Splenic artery ligation: A protection against hepatic ischemia/reperfusion injury in partially hepatectomized rats

Aim: In liver resection, the temporary occlusion of the hepatoduodenal ligament (Pringle maneuver) is often used. However, the maneuver causes ischemia/reperfusion (I/R) injury in the remnant liver. Heme oxygenase (HO)‐1 has a cytoprotective role against this injury. Our aim is to investigate whether splenic artery ligation induces HO‐1 expression in the liver and ameliorates the hepatic I/R injury in partially hepatectomized rats. Methods: Rats underwent splenic artery ligation by occluding the main splenic artery. Two days later, the total hepatic ischemia (Pringle maneuver) was conducted, and then a two‐thirds partial hepatectomy (PH) was performed just before the start of reperfusion. HO inhibitor was twice injected s.c. at 3 and 16 h before the Pringle maneuver. HO‐1 levels were determined by western blotting. Liver injury was biochemically assessed. Results: In normal rats, HO‐1 was highly expressed in the spleen, but not in the liver. Splenic artery ligation induced HO‐1 in the livers. When rats underwent 20 and 30 min of Pringle maneuver/PH, survival rates were 28% and 8%, respectively. Splenic artery ligation significantly improved both the survival rates: 73% and 56%, respectively. Under these conditions, administration of HO‐1 inhibitor at least partly negated the efficacy of splenic artery ligation. Splenic artery ligation also increased the recovery rate of the remnant liver mass and platelet counts in Pringle maneuver/PH‐treated rats. Conclusion: Splenic artery ligation was significantly effective on the hepatic I/R injury in partially hepatectomized rats. Induction of HO‐1 may be at least partly involved in the improvement of this injury.     

Effects of Wy14643 on hepatic ischemia reperfusion injury in rats

AIM： To investigate the effects and possible mechanisms of Wy14643 on hepatic ischemiareperfusion （I/R） injury in rats. METHODS： Thirty male Sprague-Dawley rats weighing 220-280 g were randomly divided into five experimental groups： sham group （G1, n = 6）： a sham operation was performed （except for liver I/R）, I/R-untreated group （G2, n = 6）： rats underwent liver ischemia for 90 min followed by reperfusion for 4h; and I/R ＋ Wy14643 groups （G3, G4, G5; n = 6）： after the same surgical procedure as in group 2, animals were pretreated with Wy14643 at the dose of 1, 5 and 10 mg/kg 1 h before ischemia, respectively. Hepatic ischemia-reperfusion （I/R） was induced by clamping blood supply to the left lateral and median lobes of the liver for 90 min, and atraumatic clamp was removed for 4 h reperfusion. Blood samples and liver tissues were obtained at the end of reperfusion to assess serum and hepatic tissue homogenate aminotransferase （ALT）, aspartate aminotransferase （AST）, myeloperoxidase （MPO）, serum interleukin- 1β（IL-1β） and tumor necrosis factor alpha （TNF-α）, as well as activity of superoxide dismutase （SOD） and content of malondialdehyde （MDA） in the hepatic tissue homogenate. RESULTS： Hepatic I/R induced a significant increase in the serum levels of ALT, AST, TNF-α, IL-1β and MPO, as well as the levels of ALT, AST and MDA in the liver tissue homogenate, which were reduced by pretreatment with Wy14643 at the dose of 1, 5 and 10 mg/kg, respectively. The activity of SOD in the liver tissue homogenate was decreased after hepatic I/R, which was enhanced by Wy14643 pretreatment. In addition, serum and liver tissue homogenate ALT and AST in the Wy14643 10 mg/kg group were lower than in the Wy14643 1 mg/kg and 5 mg/kg groups, respectively. CONCLUSION： Wy14643 pretreatment exerts significant protection against hepatic I/R injury in rats. The protective effects are possibly associated with enhancement of anti-oxidant and inhibition inflammation res     

Doxorubicin preconditioning: a protection against rat hepatic ischemia-reperfusion injury

Doxorubicin produces clinically useful responses in a variety of human cancers. However, the toxicity of doxorubicin has limited its usefulness. This side effect is mainly due to the doxorubicin-mediated free radical formation. Administration of doxorubicin (10 mg/kg body weight) to rats intravenously induces heme oxygenase-1 (HO-1) in the liver. The levels of HO-1 protein were first detected at 6 hours and peaked at about 18 to 24 hours after the injection. It is known that HO-1 plays a protective role against the oxidative injury. Therefore, we have examined the protective effect of doxorubicin preconditioning against the hepatic ischemia-reperfusion injury. Partial hepatic ischemia was produced in the left and medium lobes for 45 minutes followed by 120 minutes reperfusion. When low doses of doxorubicin (1 mg/kg body weight) was intravenously administered to rats 2 days before the ischemia, the serum alanine transaminase (ALT) levels in the preconditioning rat were clearly improved compared with those in the rat without preconditioning. Under this situation, zinc-protoporphyrin IX, a specific inhibitor of HO-1, was injected subcutaneously to rats at 3 and 16 hours before the ischemia, the ALT levels were not improved by doxorubicin preconditioning. Histopathologic examination also supported these results. Although the HO-1 protein level was fairly low 2 days after the doxorubicin administration, significant amounts of HO-1 protein were detected. Our results indicated that the induction of HO-1 played a protective role against hepatic ischemia-reperfusion injury and that doxorubicin preconditioning is more clinically useful than other preconditioning methods.     

大白兔肝缺血再灌注损伤时血浆内毒素的变化及对肝肾功能的影响

目的探讨肝缺血再灌注损伤过程中,肠源性内毒素的动态变化和继发性肝肾功能损害。方法取27只健康成年新西兰大白兔,体重1.4~2.3?,随机分为对照组7只,另外20只作为实验组。以缺血10min(I10min)、缺血20min(I20min)、缺血30min(I30min)和分别再灌注30min(R30min)随机分为3组。对照组取门、腔静脉血测肝肾功能及血浆内毒素,实验组阻断第一肝门造成不同的缺血时段,松开血管夹再灌注30min,其余实验同对照组。结果实验组中血浆谷草转氨酶、谷丙转氨酶、尿素氮、肌苷含量及内毒素浓度均有升高,在I10min/R30min组即有升高,但与对照组相比差异无显著性(P>0.05);而后随着缺血时间的延长这些指标继续明显升高,至I30min/R30min组达最高值,与对照组比差异有显著性(P<0.05~0.01)。肾组织电镜观察发现I10min/R30min组肾脏超微结构无明显改变,而I20min/R30min组和I30min/R30min组肾脏超微结构损害明显。结论肝门阻断后门静脉系统淤血,致肠源性内毒素产生和移位;肝门再开放造成肝缺血再灌注损伤,且随着缺血时间的延长,门、腔静脉血中内毒素水平进行性升高,肝功能进一步损害,最终引起肝肾综合征。     

血红素氧合酶对大鼠肝脏缺血再灌注损伤细胞凋亡及相关基因的影响

目的通过应用血红素氧合酶（HO）的诱导剂氯高血红素和抑制剂锌原卟啉（ZnPP），探讨HO对大鼠肝脏缺血再灌注（IR）细胞凋亡及其相关基因的影响。方法将96只Sprague—Dawley大鼠采用钳夹法制备肝脏IR模型，随机分为假手术组、IR组、氯高血红素组和ZnPP组，检测再灌注0、1．5、4h和8h各个时间点大鼠肝脏功能以及病理学改变，流式细胞法测定肝细胞凋亡率、TUNEL法观察再灌注后4h大鼠肝细胞的凋亡情况，Western blot法检测再灌注后8h Bcl-2和Caspase-3的表达。结果在IR组各时间点均可见ALT和AST增高，病理学检查可见肝细胞肿胀，肝窦变窄，嗜中性粒细胞浸润和片状坏死等变化，肝组织中细胞凋亡率明显升高，Bcl-2的表达减少，而Caspase-3的表达增加。在氯高血红素组再灌注后1．5、4h和8h ALT和AST值明显降低，肝脏病理学改善，凋亡细胞减少及细胞凋亡率降低，肝脏Bcl-2的表达增加，Caspase-3的表达减少。ZnPP组则显示与之相反的结果。结论HO在肝脏IR损伤中具有保护作用，这种保护作用可能与抑制细胞凋亡有关。     

The multiple functions of heme oxygenase-1 in the liver

Heme oxygenases (HO) are essential enzymes which degrade heme into carbon monoxide (CO), biliverdin and free iron. Due to its anti-inflammatory, anti-apoptotic and, as recently described, anti-viral properties the inducible HO isoform HO-1 is an important molecule which could find its way into therapy of gastrointestinal diseases. Acute and chronic liver injuries including acute liver failure, alcoholic or viral hepatitis, chronic inflammation, fibrosis, cirrhosis, and hepatocellular carcinoma are life threatening diseases and as a consequence might result in the necessity of liver transplantation. HO-1 as well as its reaction products of heme degradation has been linked to cytoprotection. HO-1 induction in rodent models of acute and chronic hepatic inflammation resulted in improvement of liver damage and down-regulation of pro-inflammatory cytokine levels. Furthermore HO-1 induction interfered with fibrosis progression in mice and partially resolved existing fibrosis. Likewise, HO-1 induction interfered with replication of hepatitis viruses B and C, which frequently are the reason for chronic hepatitis and subsequent tumor growth. Liver transplantation is limited by ischemia/reperfusion (I/R) injury, which is characterized by hypoxia and nutrient deficiency resulting in oxidative stress, apoptosis and immune activation. Induction of HO-1 and application predominantly of CO have been shown to interfere with I/R liver injury and to improve recipient and graft survival. On the other hand HO-1 has been shown to be over-expressed in various tumors, including hepatocellular carcinoma (HCC). Due to its anti-apoptotic properties this bears the risk to promote tumor growth. Anti-apoptotic effects are predominantly mediated by CO. This review aims to summarize beneficial as well as detrimental effects of HO-1 and its products within the liver.     

Effect of notoginsenoside R1 on hepatic microcirculation disturbance induced by gut ischemia and reperfusion

AIM： To assess the effect of notoginsenoside R1 on hepatic microcirculatory disturbance induced by gut ischemia/reperfusion （I/R） in mice. METHODS： The superior mesenteric artery （SMA） of C57/BL mice was ligated for 15 min to induce gut ischemia followed by 30-rain reperfusion. In another set of experiments, R1 was continuously infused （10 mg/kg per hour） from 10 min before I/R until the end of the investigation to study the influence of R1 on hepatic microcirculatory disturbance induced by gut I/R. Hepatic microcirculation was observed by inverted microscopy, and the vascular diameter, red blood cell （RBC） velocity and sinusoid perfusion were estimated. Leukocyte rolling and adhesion were observed under a laser confocal microscope. Thirty and 60 min after reperfusion, lactate dehydrogenase （LDH）, alanine aminotransferase （ALl＇） and aspartate transaminase （AST） in peripheral blood were determined. The expression of adhesion molecules CD11b/CD18 in neutrophils and tumor necrosis factor- alpha （TNF-α）, interleukin-6 （IL-6） and monocyte chemotactic protein-1 （MCP-1） in plasma were evaluated by flow Oltometry. E-selectin and intercellular adhesion molecule-1 （ICAM-1） in hepatic tissue were examined by immunofluorescence.RESULTS： After gut I/R, the diameters of terminal portal venules and central veins, RBC velocity and the number of perfused sinusoids were decreased, while the leukocyte rolling and adhesion, the expression of E-selectin in hepatic vessels and CD18 in neutrophils, IL-6, MCP-1, LDH, ALT and AST were increased. R1 treatment attenuated these alterations except for IL-6 and MCP-1. CONCLUSION： R1 prevents I/R-induced hepatic microcirculation disturbance and hepatocyte injury, The effect of R1 is related to its inhibition of leukocyte rolling and adhesion by inhibiting the expression of E-selectin in endothelium and CD18 in neutrophils.     

Modulation of liver oxidant-antioxidant system by ischemic preconditioning during ischemia／reperfusion injury in rats

AIM: To investigate effects of ischemic pre-conditioning on the liver endogenous oxidant-antioxidant system during ischemia/reperfusion injury. METHODS: Twenty-four male Sprague-Dawley rats were randomly divided into sham-operated (Sham), ischemia/ reperfusion (I/R), ischemic pre-conditioning plus ischemia/ reperfusion (IPC) groups. Serum ALT, AST and hyaluronic acid levels were assayed and pathologic alterations observed. Liver malondialdehyde (MDA) contents, endogenous antioxidant enzymes, superoxidase dismutase (SOD), catalase (CAT), gultathionine peroxidase (GSH-Px) activities, neutrophils accumulation marker, myeloperoxidase (MPO) activities were measured respectively. RESULTS: Compared with I/R group, sinusoidal endothelial cells as well as hepatocytes damages, as assessed biochemically and histochemically, were improved significantly in IPC group; neutrophils infiltration was also markedly reduced. In IPC group, liver peroxidation, as measured by MDA contents, was significantly decreased when compared with I/R group; endogenous antioxidant enzymes, SOD, CAT and GSH-Px activities were markedly higher than that in I/R group. CONCLUSION: Ischemic pre-conditioning exerts protective effects on both hepatic sinusoidal endothelial cells and hepatocytes during liver I/R injury. Its mechanisms may involve dimunition of neutrophils infiltration and modulation of the imbalance of endogenous oxidant-antioxidant system in the organism.     

Heme oxygenase-1 induction by hemin protects liver cells from ischemia/reperfusion injury in cirrhotic rats

AIM： To investigate the potential protective effect of HO-1 on cirrhotic liver cells in rats.
METHODS： Male Wistar rats included in the current study were randomly divided into 5 groups as follows： normal （N） group; liver cirrhotic （LC） group; sham （S） group; I/R group and I/R ＋ hemin group. The model for inducing liver cirrhosis in rats was established according to a previously published protocol. Following this the segmental hepatic ischemia reperfusion operation was carried out. The rats were treated with 30 l~mol/kg hemin （HO-1 inducer, ferric portoporphyrin IX chloride） i.p. or 0.9% NaCI （control） 24 h and 12 h before hepatic ischemia for 30 min or sham laparotomy. Blood was collected for serum enzymatic measurement 6 and 12 h after reperfusion or sham laparotomy. HO-1, NF-κB and caspase-3 expressions were assessed by immunohistochemical analysis.
RESULTS： The expressions of proteins are inversely correlated to the gray values. HO-1 expression in the I/R ＋ hemin group was increased significantly than I/R group at 6 h and 12 h after hepatic I/R （6 h： 112.0± 8.3 vs 125.1± 5.7, P 〈 0.01; 12 h： 120.8± 11.0 vs 132.4 ± 6.2, P 〈 0.01）. Hemin improved serum manganese superoxide dismutase （MnSOD） （6 h： 131.3 ± 17.6 vs 107.0 ± 13.9, P 〈 0.01; 12 h： 141.4 ：E 12.5 vs 118.3± 10.2, P 〈 0.01）, lessened liver cell injury, decreased caspase-3（6 h： 166.7 ± 8.1 vs 145.5 ± 14.6, P 〈 0.01; 12 h： 172.8± 3.8 vs 148.0 ±6.5, P 〈 0.01） and NF-κB expression （6 h： 150.2 ± 8.6 vs 139.7 ±6.0, P 〈 0.01; 12 h： 151.1 ± 5.9 vs 148.1± 5.3, P 〉 0.05） and serum alanine aminotransferase （ALT） （6 h： 413.3± 104.1 vs 626.8 ±208.2, P 〈 0.01; 12 h： 322.2 ± 98.8 vs 425.8 ± 115.4, P 〈 0.05）, aspartate aminotransferase （AST） （6 h： 665.2 ± 70.1 vs 864.3± 70.4, P 〈 0.01; 12 h： 531.1 ± 98.6 vs 664.4± 115.6, P 〈 0.01）, malondialdehyde （MDA） levels （6 h： 11.1 ± 2.17 vs 13.5 ±2.01, P 〈 0.01; 12 h： 9.36     

Prophylaxis with carnosol attenuates liver injury induced by intestinal ischemia/reperfusion

AIM： To investigate the possible protective effects of carnosol on liver injury induced by intestinal ischemia reperfusion （I/R）.
METHODS： Rats were divided randomly into three experimental groups： sham, intestinal I/R and carnosol treatment （n = 18 each）. The intestinal I/R model was established by clamping the superior mesenteric artery for 1 h. In the carnosol treatment group, surgery was performed as in the intestinal I/R group, with intraperitoneal administration of 3 mg/kg carnosol 1 h before the operation. At 2, 4 and 6 h after reperfusion, rats were killed and blood, intestine and liver tissue samples were obtained. Intestine and liver histology was investigated. Serum levels of aspartate aminotransferase （AST）, alanine aminotransferase （ALT） and interleukin （IL）-6 were measured. Liver tissue superoxide dismutase （SOD） and myeloperoxidase （IvIPO） activity were assayed. The liver intercellular adhesion molecule-1 （ICAM-1） and nuclear factor κB （NF-κB） were determined by immunohistochemical analysis and western blot analysis.
RESULTS： Intestinal I/R induced intestine and liver injury, characterized by histological changes, as well as a significant increase in serum AST and ALT levels. The activity of SOD in the liver tissue decreased after I/R, which was enhanced by carnosol pretreatment. In addition, compared with the control group, carnosol markedly reduced liver tissue MPO activity and serum IL-6 level, which was in parallel with the decreased level of liver ICAI-1 and NF-κB expression.
CONCLUSION： Our results indicate that carnosol pretreatment attenuates liver injury induced by intestinal I/R, attributable to the antioxidant effect and inhibition of the NF-κB pathway.     

Heme oxygenase-1 and gut ischemia/reperfusion injury: A short review

Ischemia/reperfusion (I/R) injury of the gut is a significant problem in a variety of clinical settings and is associated with a high morbidity and mortality. Although the mechanisms involved in the pathogenesis of gut I/R injury have not been fully elucidated, it is generally believed that oxidative stress with subsequent inflammatory injury plays an important role. Heme oxygenase (HO) is the rate-limiting enzyme in the catabolism of heme, followed by production of CO, biliverdin, and free iron. The HO system is believed to confer cytoprotection by inhibiting inflammation, oxidation, and apoptosis, and maintaining microcirculation. HO-1, an inducible form of HO, serves a vital metabolic function as the rate-limiting step in the heme degradation pathway, and affords protection in models of intestinal I/R injury. HO-1 system is an important player in intestinal I/R injury condition, and may offer new targets for the management of this condition.     

Protective Effect of L-arginine Against Necrosis and Apoptosis Induced by Experimental Ischemic and Reperfusion in Rat Liver

Background/Aim:

To study the effect of L-arginine on apoptosis and necrosis induced by 1-h ischemia followed by 3-h reperfusion.

Materials and Methods:

Adult Wistar rats underwent 60 min of partial liver ischemia followed by 3-h reperfusion. Eighteen Wistar rats were divided into sham-operated control group (I) (n = 6), ischemia and reperfusion (I/R) group (0.9% saline (5 mL/kg, orally) for 7 days) (II) (n = 6), and L-arginine-treated group (10 mg/kg body weight daily orally for 7 days before inducing ischemia-reperfusion maneuver) (III) (n = 6). Apoptotic and necrotic hepatocytes, nitric oxide levels in hepatocytes, Bcl-2 mRNA, and Bcl-2 protein were measured. Liver injury was assessed by plasma alanine transaminases (ALT), aspartate transaminases (AST), liver histopathology, and electron microscopy.

Results:

An ischemic and reperfusion hepatocellular injury occurred as was indicated by increased serum ALT, AST, histopathology, and electron microscopy. Apoptosis and necrosis associated marker gene Bcl-2 mRNA and protein expression were decreased in I/R group. Pretreatment with L-arginine significantly decreased serum ALT and AST level and apoptotic and necrotic cells after 1 h ischemia followed by 3 h of reperfusion. Nitric oxide production in hepatocytes was increased twofold by L-arginine treatment when compared with I/R group. Histopathology and transmission electron microscopy (TEM) studies showed markedly diminished hepatocellular injury in L-arginine-pretreated rats during the hepatic I/R.

Conclusion:

Thus, it may be concluded that L-arginine afforded significant protection from necrosis and apoptosis in I/R injury by upregulated Bcl-2 gene and nitric oxide production.     

Nuclear factor-kappaB decoy oligodeoxynucleotides attenuates ischemia/reperfusion injury in rat liver graft

AIM: To evaluate the protective effect of NF-kappaB decoy oligodeoxynucleotides (ODNs) on ischemia/reperfusion (I/R) injury in rat liver graft. METHODS: Orthotopic syngeneic rat liver transplantation was performed with 3 h of cold preservation of liver graft in University of Wisconsin solution containing phosphorothioated double-stranded NF-kappaB decoy ODNs or scrambled ODNs. NF-kappaB decoy ODNs or scrambled ODNs were injected intravenously into donor and recipient rats 6 and 1 h before operation, respectively. Recipients were killed 0 to 16 h after liver graft reperfusion. NF-kappaB activity in the liver graft was analyzed by electrophoretic mobility shift assay (EMSA). Hepatic mRNA expression of TNF-alpha, IFN-gamma and intercellular adhesion molecule-1 (ICAM-1) were determined by semiquantitative RT-PCR. Serum levels of TNF-alpha and IFN-gamma were measured by enzyme-linked immunosorbent assays (ELISA). Serum level of alanine transaminase (ALT) was measured using a diagnostic kit. Liver graft myeloperoxidase (MPO) content was assessed. RESULTS: NF-kappaB activation in liver graft was induced in a time-dependent manner, and NF-kappaB remained activated for 16 h after graft reperfusion. NF-kappaB activation in liver graft was significant at 2 to 8 h and slightly decreased at 16 h after graft reperfusion. Administration of NF-kappaB decoy ODNs significantly suppressed NF-kappaB activation as well as mRNA expression of TNF-alpha, IFN-gamma and ICAM-1 in the liver graft. The hepatic NF-kappaB DNA binding activity [presented as integral optical density (IOD) value] in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (2.16+/-0.78 vs 36.78+/-6.35 and 3.06+/-0.84 vs 47.62+/- 8.71 for IOD value after 4 and 8 h of reperfusion, respectively, P<0.001). The hepatic mRNA expression level of TNF-alpha, IFN-gamma and ICAM-1 [presented as percent of beta-actin mRNA (%)] in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (8.31+/-3.48 vs 46.37+/-10.65 and 7.46+/- 3.72 vs 74.82+/-12.25 for hepatic TNF-alpha mRNA, 5.58+/-2.16 vs 50.46+/-9.35 and 6.47+/-2.53 vs 69.72+/-13.41 for hepatic IFN-gamma mRNA, 6.79+/-2.83 vs 46.23+/-8.74 and 5.28+/-2.46 vs 67.44+/-10.12 for hepatic ICAM-1 mRNA expression after 4 and 8 h of reperfusion, respectively, P<0.001). Administration of NF-kappaB decoy ODNs almost completely abolished the increase of serum level of TNF-alpha and IFN-gamma induced by hepatic ischemia/reperfusion, the serum level (pg/mL) of TNF-alpha and IFN-gamma in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (42.7+/-13.6 vs 176.7+/-15.8 and 48.4+/-15.1 vs 216.8+/-17.6 for TNF-alpha level, 31.5+/-12.1 vs 102.1+/-14.5 and 40.2+/-13.5 vs 118.6+/-16.7 for IFN-gamma level after 4 and 8 h of reperfusion, respectively, P<0.001). Liver graft neutrophil recruitment indicated by MPO content and hepatocellular injury indicated by serum ALT level were significantly reduced by NF-kappaB decoy ODNs, the hepatic MPO content (A655) and serum ALT level (IU/L) in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (0.17+/-0.07 vs 1.12+/-0.25 and 0.46+/-0.17 vs 1.46+/-0.32 for hepatic MPO content, 71.7+/-33.2 vs 286.1+/-49.6 and 84.3+/-39.7 vs 467.8+/-62.3 for ALT level after 4 and 8 h of reperfusion, respectively, P<0.001). CONCLUSION: The data suggest that NF-kappaB decoy ODNs protects against I/R injury in liver graft by suppressing NF-kappaB activation and subsequent expression of proinflammatory mediators.     

Protective effect of curcumin against liver warm ischemia/reperfusion injury in rat model is associated with regulation of heat shock protein and antioxidant enzymes

AIM： To investigate the hypothesis that the protective effects of curcumin in hepatic warm ischemia/reperfusion （I/R） injury are associated with increasing heat shock protein 70 （Hsp70） expression and antioxidant enzyme activity. METHODS： Sixty Sprague-Dawley male rats were randomly divided into sham, I/R, C ＋ I/R groups. The model of reduced-size liver warm ischemia and reperfusion was used. Curcumin （50 mg/kg） was administered by injection through a branch of superior mesenteric vein at 30 min before ischemia in C ＋ I/R group. Five rats were used to investigate the survival during 1 wk after operation in each group. Blood samples and liver tissues were obtained in the remaining animals after 3, 12, and 24 h of reperfusion to assess serum alanine aminotransferase （ALT）, aspartate aminotransferase （AST）, liver tissue NO2- ＋ NO3-, malondialdehyde （MDA） content, superoxide dismutase （SOD）, catalase （CAT）, nitricoxide synthase （NOS） and myeloperoxidase （MPO） activity, HspT0 expression and apoptosis ratio. RESULTS： Compared with I/R group, curcumin pretreatment group showed less ischemia/reperfusioninduced injury. CAT and SOD activity and Hsp70 expression increased significantly. A higher rate of apoptosis was observed in I/R group than in C ＋ I/R group, and a significant increase of MDA, NO2^- ＋ NO3^- and MPO level in liver tissues and serum transaminase concentration was also observed in I/R group compared to C ＋ I/R group. Curcumin also decreased the activity of inducible NO synthase （iNOS） in liver after reperfusion,but had no effect on the level of endothelial NO synthase （eNOS） after reperfusion in liver. The 7 d survival rate was significantly higher in C ＋ I/R group than in I/R group. CONCLUSION： Curcumin has protective effects against hepatic I/R injury. Its mechanism might be related to the overexpression of Hsp70 and antioxidant enzymes.     

Protective role of an endothelin-converting enzyme inhibitor (FR901533) in hepatic ischemia/reperfusion injury.

INTRODUCTION: There is evidence that endothelin (ET) contributes to disturbances of the hepatic microcirculation after warm ischemia/reperfusion (I/R) by causing vasoconstriction and by enhancing leukocyte endothelium interactions. The aim of this study was to investigate a possible protective role of the endothelin converting enzyme (ECE) inhibitor FR901533 in this setting. METHODS: In an in vivo model (42 Wistar rats), hepatic ischemia was induced for 30 min by Pringle's maneuver. Sham operated (I), untreated ischemic (II), and treatment (III) groups with FR901533 (1 mg/kg bw iv) were investigated. The effect of FR901533 in I/R was assessed by in vivo microscopy (30-90 min after reperfusion), measurement of local tissue pO2 (30 and 60 min after reperfusion), and determination of AST/ALT levels (2 h, 6 h, and 2, 6, and 14 days after reperfusion). RESULTS: In the untreated ischemic group (II) sinusoidal constriction to 76.3 +/- 4.2% of basic diameters was observed, leading to significant decreases in perfusion rate (82.3 +/- 3.6% of sham group) and in liver tissue pO(2) (43.5 +/- 3.2% of sham group) (P < 0.05). In addition, we found an increased percentage of stagnant leukocytes in sinusoids (138.3 +/- 9.8) and sticking leukocytes in postsinusoidal venules (155.2 +/- 3.3% of sham group) (P < 0.05). Hepatocellular damage (AST/ALT increase to 430.6 +/- 47.7 U/L/200.2 +/- 23.8 U/L, pre: 27.4 +/- 2.7 U/L/28.1 +/- 2.7 U/L) was detected 6 h after reperfusion (P < 0.05). Administration of the ECE inhibitor before ischemia significantly reduced I/R injury. Sinusoidal diameters were maintained (102.2 +/- 1.7%), while perfusion rate (93.1 +/- 1.8%) and tissue pO2 (105.3 +/- 2.7%) increased significantly (P < 0.05). Hepatocellular damage was decreased (AST/ALT levels after 6 h of reperfusion: 166.6 +/- 26.3 U/L/132.4 +/- 22.5 U/L, P < 0.05) and leukocyte sticking and rolling were significantly reduced (P < 0.05). CONCLUSION: Our results provide evidence that the new therapeutic approach with an ECE inhibitor is effective in reducing hepatic I/R injury.     

The protective effect of erythropoietin on ischaemia/reperfusion injury of liver

BackgroundBesides its haematopoietic effect, erythropoietin (EPO) has multiple protective effects, i.e. antiapoptotic, antioxidant and angiogenic properties. The neuroprotective effects of EPO against ischaemia have all been demonstrated in cell culture and animal models. The aim of the study was to evaluate the effect of erythropoietin on ischaemia-reperfusion injury (I/R injury) of the liver.MethodsForty-eight adult male Sprague-Dawley rats weighing 250–300 g were divided into three groups: group I, hepatic ischaemia-reperfusion (Hepatic I/R); group II, hepatic ischaemia-reperfusion + EPO (Hepatic I/R+ EPO); group III, sham. Hepatic ischaemia was created by placing a microvascular clamp on the hepatic pedicle for 45 minutes. EPO was given to group II at a dose of 1000 U/kg 120 minutes before the onset of the ischaemia. Blood samples and liver tissues were obtained after 45 minutes of reperfusion from half of the rats in each group. The remaining rats were killed after a 24-hour observation period and blood and tissue samples were obtained. Blood alanine aminotransferase, tumour necrosis factor-α (TNF-α), interleukin-2 (IL-2) and liver tissue malondialdehyde (MDA) levels were determined. Liver tissue histopathology was also evaluated by light microscopy.ResultsIn rats with hepatic ischaemia, serum levels of ALT, TNF-α, IL-2 and liver tissue levels of MDA were reduced by the administration of erythropoietin and the histopathological score was also less severe.ConclusionThis study demonstrates that pre-ischaemic administration of EPO has protective effects on hepatic I/R injury.     

Hepatoprotective effect of endogenous nitric oxide during ischemia-reperfusion in the rat

The aim of this study was to evaluate the protective or deleterious effects of endogenous nitric oxide (NO) on liver cells during hepatic ischemia-reperfusion (IR) in the rat. Injury to hepatocytes and endothelial cells was evaluated by determining cytolysis-marker activity in plasma (alanine transaminase [ALT]; aspartate transaminase [AST]) and plasma hyaluronic acid (HA) concentration. Clamping the hepatic pedicle for 45 minutes caused a significant increase in plasma AST and ALT activity after 30 minutes of reperfusion, which reached a maximum (+270% and +740%, respectively) after 6 hours of reperfusion. Plasma HA concentration was significantly higher (+130%) only after 6 hours of reperfusion. Administration of a nonselective NO synthase (NOS) inhibitor, Nomega-nitro-L-arginine (L-NNA; 10 mg/kg iv), 30 minutes before IR, caused marked aggravation of postischemic liver injury, as shown by plasma ALT and AST activity and HA concentration. This deleterious effect was partially prevented by the simultaneous injection of L-arginine, the endogenous NO precursor (100 mg/kg iv). Interestingly, L-arginine alone limited postischemic damage (AST, -25%; ALT, -45%; HA, -21% vs. untreated IR rats at 6 hours reperfusion). Pretreatment with the Guanosine 3':5'-cyclic monophosphate-independent vasodilator, prazosin, partially reversed L-NNA effects, but it did not protect untreated IR animals. Pretreatment with aminoguanidine, a selective inhibitor of inducible NOS, did not aggravate hepatic IR injury. Thus, endogenous NO, probably produced by an early and transient activation of a constitutive NOS, protects both hepatocytes and endothelial cells against liver ischemia-reperfusion injury, and this effect is not entirely a result of vasorelaxation.     

Ex vivo exposure to carbon monoxide prevents hepatic ischemia/reperfusion injury through p38 MAP kinase pathway

A direct role of carbon monoxide (CO), an effector-signaling molecule during heme oxygenase-1 (HO-1) catalysis of heme, in the protection against hepatic ischemia/reperfusion (I/R) injury needs to be established. This study was designed to determine the effects and downstream mechanisms of CO on cold I/R injury in a clinically relevant isolated perfusion rat liver model. After 24 hours of cold storage, rat livers perfused ex vivo for 2 hours with blood supplemented with CO (300 parts per million) showed significantly decreased portal venous resistance and increased bile production, as compared with control livers perfused with blood devoid of CO. These beneficial effects correlated with improved liver function (serum glutamic oxaloacetic transaminase levels) and diminished histological features of hepatocyte injury (Banff's scores). The CO-mediated cytoprotective effects were nitric oxide synthase- and cyclic guanine monophosphate-independent, but p38 mitogen-activated protein kinase (MAPK)-dependent. Moreover, adjunctive use of zinc protoporphyrin, a competitive HO-1 inhibitor, has shown that exogenous CO could fully substitute for endogenous HO-1 in preventing hepatic I/R insult. This study performed in a clinically relevant ex vivo cold ischemia model is the first to provide the evidence that HO-1-mediated cytoprotection against hepatic I/R injury depends on the generation of, and can be substituted by, exogenous CO. The p38 MAPK signaling pathway represents the key downstream mechanism by which CO prevents the I/R insult. In conclusion, regimens that employ exogenous CO should be revisited, as they may have potential applications in preventing/mitigating I/R injury, and thus expanding the liver donor pool for clinical transplantation.