Hepatic ischemic preconditioning in mice is associated with activation of NF-kappaB,p38 kinase,and cell cycle entry

A brief period of hepatic ischemia protects the liver against subsequent ischemia-reperfusion (IR) injury, but the mechanism of such preconditioning is poorly understood. We examined whether preconditioning activated nuclear factor kappa B (NF-kappaB), the stress-activated protein kinases (SAPK), c-Jun N-terminal kinase-1 (JNK-1) and p38, and entry into the cell cycle. We used a murine model of partial hepatic ischemia. Preconditioning was performed by clamping the vasculature for 2 to 20 minutes, and allowing reperfusion for 10 minutes before 90-minute ischemia or IR. As assessed by serum alanine aminotransferase (ALT) levels and liver histology, preconditioning periods of 5 and 10 minutes were highly protective against IR injury, whereas 2-, 15-, and 20-minute intervals were ineffective. Preconditioning was associated with entry of hepatocytes into the cell cycle within 2 hours of subsequent IR, as indicated by proliferating cell nuclear antigen (PCNA) nuclear staining, induction of cyclin D1 and numerous mitotic figures; in the absence of preconditioning, such changes were not seen until 24 hours. Preconditioning increased nuclear binding of NF-kappaB within 30 minutes of the subsequent ischemic interval, paralleled by degradation of inhibitory (binding) protein for NF-kappaB (IkappaBalpha). Ischemic preconditioning also activated p38 kinase and JNK-1, which are known to converge on cyclin D1 regulation. The protective effect of the preconditioning regimen was more closely associated with p38 kinase than JNK-1 activation. In conclusion, the hepatoprotective effects of ischemic preconditioning are associated with activation of NF-kappaB and SAPKs that are associated with entry of hepatocytes into the cell cycle, a critical biological effect that favors survival of the liver against ischemic and IR injury.     

Splenectomy ameliorates hepatic ischemia and reperfusion injury mediated by heme oxygenase-1 induction in the rat

BACKGROUND/AIMS: Ischemia/reperfusion (I/R) induces severe organic injury. I/R injury seems to be mainly caused by oxidative stress. The aim of this study was to determine the role of the spleen in experimental hepatic I/R injury in the rat. Stress protein heme oxygenase (HO)-1 plays a protective role against the oxidative injury. In normal state, HO-1 is highly expressed in the spleen. METHODS: Liver HO-1 expression was assessed by Western blot before and after splenects. Liver injury was assessed by measurement of ALT and AST and by histopathology. RESULTS: Although HO-1 was not detected in normal liver, levels of HO-1 protein gradually increased and peaked on 3 days after splenectomy. When splenectomy was performed 3 days prior to the hepatic (45-min) ischemia followed by (2-h) reperfusion, the levels of serum aspartate transaminase (AST) and alanine transaminase (ALT), as markers for hepatic injury, were improved compared to the rats with I/R alone. In addition, prior administration of zinc-protoporphyrin IX, a specific inhibitor of HO, suppressed the protective effect of the splenectomy on the subsequent hepatic I/R injury. Histopathological examination also confirmed these results. CONCLUSIONS: Our findings suggest that the elevated HO-1 levels by splenectomy play a protective role against hepatic I/R injury.     

Low-dose TNF-alpha protects against hepatic ischemia-reperfusion injury in mice: implications for preconditioning

Tumor necrosis factor alpha (TNF-alpha) is implicated in the pathogenesis of hepatic ischemia reperfusion injury but can also prime hepatocytes to enter the cell cycle. Ischemic preconditioning protects against ischemia-reperfusion (IR) liver injury and is associated with activation of nuclear factor kappaB (NF-kappaB) and cell cycle entry. We examined the pattern of TNF-alpha release during hepatic IR in the presence or absence of ischemic preconditioning, and we tested whether a single low-dose injection of TNF could mimic the biologic effects of ischemic preconditioning. In na?ve mice, hepatic and plasma levels of TNF-alpha rose during hepatic ischemia, reaching high levels after 90 minutes; values remained elevated during reperfusion until 44 hours. Following the ischemic preconditioning stimulus, there was an early rise in hepatic and serum TNF-alpha levels, but, during a second prolonged ischemic interval peak, TNF-alpha values were lower than in na?ve mice and declined to negligible levels by 2 hours reperfusion. An injection with 1 microg or 5 microg/kg body weight TNF-alpha 30 minutes prior to hepatic IR substantially reduced liver injury determined by liver histology and serum alanine aminotransferase (ALT) levels. As in ischemic preconditioning, TNF-alpha pretreatment activated NF-kappaB DNA binding, STAT3, cyclin D1, cyclin-dependent kinase 4 (cdk4) expression, and cell cycle entry, determined by proliferating cell nuclear antigen (PCNA) staining of hepatocyte nuclei. In conclusion, the hepatoprotective effects of "preconditioning" can be simulated by TNF-alpha injection, which has identical downstream effects on cell cycle entry. We propose that transient increases in TNF-alpha levels may substitute for, as well as, mediate the hepatoprotective effects of ischemic preconditioning against hepatic IR injury.     

Impact of hyperthermic preconditioning on postischemic hepatic microcirculatory disturbances in an isolated perfusion model of the rat liver

Sublethal hyperthermia and the following recovery from this heat exposure, referred to as hyperthermic preconditioning, elicits a transient state of tolerance to oxidative insults through an intracellular protective response: stress response. The impact of hyperthermic preconditioning on hepatic microcirculatory disturbance, which is one of the determinants of ischemia/reperfusion-induced injury of the liver, was investigated by using intravital fluorescence microscopy. Thirty minutes of ischemia and a subsequent 120 minutes of reperfusion was induced in an in situ isolated perfusion model of Sprague-Dawley rats. Heat stress was given by whole-body hyperthermia, and a subsequent recovery was allowed for 18 or 48 hours, respectively. Postischemic decrease in sinusoidal perfusion rate and sinusoidal diameter, leukocyte stagnation in sinusoids, and leukocyte adhesion in postsinusoidal venules were significantly attenuated in both hyperthermia-pretreated groups. A recovery of bile production, a reduction of liver enzyme release, and an attenuation of tissue edema and histological damage were also observed. A marked expression of heat shock protein (HSP) 70 and heme oxygenase (HO-1)/HSP32 was correlatively observed in the liver tissue coincident with the induction of these protective effects. Hyperthermic preconditioning provides a continuous long-term and constant inhibitory effect (up to 48 hours after heat exposure) on postischemic injury of the liver through the attenuation of microcirculatory disturbances. These beneficial effects might be associated with a concomitant increase in HSP70 and HO-1/HSP32 expression.     

Protective effects of glutamine preconditioning on ischemia-reperfusion injury in rats

BACKGROUND:Hepatic ischemia-reperfusion injury is a common phenomenon in hepatic surgical procedures and can result in further severe damage.This study aimed to investigate the protective effects of glutamine preconditioning on hepatic ischemia-reperfusion injury in rats and its dose-dependency. METHODS:Thirty-two healthy male Wistar rats were randomly divided into four groups(n=8 per group).One group received 0.9%NaCl(control)and the other three received glutamine(Gln groups)4 hours before ischemia.The Gln...     

Oxidative injury in rat fatty liver exposed to ischemia-reperfusion is modulated by nutritional status

BACKGROUND AND AIMS: Oxidative stress contributes to ischemia-reperfusion injury in fatty livers. This study aimed to determine whether glycogen depletion influences this oxidative injury and whether the administration of glucose can be protective. METHODS: Rats with choline deficiency-induced fatty liver underwent hepatic ischemia-reperfusion. Experimental groups: (1) fed rats; (2) 18 h fasted rats; (3) 18 h fasted rats supplemented with glucose prior to surgery. The thiobarbituric acid-reactive substances, protein carbonyls and total glutathione concentrations were measured in liver tissue and isolated mitochondria as parameters of oxidative stress before and after ischemia and during reperfusion. The mitochondrial F1-ATPase content and the serum alanine transaminase were also determined. RESULTS: With respect to fed rats, fasted rats exhibited an increased oxidative injury in both liver tissue and isolated mitochondria throughout the experiment with the only exception of thiobarbituric acid-reactive substances, which were not affected by the nutritional status in liver tissue. Fasted rats showed a significantly lower F1-ATPase content and higher alanine transaminase levels. Glucose supplementation significantly reduced the fasting-associated exacerbation of oxidative stress and liver injury and the F1-ATPase exhaustion. CONCLUSIONS: These data indicate that the pre-existing hepatic glycogen content modulates the oxidative damage in rat fatty livers exposed to ischemia-reperfusion injury and that the energetic substrate supplementation may represent a clinically feasible protective strategy.     

荷载HO-1基因的脑靶向纳米粒对缺血性脑病大鼠海马神经元的保护作用

目的探讨荷载血红素加氧酶-1（HO-1）基因的乳铁蛋白与聚乙二醇修饰的阳离子壳聚糖（Lf-PEGQMC）纳米粒对缺血性脑病大鼠海马神经元的保护作用。方法将24只SD大鼠随机分为假手术组、缺血性脑病组、缺血性脑病＋HO-1基因组（纯基因组）、缺血性脑病＋荷载HO-1基因的Lf-PEG-QMC纳米粒干预组（纳米粒组）,每组各6只。缺血性脑病组、纯基因组、纳米粒组采用四动脉阻塞法制备缺血性脑病模型,假手术组仅分离而不结扎双侧颈总动脉。缺血性脑病组于缺血15 min后松开血管夹进行再灌注;纯基因组缺血15 min后再灌注时由尾静脉注射HO-1基因质粒20μg/kg;纳米粒组缺血15 min后再灌注时由尾静脉注射荷载HO-1基因的Lf-PEGQMC纳米粒（按基因剂量为20μg/kg给药）;假手术组不行再灌注操作。再灌注后第5天将各组大鼠麻醉后取脑组织制作切片,采用HE染色法在倒置显微镜下（×400）检测海马CA1区神经元存活数量,计算神经元存活率。结果假手术组、缺血性脑病组、纯基因组、纳米粒组神经元存活率分别为100%、11.0%、16.6%、57.6%,缺血性脑病组、纯基因组与纳米粒组神经元存活率均低于假手术组（P〈0.05）,纳米粒组神经元存活率高于缺血性脑病组及纯基因组（P〈0.05）,纯基因组与缺血性脑病组相比无统计学差异。结论 Lf-PEG-QMC纳米粒能有效携带HO-1基因跨越BBB并在脑组织中表达,有助于HO-1发挥对缺血性脑病海马神经元的保护作用。     

Dual role of tumor necrosis factor-alpha in hepatic ischemia-reperfusion injury: studies in tumor necrosis factor-alpha gene knockout mice

Although hepatic ischemia-reperfusion (IR) injury is partially mediated by tumor necrosis factor-alpha (TNF), we recently found that low-dose TNF before IR is hepatoprotective. We examined the seemingly conflicting roles of TNF in mediating liver injury in a partial hepatic IR model using TNF gene knockout (TNF ko) mice to allow TNF replacement at specified times. Compared with wild-type mice, TNF ko mice exhibit minimal alanine aminotransferase release and few hepatonecrotic lesions during the early (time, 2 hours) and late (time, 24 hours) phases of IR. TNF ko mice differed from wild-type mice in that TNF ko mice exhibited no activation or induction of nuclear factor-kappa B, p38, cyclin D1, or proliferating cell nuclear antigen after IR. A single low-dose TNF injection 1 minute before the onset of hepatic ischemia restored hepatic IR injury in TNF ko mice. To clarify the importance of TNF for hepatoprotection, preconditioning (10 minutes of ischemia and 10 minutes of reperfusion) was performed before the onset of IR for TNF ko mice whose capacity to undergo IR injury had been restored by TNF replacement. Ischemic preconditioning failed to protect these mice from TNF-augmented IR injury; however, following the administration of intravenous TNF (1 microg per kg body weight, which mimics the early increase in hepatic and plasma TNF levels that is mobilized by ischemic preconditioning), significant hepatoprotection against both the early and late phases of TNF-augmented IR injury was observed. In conclusion, TNF appears to mediate both the early and late phases of liver injury in hepatic IR, but it also is an essential mediator of hepatoprotective effects brought about by ischemic preconditioning.     

Neutrophil-mediated liver injury during hepatic ischemia-reperfusion in rats

BACKGROUND: Neutrophil plays an important role in hepatic ischemia-reperfusion injury. We investigated neutrophil infiltration in liver tissue, Kupffer cells' role in neutrophil accumulation, and apoptosis and regeneration of hepatocytes in liver ischemia-reperfusion injury. METHODS: Vascular microclamps were placed across the pedicles of the median and left lateral lobes for 90 minutes after 30% hepatectomy with the resection of caudate, right lateral and quadrate lobes and papillary process. Gadolinium chloride (GdCl3) was used to destroy Kupffer cells. Neutrophil activity was inhibited with Urge-8, a monoclonal antibody against neutrophil produced in our laboratory. GdCl3 (10 mg/kg) and Urge-8 (50 mg/kg) were given intravenously in respective groups. Ischemia control, GdCl3 and Urge-8 groups were compared. RESULTS: Following hepatic reperfusion, serum interleukin-8 (IL-8) levels and hepatic neutrophil counts peaked at 3 hours, and peak concentrations of alanine aminotransferase (ALT) occurred at 6 hours. Animals of the control group showed increases in neutrophil infiltration in liver tissue, liver enzyme levels, and apoptosis index of hepatocytes and decreases in overall survival rate and proliferating cell nuclear antigen (PCNA) expression of hepatocytes. The survival rates and PCNA proportion of hepatocytes were higher and the levels of hepatic neutrophil infiltration, liver enzymes, and hepatocyte apoptosis after reperfusion were lower in the GdCl3 and Urge-8 groups than those in the ischemia control group. CONCLUSIONS: Blockades of Kupffer cells' activity and neutrophil infiltration by GdCl3 and Urge-8 eliminate neutrophil-mediated hepatic injury and enhance subsequent hepatic regeneration during liver ischemiareperfusion.     

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.     

New drug delivery system for liver sinusoidal endothelial cells for ischemia-reperfusion injury

AIM: To investigate the cytoprotective effects in hepatic ischemia-reperfusion injury, we developed a new formulation of hyaluronic acid (HA) and sphingosine 1-phophate.METHODS: We divided Sprague-Dawley rats into 4 groups: control, HA, sphingosine 1-phosphate (S1P), and HA-S1P. After the administration of each agent, we subjected the rat livers to total ischemia followed by reperfusion. After reperfusion, we performed the following investigations: alanine aminotransferase (ALT), histological findings, TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining, and transmission electron microscopy (TEM). We also investigated the expression of proteins associated with apoptosis, hepatoprotection, and S1P accumulation.RESULTS: S1P accumulated in the HA-S1P group livers more than S1P group livers. Serum ALT levels, TUNEL-positive hepatocytes, and expression of cleaved caspase-3 expression, were significantly decreased in the HA-S1P group. TEM revealed that the liver sinusoidal endothelial cell (LSEC) lining was preserved in the HA-S1P group. Moreover, the HA-S1P group showed a greater increase in the HO-1 protein levels compared to the S1P group.CONCLUSION: Our results suggest that HA-S1P exhibits cytoprotective effects in the liver through the inhibition of LSEC apoptosis. HA-S1P is an effective agent for hepatic ischemia/reperfusion injury.     

茶多酚对大鼠肝脏缺血-再灌注损伤的保护作用

目的探讨茶多酚对大鼠肝脏缺血再灌注损伤的保护作用及其机制。方法健康雄性SD大鼠30只，随机均分为4组：假手术正常对照组6只；肝脏缺血再灌注损伤模型组8只；茶多酚低浓度于预组8只（75mg／kg）；茶多酚高浓度于预组8只（150mg／kg）．缺血30min再灌注60min检测肝组织MDA含量、GSH-PX活性及血浆ALT含量。光镜下比较各组肝组织损伤情况。结果肝缺血再灌注模型组ALT、MDA含量明显高于假手术正常对照组（P〈0．01），GSH-PX活力则降低（P〈0．01）；茶多酚低浓度及高浓度组ALT，MDA含量均明显低于肝缺血再灌注模型组（P〈0．01），而GSH-Px活力均高于肝缺血再灌注模型组（P〈0．01）；光镜观察茶多酚低浓度及高浓度预处理组肝细胞损伤明显小于肝缺血再灌注模型组。结论茶多酚对大鼠肝脏缺血再灌注损伤具有显著的保护作用。     

Ischemia-reperfusion injury in rat fatty liver: role of nutritional status

Fatty livers are more sensitive to the deleterious effects of ischemia-reperfusion than normal livers. Nutritional status greatly modulates this injury in normal livers, but its role in the specific setting of fatty liver is unknown. This study aimed to determine the effect of nutritional status on warm ischemia-reperfusion injury in rat fatty livers. Fed and fasted rats with normal or fatty liver induced by a choline deficient diet underwent 1 hour of lobar ischemia and reperfusion. Rat survival was determined for 7 days. Serum transaminases, liver histology and cell ultrastructure were assessed before and after ischemia, and at 30 minutes, 2 hours, 8 hours, and 24 hours after reperfusion. Survival was also determined in fatty fasted rats supplemented with glucose before surgery. The preischemic hepatic glycogen was measured in all groups. Whereas survival was similar in fasted and fed rats with normal liver (90% vs. 100%), fasting dramatically reduced survival in rats with fatty liver (14% vs. 64%, P <.01). Accordingly, fasting and fatty degeneration had a synergistic effect in exacerbating liver injury. Mitochondrial damage was a predominant feature of ultrastructural hepatocyte injury in fasted fatty livers. Glucose supplementation partially prevented the fasting-induced depletion of glycogen and improved the 7-day rat survival to 45%. These data indicate that rat fatty livers exposed to normothermic ischemia-reperfusion injury are much more sensitive to fasting than histologically normal livers. Because glucose supplementation improves both the hepatic glycogen stores and the rat survival, a nutritional repletion procedure may be part of a treatment strategy aimed to prevent ischemia-reperfusion injury in fatty livers.     

Anticoagulant pretreatment attenuates production of cytokine-induced neutrophil chemoattractant following ischemia-reperfusion of rat liver

We investigated whether anticoagulation would diminish ischemia-reperfusion injury of the liver. Liver ischemia was induced in rats by occluding the portal vein for 30 min. Anticoagulant was injected intravenously 10 min before occlusion. Serum concentrations of cytokine-induced neutrophil chemoattractant (CINC) in untreated rats increased following reperfusion, reaching a peak at 6 hr, then decreasing gradually to control levels by 24 hr. CINC levels in rats pretreated with heparin (50 units/kg), AT-III (250 units/kg), or DEGR-Xa (10 mg/kg) peaked at 3 hr after reperfusion and declined to baseline within 12 hr; peak CINC values were significantly lower than in untreated control rats. Expression of CINC mRNA in liver tissue paralleled the CINC serum levels. Both myeloperoxidase activity and the number of neutrophils in the liver were decreased in the anticoagulant groups. In addition, significant correlations were observed between the maximum values of AST, ALT, and LDH versus the peak CINC levels following ischemia-reperfusion. These results indicate that the release of CINC after ischemia-reperfusion of the liver is mediated by activation of coagulation within the hepatic microcirculation.     

肝硬变肝脏热缺血损伤耐受性的实验研究

对肝硬变肝脏缺血再灌注损伤的耐受性进行了实验研究。发现肝硬变大鼠较正常大鼠肝脏超氧化物歧化酶(SOD)明显降低、过氧化脂质丙二醛(MDA)明显升高。在缺血再灌注损伤后肝硬变大鼠肝脏SOD下降幅度较正常鼠为小,MDA水平较后者明显升高。血清ALT、LDH及肝脏组织学变化均显示肝硬变大鼠的肝细胞损伤较重。上述结果表明肝硬变肝脏对缺血再灌注损伤的耐受性较差,抗氧化能力降低可能是其主要原因之一。     

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.     

Extended preservation of rat liver graft by induction of heme oxygenase-1

Livers can be preserved only for a short period without jeopardizing the transplantation outcome. Heat shock proteins (HSPs) protect against ischemia and reperfusion injury. We studied whether their induction and, in particular, the induction of heme oxygenase 1 (HO-1), improves transplantation survival after an extended time of cold storage. Rats were subjected to heat preconditioning (42 degrees C for 20 minutes). Livers were harvested 24 hours later, preserved in cold University of Wisconsin solution for 44 hours, and transplanted in isogeneic rats (arterialized transplantation). HO-1 was specifically induced and inhibited by cobalt protoporphyrin and tin protoporphyrin, respectively. All animals receiving a graft without preconditioning and subjected to 44 hours of cold preservation died within 3 days, whereas 89% of rats who received a graft exposed to heat survived for 3 weeks (P =.0004). Preconditioning reduced serum aspartate transaminase (AST) and lactate dehydrogenase activities after reperfusion, improved bile flow, and decreased the histologic lesions of reperfusion injury. These significant effects of heat preconditioning were prevented by administration of tin protoporphyrin and could be reproduced by administration of cobalt protoporphyrin. In grafts without preconditioning, only a small fraction (<5%) of hepatocytes were positive with the terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay, and even less expressed activated caspase 3. Preconditioning tended to reduce the number of positive cells and to stimulate the expression of antiapoptotic Bcl-X(L). In conclusion, heat preconditioning and, specifically, overexpression of HO-1 improve posttransplantation survival and graft function after prolonged cold ischemia preservation. The mechanism underlying these beneficial effects does not appear to be prevention of apoptosis.     

Kupffer-cell specific induction of heme oxygenase 1 (hsp32) by the atrial natriuretic peptide--role of cGMP

BACKGROUND/AIMS: Pretreatment with atrial natriuretic peptide (ANP) attenuates ischemia-reperfusion injury of livers via cGMP. Heme oxygenase-1 (HO-1) is known as a protective mediator in ischemia-reperfusion injury. The aim of this study was to investigate whether ANP affects the expression of HO-1. METHODS: Rat livers were perfused with KH-buffer with/without ANP or 8-Br-cGMP, kept in UW solution (4 degrees C, 24 h), and reperfused. HO-1 mRNA and protein was determined by Northern and Western blot, in situ hybridization, and immunohistochemistry in livers or isolated liver cells. RESULTS: ANP significantly elevated HO-1 mRNA expression at the end of the preconditioning period and was without effects at the end of ischemia and during reperfusion. 8-Br-cGMP did not affect HO-1 mRNA expression. In situ hybridization as well as immunohistological double-staining revealed that Kupffer cells but not hepatocytes showed HO-1 mRNA and protein expression. Hepatocytes revealed no changes in HO-1 protein whereas Kupffer cells showed a marked increase in HO-1 protein after ANP treatment. Inhibition of HO-1 did not abrogate hepatoprotection conveyed by ANP. CONCLUSION: Our data show the potency of ANP to specifically induce HO-1 in Kupffer cells independently of cGMP. This increased expression of HO-1 is not involved in hepatoprotection conferred by ANP being in line with the knowledge that ANP mediates hepatoprotection via cGMP.     

实验性肝损伤大鼠肝脏HO-1的表达及CO水平变化

目的研究急性肝损伤时大鼠肝脏HO-1的表达情况和CO水平,探讨HO-1和内源性CO在大鼠急性肝损伤中的作用.方法制备急性四氯化碳肝损伤模型,采用RT-PCR和免疫组化法测定不同时间点大鼠肝脏HO-1 mRNA和蛋白的表达情况;测定各时间点肝组织SOD、MDA含量变化,同时测定股静脉血中HbCO水平和ALT、AST肝功能指标.结果HO-1mRNA在正常大鼠有弱表达,染毒3 h后表达显著增强,于24 h时间点表达最强,与对照组相比差异非常显著(P＜0.01);免疫组化结果显示;HO-1蛋白在正常大鼠表达较低或无,染毒3h后即有明显表达,16至48h的时间点内表达均显著增强,主要定位于肝实质细胞、库普细胞的胞浆内.对照组HbCO水平极低,给予四氯化碳3 h后HbCO水平开始升高,此后各时间点均明显高于对照组,差异有显著性,这与HO-1表达情况相一致.此外,染毒后大鼠血清ALT、AST和MDA明显升高,SOD活性则显著降低,和对照组相比差异均十分显著.结论大鼠急性肝损伤后出现HO-1表达持续上调和血中CO水平迅速增高,提示HO/CO系统参与急性肝损伤的病理生理过程,其表达增加可能对机体有重要调节作用.