Hepatic IR Research Articles

Mitochondrial Damage-Associated Molecular Patterns (MTDs) Are Released during Hepatic Ischemia Reperfusion and Induce Inflammatory Responses.

Ischemia / reperfusion injury (IRI) during the course of liver transplantation enhances the immunogenicity of allografts and thus impacts overall graft outcome. This sterile inflammatory insult is known to activate innate immunity and propagate organ damage through the recognition of damage-associate molecular pattern (DAMP) molecules. The purpose of the present study was to investigate the role of mitochondrial DAMPs (MTDs) in the pathogenesis of hepatic IRI. Using in vitro models we observed that levels of MTDs were significantly higher in both transplantation-associated and warm IR, and that co-culture of MTDs with human and rat hepatocytes significantly increased cell death. MTDs were also released in an in vivo rat model of hepatic IRI and associated with increased secretion of inflammatory cytokines (TNF-α, IL-6, and IL-10) and increased liver injury compared to the sham group. Our results suggest that hepatic IR results in a significant increase of MTDs both in vitro and in vivo suggesting that MTDs may serve as a novel marker in hepatic IRI. Co-culture of MTDs with hepatocytes showed a decrease in cell viability in a concentration dependent manner, which indicates that MTDs is a toxic mediator participating in the pathogenesis of liver IR injury.

Abstract 2720: In vitro evaluation of a novel chemotherapeutic agent, NecroX-7, in human Burkitt lymphoma cells

Abstract NecroX-7 is a novel small molecule of the NecroX series, its chemical compound is C25H32N4O4S2 with molecular weight 516.67 based on the indole-moiety. It prevents from oxidative stress-induced cell death. NecroX-7 prevented the release of HMGB1 or ameliorated hepatic IR injury. It is shown that NecroX-7 can inhibit NAPDH oxidase activity and osteoclast differentiation. Furthermore, NecroX-7 protected against APAP-induced hepatic injury by directly binding to NAPQI. However, the function and the underlying mechanisms of NecroX-7 in cancer cell growth have not been well established. This study was aimed at evaluating the efficacy as anticancer agent in human lymphoma cells. Human Burkitt lymphoma cell lines were treated NecroX-7 in vitro. The effects of NecroX-7 treatment were evaluated using MTS assay for cell viability and annexin-V/propidium iodide labeling for apoptosis. Cell cycle was analyzed by Flow cytometry. Western blotting was used to look for NecroX-7 induced protein changes. As results, NecroX-7 treatment efficiently inhibited cell growth at dose- and time- dependent and induced apoptosis in cell lines. Apoptosis was showed by accumulation of annexin-V and PI-positive cells, caspase-3 activation and induction of PARP cleavage. The IC50 values of NecoX-7 in cell lines were around 50 uM. NecroX-7 induced G2/M phase cell cycle arrest in Ramos and Daudi cells.These results suggest that NecroX-7 is a potential anticancer agent for treatment of human Burkitt lymphoma cells and provide a rational for preclinical and clinical evaluation of NecoX-7 for lymphoma therapy. Citation Format: Eun- Jung Kim, Eun Sol Lee, Keon- Il Im, Soon Ha Kim, Jung- Yeon Lim, Nayoun Kim, Seok- Goo Cho. In vitro evaluation of a novel chemotherapeutic agent, NecroX-7, in human Burkitt lymphoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2720. doi:10.1158/1538-7445.AM2014-2720

Growth hormone abolishes beneficial effects of calorie restriction in long-lived Ames dwarf mice

Disruption of the growth hormone (GH) axis promotes longevity and delays aging. In contrast, GH over-expression may lead to accelerated aging and shorter life. Calorie restriction (CR) improves insulin sensitivity and may extend lifespan. Long-lived Ames dwarf (df/df) mice have additional extension of longevity when subjected to 30% CR. The aim of the study was to assess effects of CR or GH replacement therapy separately and as a combined (CR+GH) treatment in GH-deficient df/df and normal mice, on selected metabolic parameters (e.g., insulin, glucose, cholesterol), insulin signaling components (e.g., insulin receptor [IR] β-subunit, phosphorylated form of IR [IR pY1158], protein kinase C ζ/λ [p-PKCζ/λ] and mTOR [p-mTOR]), transcription factor p-CREB, and components of the mitogen-activated protein kinase (MAPK) signaling (p-ERK1/2, p-p38), responsible for cell proliferation, differentiation and survival. CR decreased plasma levels of insulin, glucose, cholesterol and leptin, and increased hepatic IR β-subunit and IR pY1158 levels as well as IR, IRS-1 and GLUT-2 gene expression compared to ad libitum feeding, showing a significant beneficial diet intervention effect. Moreover, hepatic protein levels of p-PKCζ/λ, p-mTOR and p-p38 decreased, and p-CREB increased in CR mice. On the contrary, GH increased levels of glucose, cholesterol and leptin in plasma, and p-mTOR or p-p38 in livers, and decreased plasma adiponectin and hepatic IR β-subunit compared to saline treatment. There were no GH effects on adiponectin in N mice. Moreover, GH replacement therapy did not affect IR, IRS-1 and GLUT-2 gene expression. GH treatment abolishes the beneficial effects of CR; it may suggest an important role of GH–IGF1 axis in mediating the CR action. Suppressed somatotrophic signaling seems to predominate over GH replacement therapy in the context of the examined parameters and signaling pathways.

RhoA/ROCK Signaling Triggers Innate Immune Responses in Ischemia-Stressed Liver Through Activation of c-Jun N-Terminal Kinase Pathway.

Background: The RhoA/Rho kinase (ROCK) signaling plays an important role in innate immune cell activation and the development of inflammatory diseases. However, little is known on the functional role of RhoA/ROCK signaling in the activation of innate immune responses during organ ischemia-reperfusion injury (IRI). This study was designed to explore potential molecular mechanisms of Rho/ROCK signaling in innate immune responses triggered by IR stress in a mouse liver. Methods: Using a partial hepatic warm ischemia model (90 min), C57BL/6 wide-type (WT) mice (n=6/gr) were pretreated with ROCK inhibitor H1152 (3 mg/kg, i.p.) or vehicle at 1h prior to ischemia, and then sacrificed at 6 h of reperfusion. In parallel in vitro study, bone marrow-derived WT macrophages (BMMs) were transfected with JNK1 siRNA/nonspecific siRNA (100 nM) or treated with H1152 (10μM), and then incubated with LPS (100 ng/ml). Results: Hepatic IR increased ROCK1 and c-Jun N-terminal kinase (JNK) expression and enhanced PTEN activity, whereas anti-ROCK (H1152) treatment decreased JNK and PTEN/TLR4 activation yet increased PI3K/Akt expression. Unlike in vehicle-treated WT controls, H1152 preconditioning improved hepatic function, reduced hepatocellular damage, and depressed macrophage/neutrophil trafficking, pro-inflammatory cytokine programs, and hepatocellular necrosis/apoptosis, while increasing anti-apoptotic functions in ischemic liver lobes. LPS-stimulated BMMs in vitro revealed increased expression of ROCK1, JNK, PTEN, and TLR4. However, knockdown of JNK with siRNA readily suppressed PTEN/TLR4, leading to diminished NF-κB activation and decreased expression of proinflammatory mediators. Consistent with our in vivo findings, pretreatment of LPS-stimulated BMMs with H1152 decreased JNK expression and upregulated Akt phosphorylation. Conclusion: This study demonstrates that IR-triggered RhoA/ROCK signaling induced JNK activation, leading to enhanced PTEN/TLR4 signaling. Inhibition of RhoA/ROCK protected livers from IR-mediated inflammation. Knockdown of JNK depressed PTEN yet promoted PI3K/Akt, which in turn negatively regulated TLR4 activation. By documenting the critical role of RhoA/ROCK-JNK signaling cascade in the mechanism of innate immune-driven local inflammation responses, our findings provide the rationale for a novel therapeutic strategy against IRI in organ transplant recipients.

Protective effects of pretreatment with oleanolic acid in rats in the acute phase of hepatic ischemia-reperfusion injury: role of the PI3K/Akt pathway.

Oleanolic acid (OA) has been used to treat liver disorders, but whether it can attenuate hepatic ischemia-reperfusion- (IR-) associated liver dysfunction remains unexplored. In the present study, 160 male Sprague-Dawley rats were equally divided into five groups: group SH received neither hepatic IR nor drugs; group IR received hepatic IR without drugs; group CM and group OA received 0.5% sodium carboxymethylcellulose and 100 mg/kg OA, intragastrically, once a day for seven days before the hepatic IR, respectively; on the basis of treatment in group OA, group OA+wortmannin further received 15 μg/kg of PI3K inhibitor wortmannin, intraperitoneally, 30 min before the hepatic IR. Then each group was equally divided into four subgroups according to four time points (preoperation, 0 h, 3 h, and 6 h after reperfusion). Serum ALT activity, IL-1β concentration, and hepatic phosphorylation of PI3K, Akt, and GSK-3β protein expression were serially studied. We found that OA pretreatment improved histological status and decreased serum ALT and IL-1β levels. It also increased p-PI3K, p-Akt, and p-GSK-3β protein expression at all the four time points. Prophylactic wortmannin partially reversed OA's protective effects. The data indicate that OA pretreatment protects liver from IR injury during the acute phase partially through PI3K/Akt-mediated inactivation of GSK-3β.

Protective Effect of N-Acetylserotonin against Acute Hepatic Ischemia-Reperfusion Injury in Mice

The purpose of this study was to investigate the possible protective effect of N-acetylserotonin (NAS) against acute hepatic ischemia-reperfusion (I/R) injury in mice. Adult male mice were randomly divided into three groups: sham, I/R, and I/R + NAS. The hepatic I/R injury model was generated by clamping the hepatic artery, portal vein, and common bile duct with a microvascular bulldog clamp for 30 min, and then removing the clamp and allowing reperfusion for 6 h. Morphologic changes and hepatocyte apoptosis were evaluated by hematoxylin-eosin (HE) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, respectively. Activated caspase-3 expression was evaluated by immunohistochemistry and Western blot. The activation of aspartate aminotransferase (AST), malondialdehyde (MDA), and superoxide dismutase (SOD) was evaluated by enzyme-linked immunosorbent assay (ELISA). The data show that NAS rescued hepatocyte morphological damage and dysfunction, decreased the number of apoptotic hepatocytes, and reduced caspase-3 activation. Our work demonstrates that NAS ameliorates hepatic IR injury.

The Protective Effects of Different-Time-Ischemic Preconditioning on the Reperfusion Injury in Fatty Livers in Rats

BackgroundThe present study was aimed to investigate the protective effects of different-time-ischemic preconditioning on the reperfusion injury in fatty livers in rats, and to elucidate the mechanisms underlying the protective effects and the optimal safe ischemic preconditioning time on the hepatic IR injury in steatotic livers.Methodology/Principal FindingsA rat fatty liver model was established by high-fat diet feeding. We investigated the changes in the concentration of AST, ALT, LDH and NO in the serum, and of MDA, SOD, and MPO in the liver samples in response to different ischemic preconditioning times and ischemia-reperfusion injury. Histological analysis was performed to evaluate the results of the hepatic fatty infiltration. 1) At 24 h after 15 min ischemic preconditioning with 10 min reperfusion (15 min +10 min IP), the extent and area of the necrosis was markedly higher in the fatty liver samples with respect to IR, compared to the normal liver samples. 2) In response to the treatment of 5/8 min +10 min IP, the fatty liver group showed lower levels of serological indicators and liver MDA and MPO compared to the other groups, while the SOD activity of the fatty liver group was significantly higher than the other groups (p<0.05). Compared to the corresponding IR group, all IP groups showed a significantly higher serum NO concentration (p<0.05). Among the fatty liver groups, the 5/8 min+10 min IP group showed the highest NO concentration (p<0.05).Conclusions/SignificanceFat infiltration could aggravate the ischemia-reperfusion injury in the rat liver. Furthermore, ischemic preconditioning could increase the tolerance of the fatty liver, which was induced by the high-fat diet, to hepatic ischemia-reperfusion injury in rats. The protocol of 5/8 min +10 min IP was the optimal regimen for the treatment of moderate and severe fatty livers.

Induction of heme oxygenase-1 protects mouse liver from apoptotic ischemia/reperfusion injury

Ischemia/reperfusion (I/R) injury is the main cause of primary graft dysfunction of liver allografts. Cobalt-protoporphyrin (CoPP)-dependent induction of heme oxygenase (HO)-1 has been shown to protect the liver from I/R injury. This study analyzes the apoptotic mechanisms of HO-1-mediated cytoprotection in mouse liver exposed to I/R injury. HO-1 induction was achieved by the administration of CoPP (1.5mg/kg body weight i.p.). Mice were studied in in vivo model of hepatic segmental (70%) ischemia for 60min and reperfusion injury. Mice were randomly allocated to four main experimental groups (n=10 each): (1) A control group undergoing sham operation. (2) Similar to group 1 but with the administration of CoPP 72h before the operation. (3) Mice undergoing in vivo hepatic I/R. (4) Similar to group 3 but with the administration of CoPP 72h before ischemia induction. When compared with the I/R mice group, in the I/R+CoPP mice group, the increased hepatic expression of HO-1 was associated with a significant reduction in liver enzyme levels, fewer apoptotic hepatocytes cells were identified by morphological criteria and by immunohistochemistry for caspase-3, there was a decreased mean number of proliferating cells (positively stained for Ki67), and a reduced hepatic expression of: C/EBP homologous protein (an index of endoplasmic reticulum stress), the NF-κB's regulated genes (CIAP2, MCP-1 and IL-6), and increased hepatic expression of IκBa (the inhibitory protein of NF-κB). HO-1 over-expression plays a pivotal role in reducing the hepatic apoptotic IR injury. HO-1 may serve as a potential target for therapeutic intervention in hepatic I/R injury during liver transplantation.

Hepatoprotective Effect of Inularacemosa on Hepatic Ischemia/reperfusion Induced Injury in Rats

Introduction: Hepatic Ischemia-Reperfusion (I/R) injury contributes to organ injury and dysfunction after hepatic surgery and transplantation. I/R induce Kupffer cell activation, leading to the release of pro-inflammatory cytokines that promote injury, increase adhesion molecule expression, and facilitate polymorphonuclear neutrophil injury. Inularacemosa contains high concentrations of the flavonol glycosides, which has been shown effect on cardiac function and oxidative stress against isoproterenol – induced myocardial infarction. Historically, the roots were reputed to have Anti-inflammatory and Analgesic effects. The hepatoprotective activity of the drug against hepatic Ischemia/Reperfusion injury has not been reported yet. Objective: To study the hepatoprotective effect of hydroalcholic extract of Inularacemosa at 200 and 400 mg/kg against hepatic ischemia/reperfusion injury. Methodology: 24 male wistar rats were divided in to four groups. The normal control group, model control group and extract treated group at a dose of 200 and 400 mg/kg were orally fed with distilled water as vehicle for 21 days followed by ischemia/reperfusion on twenty second day. Blood and liver samples were obtained from all the animals on 22nd day for biochemical analysis of AST, ALT, ALP and LDH and histopathological studies were also performed. Results: The results showed that the ischemia/reperfusion injury causes significant increase in the levels of AST, ALT, ALP and LDH in model control group indicating the cell damage and tissue injury whereas supplementation with hydroalcholic extract of Inularacemosa significantly reduced the elevated levels of above parameters. Histopathological analysis showed high degree of congestion and mild necrosis in model control group which was reduced to minimum levels in drug treated groups. Inularacemosa increased the free radicals scavenging activity in the early period of hepatic IR injury in rats. Conclusion: Reduced level of liver enzymes and histopathological studies evident that Inularacemosa possesses beneficial effects on the hepatocytes in hepatic I/R injury.

The Uncoordinated-5 Homolog B Receptor Affects Hepatic Ischemia Reperfusion Injury

Recent evidence has demonstrated additional roles for the neuronal guidance protein receptor UNC5B outside the nervous system. Given the fact that ischemia reperfusion injury (IRI) of the liver is a common source of liver dysfunction and the role of UNC5B during an acute inflammatory response we investigated the role of UNC5B on acute hepatic IRI. We report here that UNC5B+/− mice display reduced hepatic IRI and neutrophil (PMN) infiltration compared to WT controls. This correlated with serum levels of lactate dehydrogenase (LDH), aspartate- (AST) and alanine- (ALT) aminotransferase, the presence of PMN within ischemic hepatic tissue, and serum levels of inflammatory cytokines. Moreover, injection of an anti-UNC5B antibody resulted in a significant reduction of hepatic IR injury. This was associated with reduced parameters of liver injury (LDH, ALT, AST) and accumulation of PMN within the injured hepatic tissue. In conclusion our studies demonstrate a significant role for UNC5B in the development of hepatic IRI and identified UNC5B as a potential drug target to prevent liver dysfunction in the future.

Isoform‐selective changes in arachidonic acid (AA) metabolizing cytochrome P450 (P450) enzymes in warm hepatic ischemia (IR) reperfusion injury

The objective of this study was to investigate the effects of warm hepatic IR injury on the major P450 enzymes responsible for epoxygenation (CYP2J2 and CYP2C23) and ω/ω‐1 hydroxylation (CYP4A) of AA in rats. Four groups of rats (n = 5–6/group) were subjected to 60 min of in vivo partial (70%) hepatic ischemia or sham operation, followed by 3 or 24 hr of reperfusion. Liver microsomes and plasma samples, collected at the end of the reperfusion period, were used for quantitation of alanine aminotransferase (ALT) concentrations and the protein levels of CYP4A, CYP2C23, and CYP2J2 (Western blot), respectively. Compared with sham‐operated animals, IR caused 10‐ and 3.3‐fold increases in the ALT concentrations after 3 and 24 hr of reperfusion, respectively (P&lt;0.0001). In the 3‐hr reperfusion group, IR caused no changes in the expression of CYP4A and CYP2C23, whereas it down regulated CYP2J2 levels by 19.7% (P&lt;0.01). In the 24‐hr reperfusion group, IR caused no changes in the expression of CYP2J2, whereas it upregulated CYP4A by 46.5% (P&lt;0.05) and CYP2C23 by 56.1% (P&lt;0.05). These results suggest that warm hepatic IR causes isoform‐selective changes in the AA‐metabolizing enzymes, potentially altering hepatic disposition of P450‐mediated metabolites of AA. Financial Support: Texas Higher Education Coordinating Board and Texas Tech School of Pharmacy Vascular Drug Research Center.

Ischemia and Reperfusion Liver Injury is Reduced in the Absence of Toll-like Receptor 4

Background/Aims: Toll-like receptor 4 (TLR4) is expressed on hepatic non-parenchymal cells and hepatocytes. Hepatic signaling through TLR4 is critical in the pathogenesis of ischemia reperfusion injury (IRI) and leads to the release of cytokines. The role of bone marrow-derived TLR4 in the early reperfusion stage is unclear. Methods: We used wild type mice (WT), TLR4deficient (TLR4ko) mice and chimeras to dissociate between the role of TLR4 expression in the liver (TLR4ko/WT) and in the immuno-hematopoietic system (WT/TLR4ko) in mouse hepatic IR injury model. Mice were subjected to in vivo partial IRI (70% for 60 min). Results: Compared with WT IR livers, TLR4ko IRI mice (4 hours) showed a significant reduction in serum liver enzyme, hepatic TNF-α and interleukin-1β levels. Fewer apoptotic hepatocytes cells were identified by morphological criteria and immunohistochemistry for caspase-3. In TLR4ko mice, decreased hepatic CJUN and NF-ĸB expression during IRI was noted compared with WT mice. Chimeric mice having either TLR4 bone-marrow or non-bone marrow derived cells following IRI exhibited almost similar hepatic injury as WT mice in the immediate reperfusion stage. Conclusion: Both TLR4 bone marrow-derived and non-bone marrow-derived cells are necessary in the initial process of hepatic injury. Activating TLR4-dependent signaling is required for IRI. The absence of the TLR4 gene plays a pivotal role in reducing hepatic IR injury.

Phosphorylation of Vasodilator-Stimulated Phosphoprotein (VASP) Dampens Hepatic Ischemia-Reperfusion Injury

Recent work has demonstrated that the formation of platelet neutrophil complexes (PNCs) affects inflammatory tissue injury. Vasodilator-stimulated phosphoprotein (VASP) is crucially involved into the control of PNC formation and myocardial reperfusion injury. Given the clinical importance of hepatic IR injury we pursued the role of VASP during hepatic ischemia followed by reperfusion. We report here that VASP −/− animals demonstrate reduced hepatic IR injury compared to wildtype (WT) controls. This correlated with serum levels of lactate dehydrogenase (LDH), aspartate (AST) and alanine (ALT) aminotransferase and the presence of PNCs within ischemic hepatic tissue and could be confirmed using repression of VASP through siRNA. In studies employing bone marrow chimeric mice we identified hematopoietic VASP to be of crucial importance for the extent of hepatic injury. Phosphorylation of VASP on Ser153 through Prostaglandin E1 or on Ser235 through atrial natriuretic peptide resulted in a significant reduction of hepatic IR injury. This was associated with a reduced presence of PNCs in ischemic hepatic tissue. Taken together, these studies identified VASP and VASP phosphorylation as crucial target for future hepatoprotective strategies.

Organ-Specific Defects in Insulin-Like Growth Factor and Insulin Receptor Signaling in Late Gestational Asymmetric Intrauterine Growth Restriction in Cited1 Mutant Mice

Late gestational placental insufficiency resulting in asymmetric intrauterine organ growth restriction (IUGR) is associated with an increased incidence of diabetes, cardiovascular and renal disease in adults. The molecular mechanisms mediating these defects are poorly understood. To explore this, we investigated the mechanisms leading to IUGR in Cited1 knockout mice, a genetic model of late gestational placental insufficiency. We show that loss of placental Cited1 leads to asymmetric IUGR with decreased liver, lung, and kidney sizes and preservation of fetal brain weight. IGF and insulin signaling regulate embryonic organ growth. IGF-I and IGF-II protein and mRNA expression are reduced in livers, lungs, and kidneys of embryonic d 18.5 embryos with IUGR. Decreased IGF-I is associated with reduced activating phosphorylation of the type 1 IGF receptor (pIGF-IR) in the kidney, whereas reduced IGF-II is associated with decreased phosphorylation of the insulin receptor (pIR) in the lung. In contrast, decreased pIR is associated with reduced IGF-I but not IGF-II in the liver. However, pancreatic β-cell mass and serum insulin levels are also decreased in mice with IUGR, suggesting that hepatic IR signaling may be regulated by alterations in fetal insulin production. These findings contrast with observations in IUGR fetal brains in which there is no change in IGF-IR/IR phosphorylation, and IGF-I and IGF-II expression is actually increased. In conclusion, IUGR disrupts normal fetal IGF and insulin production and is associated with organ-specific defects in IGF-IR and IR signaling that may regulate asymmetric IUGR in late gestational placental insufficiency.

Rosiglitazone attenuates tumor necrosis factor-α-induced protein-tyrosine phosphatase-1B production in HepG2 cells.

Tumor necrosis factor (TNF)-α impairs insulin signaling and plays an important role in the development of insulin resistance. The underlying molecular mechanism by which TNF-α regulates hepatic protein-tyrosine phosphatase (PTP)-1B expression is not well understood. Rosiglitazone is used as a drug to improve insulin sensitivity in vivo. However, its effect on TNF-α-induced PTP-1B expression remains to be explored. In the present study, we sought to identify the mechanism of TNF-α-regulated hepatic PTP-1B expression and evaluate the effect of rosiglitazone on TNF-α-induced hepatic PTP-1B upregulation. TNF-α up-regulates PTP- 1B expression in a dose-dependent manner and decreases insulin-stimulated phosphorylation of IR and insulin receptor substrate-1 in HepG2 cells. TNF-α increases p65 protein level and nuclear factor κB (NF-κB) activity. Inhibition of NF-κB activation by pyrrolidine dithiocarbamate impairs TNF-α-induced PTP-1B upregulation. Rosiglitazone significantly blocks TNF-α-induced PTP-1B upregulation and NF-κB activation. Our data strongly suggest that TNF-α-induced PTP-1B overexpression may contribute to hepatic IR in obesity and diabetes, and NF-κB is involved in rosiglitazone attenuated PTP- 1B upregulation by TNF-α.

Iloprost, a prostacyclin (PGI2) analogue, reduces liver injury in hepatic ischemiareperfusion in rats

To evaluate the effects of iloprost a prostacyclin analogue on the hepatic IR injury in rats. Forty male Sprague-Dawley rats (250-300 g) were divided into four groups each containing 10 rats;(1)--controls: data from unmanipulated animals; (2) sham group: rats subjected to the surgical procedure, except for liver I/R, and given saline; (3) I/R group: rats that underwent liver ischemia for 45 min followed by reperfusion for 45 min; (4) IR/ Iloprost group: rats pretreated with iloprost (10 microg kg-1, i.v). Liver tissues were taken to determine SOD, CAT, GSH, and MDA levels and for biochemical and histological evaluation. The plasma ALT and AST levels were increased in group 3 than in group 4. MDA values and the liver injury score decreased, while the SOD, CAT, and GSH values increased in group 4 compared to group 3. In group 3, hepatocytes were swollen with marked vacuolization. In group 4, there were regular sinusoidal structures with normal morphology without any signs of congestion. We demonstrated hepatoprotective effects of iloprost against severe ischemia and reperfusion injury in rat liver.

Human Heat Shock Protein 27 Overexpressing Mice Are Protected Against Hepatic Ischemia and Reperfusion Injury

Hepatic ischemia reperfusion injury (IRI) is a major clinical problem during the perioperative period and occurs frequently after major hepatic resection or liver transplantation. Our laboratory previously demonstrated that exogenous A1 adenosine receptor activation protects against renal IRI by upregulation and phosphorylation of heat shock protein 27 (HSP27). This study used mice overexpressing human HSP27 (huHSP27 OE) to determine whether these mice are protected against liver IRI. After hepatic IR, the huHSP27 OE mice had significant protection against liver injury (reduced alanine transferase) and necrosis (hematoxylin-eosin staining) compared with the HSP27 WT mice. The huHSP27 OE mice also showed less induction of proinflammatory messenger RNA MIP-2, reduced neutrophil infiltration, and decreased apoptosis (caspase 3 fragmentation and DNA laddering) compared with the HSP27 WT mice. Finally, the huHSP27 OE mice showed significantly less disruption of filamentous actin in hepatocytes and bile canaliculi of the ischemic lobes compared with the HSP27 WT mice. Depletion of Kupffer cells with gadolinium chloride provided significant protection against liver IRI in HSP27 WT mice but not in huHSP27 OE mice suggesting that the overexpression of huHSP27 in the Kupffer cells may be responsible for the hepatic protection observed in huHSP27 OE mice. Our results show that the overexpression of huHSP27 in Kupffer cells of the liver may be responsible for the protection against hepatic IRI in vivo by reducing necrosis and apoptosis and by stabilizing F-actin with subsequent reductions in inflammation and proinflammatory neutrophil infiltration. Harnessing the mechanisms of cytoprotection with HSP27 may lead to new therapies for the management of perioperative hepatic IRI.

Hepatic insulin signaling regulates VLDL secretion and atherogenesis in mice

Type 2 diabetes is associated with accelerated atherogenesis, which may result from a combination of factors, including dyslipidemia characterized by increased VLDL secretion, and insulin resistance. To assess the hypothesis that both hepatic and peripheral insulin resistance contribute to atherogenesis, we crossed mice deficient for the LDL receptor (Ldlr-/- mice) with mice that express low levels of IR in the liver and lack IR in peripheral tissues (the L1B6 mouse strain). Unexpectedly, compared with Ldlr-/- controls, L1B6Ldlr-/- mice fed a Western diet showed reduced VLDL and LDL levels, reduced atherosclerosis, decreased hepatic AKT signaling, decreased expression of genes associated with lipogenesis, and diminished VLDL apoB and lipid secretion. Adenovirus-mediated hepatic expression of either constitutively active AKT or dominant negative glycogen synthase kinase (GSK) markedly increased VLDL and LDL levels such that they were similar in both Ldlr-/- and L1B6Ldlr-/- mice. Knocking down expression of hepatic IR by adenovirus-mediated shRNA decreased VLDL triglyceride and apoB secretion in Ldlr-/- mice. Furthermore, knocking down hepatic IR expression in either WT or ob/ob mice reduced VLDL secretion but also resulted in decreased hepatic Ldlr protein. These findings suggest a dual action of hepatic IR on lipoprotein levels, in which the ability to increase VLDL apoB and lipid secretion via AKT/GSK is offset by upregulation of Ldlr.

Suppressor of cytokine signaling expression with increasing severity of murine hepatic ischemia-reperfusion injury

Preservation of function requires tight regulation of the cellular events initiated when hepatic ischemia is followed by reperfusion (IR). One important mechanism modulating the cytokine-directed response to injury is Suppressors of Cytokine Signaling. SOCS1 and SOCS3 ensure appropriate intensity and duration of cytokine signaling through negative feedback on JAK-STAT signaling. The contribution of SOCS1 and SOCS3-mediated regulation to the evolution of hepatic IR injury is unknown. C57Blk6 mice were subjected to mild (20 min) or severe (90 min) hepatic ischemia. Liver was analyzed for cytokine and SOCS1/3 induction as well as JAK-STAT activation at intervals after reperfusion. Tnf, Il-1beta, and Il-6 expression paralleled increasing injury severity. Despite early phosphorylation of both STAT1 and STAT3 after severe injury, only nuclear translocation of activated STAT3, suggesting that the induction of target genes through JAK-STAT after IR is predominantly via STAT3. Socs3 was expressed across the injury spectrum while Socs1 was induced only in the face of severe IR injury. Severe IR in Il-6 deficient mice confirmed that Il-6, acting via STAT3, serves as a primary inducer of both regulatory mechanisms. Under the influence of IL-6-mediated STAT3 signaling, Socs1 serves as a complimentary regulatory mechanism when Socs3 is insufficient to limit cytokine-mediated inflammation after hepatic IR.

Abstract 251: Endogenous Endocrine Function of Cardiac-derived Nitric Oxide Yields Distant Organ Protection Against Ischemic Injury

Background : eNOS-derived nitric oxide (NO) has long been considered a paracrine signaling molecule only capable of affecting nearby cells due to its short half-life in blood (&lt;2ms). We have recently shown that nitrite, previously thought to be an inert oxidation product of NO, is an important storage form converted to NO under hypoxic conditions via reductase activity. Currently, no studies have revealed endogenously generated NO to possess a clearly defined endocrine function, we therefore evaluated whether generation of NO in the heart could modulate remote physiological actions. Methods: Mice with cardiac-specific (αMHC promoter) eNOS overexpression (CS-eNOS-Tg) and WT littermates were analyzed for NO metabolites in the heart, plasma, and liver (NO 2 − /NO 3 − - ion chromatography; nitroso/NO-heme - gas phase chemiluminescence). WT and CS-eNOS-Tg mice were subjected to 60 min hepatic ischemia and following 5 hr reperfusion (IR) serum ALT and AST were analyzed. Results: Overexpression of eNOS restricted to the heart resulted in a significant increase in nitrite in the heart (2.2 fold), plasma (3.2 fold) and liver (6.5 fold). CS-eNOS-Tg displayed a significant reduction in hepatic IR injury (4.3 fold reduction in ALT and a 3.5 fold reduction in AST). Conclusions: This represents the first report demonstrating that eNOS-derived NO is transported in the blood and stored in remote organs. Importantly, increases in nitrite levels resulted in protection against IR injury. These findings suggest NO possesses endocrine function via transport of nitrite in the blood to distant organs and that transported nitrite exhibits physiological activity.