min after intravenous FTY720 administration (1 mg/kg), mean arterial pressure (MAP) rose to 114+/-3.3% of baseline (P <0.01) before returning to the normal range within 30-45 min. Lower doses of FTY720 (0.3 and 0.5 mg/kg, i.v.) did not affect MAP. Renal cortical perfusion, renal artery blood flow, and renal vascular resistance were not altered by FTY720 at i.v. doses up to 1 mg/kg. Animals that received FTY720 (5 mg/kg/day) for 3 weeks showed a significant reduction in body weight (-4.8+/-1% of baseline at 3 weeks, P <0.001); however, weight-adjusted creatinine clearance, 24 h urine production, and urine osmolality were not different from those in control animals (0.71+/-0.1 vs. 0.74+/-0.1 ml/min/100 g, 2.63+/-0.2 vs. 3.12+/-0.2 ml/100 g, and 2003+/-33 vs. 1966+/-56 mOsm/kg, respectively). FTY720 at the same repeat oral dosage was, nevertheless, associated with a significantly lower 24 h sodium excretion and a significantly lower fractional excretion of sodium compared with those in control animals (223.4+/-35 vs. 304.5+/-50 micromol/100 g and 1.75+/-0.3 vs. 2.23+/-0.3%, respectively; P <0.05). CONCLUSIONS: Our data indicate that, at least in the short term, oral FTY720 does not cause any significant adverse effects on renal or hepatic hemodynamics, nor does it cause any reduction in glomerular perfusion and thus may provide reasonable rescue/add-on therapy in calcineurin-inhibitor treated transplant recipients. At high repeat oral dosages, however, FTY720 may alter renal handling of sodium.     

FTY720预防大鼠肾脏缺血再灌注损伤的实验研究

目的:研究新型免疫抑制剂FTY720对大鼠肾脏缺血再灌注损伤(IRI)的预防作用.方法:制备大鼠肾脏IRI模型,从下腔静脉注入不同剂量的FTY720,观察术后第1、2、3、5、7 d血清肌酐值(Scr)和术后第2、7 d外周血淋巴细胞数(PLC)的变化,并在术后第2 d取肾脏作组织学检查观察急性肾小管坏死的情况.结果:FTY720处理组动物术后Scr水平显著低于对照组并呈剂量依赖性;FTY720处理组术后第2 d的PLC显著低于对照组;组织学检查显示FTY720处理组肾脏的缺血性损伤轻于对照组.结论:FTY720可以减少PLC,对大鼠肾脏IRI有预防作用.     

Ischemic injury in cirrhotic livers: an experimental study of the temporary arrest of hepatic circulation.

In order to prevent massive bleeding at hepatectomy, the temporary arrest of hepatic circulation is often performed but it is not clear whether this arrest of circulation is tolerated equally by the cirrhotic liver and the normal liver. We investigated biochemically the detailed effects of ischemia on cirrhotic livers in rats with experimentally induced liver cirrhosis. Thioacetoamide was used to prepare the rat cirrhotic liver model (LC group, n = 6). After laparotomy, the vessels to the left lateral lobe were clamped for 30 min and then declamped. Changes in AST isozymes and the aminogram in the blood were examined after ischemia. Postischemic changes in hepatic adenine nucleotides (AdN) and the brain aminogram were also examined. These were compared with those of normal liver ischemia (N group, n = 6) at 24 hr after recirculation. In the LC group, serum levels of mitochondrial AST, a parameter of necrotic cells, were significantly higher than those of the N group. Hepatic AdN levels decreased to 60.6% of the original levels after ischemic injury but those of the N group remained at 95.4% of the original level. Since AdN in tissue is accepted as a reliable parameter of viable cells, cirrhotic livers subjected to ischemia might have more necrotic cells than normal livers. Sequential analysis of serum aminograms of the LC group after ischemia revealed that the ratio of Val+Leu+Ileu/Tyr+Phe decreased to near 1.0 but that of the N group always remained higher than 3.0. Based on these results, it was concluded that ischemic injury in cirrhotic livers is more hazardous than that in normal livers.     

FTY720 for treatment of ischemia-reperfusion injury following complete renal ischemia; impact on long-term survival and T-lymphocyte tissue infiltration

Background

Organ dysfunction due to ischemia-reperfusion (I/R) injury is a common problem in transplant, liver, trauma, and heart surgery. I/R injury is mediated by upregulated expression of endothelial cell surface adhesion molecules and subsequent adhesion and activation of circulating leukocytes. The purpose of this study was to evaluate the effect of an intraoperative administration of FTY720 in an animal model with controlled bilateral warm kidney ischemia compared to steroids or placebo application.

Methods

Male C57BL6/J mice (n = 72, weight 25 to 30 g) were exposed to 30 minutes of bilateral kidney ischemia and followed by a 48 hour observation period. FTY720 (1 mg/kg body weight [BW]), steroids (5 mg/kg BW), or saline solution were administered. In addition, a sham-operated control group was included. At the termination of the experiments, all surviving animals were humanely killed. The impact of the various drugs on overall animal survival, timing of death, peripheral T-cell count, and T-lymphocyte infiltration in the kidneys was determined.

Results

Following bilateral kidney I/R injury, FTY720 was associated with a significant improved animal survival (85.7%) compared with steroids (50%) or controls (42.4%). FACS analysis showed significant T-lymphocyte depletion in peripheral blood in the FTY720 but not in the other groups. T-lymphocyte tissue concentration in liver and kidney tissue did not show statistically significant differences following FTY720, steroid, or saline treatment.

Conclusion

FTY720, when administered intraoperatively, improved survival significantly in mice submitted to bilateral kidney ischemia but did not have any significant impact on the parenchymal T-lymphocyte infiltration in the ischemic organ.     

大鼠肝脏缺血-再灌注损伤时齐墩果酸对氧自由基的影响

目的 探讨齐墩果酸(OA)对大鼠肝脏缺血-再灌注损伤时氧自由基的影响.方法 128只雄性SD大鼠随机分为假手术组(SH组)、缺血-再灌注组(IR组)、羧甲基纤维素钠(CMC-Na组)和OA组.建立70%肝脏缺血-再灌注模型,测定肝脏缺血60 min再灌注0、3、6、12 h后血清ALT活性和肝组织丙二醛(MDA)水平、超氧化物歧化酶(SOD)活性、谷胱甘肽(GSH)水平.结果 再灌注3、6、12 h,IR组、CMC-Na组、OA组血清ALT活性、肝组织MDA水平分别显著高于SH组(P<0.05),肝组织SOD活性、GSH水平分别明显低于SH组(P<0.05);OA组ALT活性、MDA水平分别较IR组和CMC-Na组明显降低(P<0.05),SOD活性和GSH水平分别较IR组和CMC-Na组显著升高(P<0.05).结论 OA对肝脏缺血-再灌注损伤具有一定保护作用,其抗肝脏缺血-再灌注损伤作用与抑制自由基的生成和释放有关.     

Preconditioned hyperbaric oxygenation protects the liver against ischemia-reperfusion injury in rats

Hyperbaric oxygen (HBO) therapy is an effective adjunct in treating ischemia-reperfusion (I/R) injury of brain, small intestine, testis, and crushing extremities. This study was designed to test the hypotheses that preconditioning the rats with HBO could protect the liver against subsequent I/R injury. Daily treatment with one-dose HBO (90 min, 2.5 ATA) was brought about for male Sprague Dawley rats for 1 to 3 days before an I/R injury of liver. Hepatic expression of heat-shock protein 70 (Hsp70), total concentration of glutathione (GSH), activity of catalase, superoxide dismutase (SOD), and serum AST and ALT were estimated before and after HBO, as well as after I/R injury. The results showed that activity of hepatic catalase was decreased by one dose, but not three doses, of HBO as compared with baseline data. However, hepatic Hsp70 expression fluctuated insignificantly. AST and ALT increase less in rats preconditioned with one-dose HBO as compared with those without HBO or with three-dose HBO. Our results showed preconditioning by one-dose HBO protects rat liver against subsequent ischemia-reperfusion injury.     

Akt activation protects rat liver from ischemia/reperfusion injury

BACKGROUND: Apoptosis as well as necrosis may play an important role in hepatic ischemia/reperfusion (I/R) injury. Akt, a serine-threonine protein kinase, is known to promote cell survival. We investigated whether gene transfer of constitutively active or dominant negative Akt could affect hepatic I/R injury. MATERIALS AND METHODS: Hepatic I/R injury was induced in rats by Pringle's maneuver for 20 min followed by reperfusion. Adenoviruses encoding a constitutively active form of Akt (myrAkt), a dominant negative form of Akt (dnAkt), or beta-galactosidase (LacZ) were injected through the tail vein 72 h before hepatic I/R. RESULTS: Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining demonstrated a significant increase in the positive cells 240 min after reperfusion. Immunoblotting with phospho-Akt antibody showed phosphorylation of Akt from 90 to 180 min after reperfusion. The expression of myrAkt reduced the number of TUNEL-positive cells and hepatic necrosis around the central veins in the liver after reperfusion. This expression also significantly inhibited the increase in serum alanine aminotransferase (297 +/- 131 IU/L, P < 0.05) 120 min after I/R, compared with increases in uninfected (1761 +/- 671 IU/L), LacZ adenovirus (1528 +/- 671 IU/L)-, and dnAkt adenovirus (1342 +/- 485 IU/L)-infected rats. MyrAkt expression phosphorylated Bad and inhibited the release of cytochrome-c after reperfusion. No difference in nuclear translocation of nuclear factor (NF)-kappaB, p65 was seen among the three groups of rats, however. CONCLUSION: Adenoviral gene transfer of myrAkt could inhibit apoptotic cell death and subsequent hepatic I/R injury in the rat, through Bad, not NF-kappaB.     

硫化氢对大鼠肝缺血再灌注损伤的影响

目的 评价硫化氢对大鼠肝缺血再灌注损伤的影响.方法 健康雄性SD大鼠30只,体重220～250 g,采用随机数字表法,将其随机分为假手术组(S组)、缺血再灌注组(IR组)和不同剂量硫化氢组(H2S1～3组),每组6只.S组仅暴露肝门,不夹闭动、静脉;IR组采用夹闭左、中叶肝蒂、门静脉和肝动脉支1h恢复灌注的方法制备大鼠肝缺血再灌注模型;H2S1～3组于再灌注前5min分别腹腔注射14、28、56 μmol/kg硫化氢钠.于再灌注6h时抽取下腔静脉血样并取肝组织,采用全自动生化分析仪测定血清谷丙转氨酶(ALT)和谷草转氨酶(AST)活性,采用二硫代二硝基苯甲酸法测定肝组织谷胱甘肽(GSH)含量,光镜下观察肝组织病理学结果.结果 与S组相比,IR组血清ALT和AST活性升高,肝组织GSH含量下降(P＜0.05);与IR组相比,H2S1～3组ALT和AST活性降低,肝组织GSH含量升高(P＜0.05);H2S1～3组肝病理学损伤较IR组明显减轻.结论 H2S可减轻大鼠肝缺血再灌注损伤.     

Attenuation of liver and lung injury after hepatic ischemia and reperfusion by a cytokine-suppressive agent, FR167653

BACKGROUND: Hepatic ischemia/reperfusion (I/R) injury is an important clinical problem and leads to the release of the proinflammatory cytokines, TNF-alpha and IL-1. These cytokines play important roles in the induction of polymorphonuclear neutrophil (PMN) activation and infiltration, and induce not only localized hepatic injury but also remote organ injury, especially pulmonary injury. Using a total hepatic ischemia model in rats, we tested our hypothesis that suppression of TNF-alpha and IL-1 by FR167653 ameliorates I/R injury in the liver and lung. METHODS: Male Wistar rats, weighing 240-280 g, were divided into 3 groups, an FR group, a control group and a sham group. In the FR group, FR167653 (1 mg/kg/h) was administered continuously to the animals for 30 min prior to the onset of ischemia and for 2 h after reperfusion. The control group received normal saline. A porto-systemic shunt was placed between the cecal branch of the portal vein and the jugular vein, and total hepatic ischemia was produced for 90 min. The sham group was treated with placement of the porto-systemic shunt only. The 1-week survival rate, liver enzyme activity, hepatic tissue blood flow (HTBF), cytokine mRNA expression, myeloperoxidase (MPO) activity and histological results were studied. RESULTS: The 1-week survival rate and HTBF were significantly higher in the FR group than in the control group. Serum AST, ALT, and LDH levels were significantly lower in the FR group at 30 min, 1 h and 3 h after reperfusion. MPO levels in liver and lung tissue were also significantly lower in the FR group. The expression of IL-1beta mRNA remarkably decreased up to 6 h after reperfusion in the FR group. CONCLUSIONS: We concluded that the inflammatory cytokines, IL-1beta, play important roles in hepatic I/R injury. FR167653 might ameliorate I/R injury and be useful in liver surgery with ischemia.