Hepatic Ischemic Preconditioning Research Articles

Hepatic Ischemic Preconditioning Alleviates Ischemia-Reperfusion Injury by Decreasing TIM4 Expression.

Ischemia-reperfusion injury (IRI) of the liver is a primary cause of post-liver-surgery complications and ischemic preconditioning (IPC) has been verified to protect against ischemia-reperfusion injury. TIM-4 activation plays an important role in macrophage mediated hepatic IRI. This study aimed to determine whether IPC protects against hepatic IRI through inhibiting TIM-4 activation. In this study, a model of warm liver ischemia (90 min) and reperfusion for 6 h was used. Mice were subjected to ischemia-reperfusion injury with or without ischemic preconditioning and TIM4 blocking antibody. Western blot was determined to detect the expression of TIM4 protein and mitochondrial apoptosis-related protein expression. Liver function was evaluated using the level of alanine transaminase (ALT) and aspartate transaminase (AST), cell apoptosis and pathological examination. We found that compared with the control group, ischemic preconditioning reduced IRI by decreasing hepatocyte apoptosis, ALT, AST, CD68 and CD3 positive cells, tissue myeloperoxidase activity(MPO), and downregulating TIM-4 expression. TIM4 blocking could reduce CD68 and CD3 positive cells in liver. Furthermore, activated monocytes transfusion significantly abolished the protect effect of IPC with increased hepatocyte apoptosis, ALT, AST, CD68 and CD3 positive cells while TIM-4 knockdown monocytes lost this effect. These results suggested that IPC protects against hepatic IRI by downregulating TIM-4 and indicated TIM-4 would be a novel therapeutic target to minimize IRI.

Ischemic Preconditioning Affects Long-Term Cell Fate through DNA Damage–Related Molecular Signaling and Altered Proliferation

Despite the potential of ischemic preconditioning for organ protection, long-term effects in terms of molecular processes and cell fates are ill defined. We determined consequences of hepatic ischemic preconditioning in rats, including cell transplantation assays. Ischemic preconditioning induced persistent alterations; for example, after 5 days liver histology was normal, but γ-glutamyl transpeptidase expression was observed, with altered antioxidant enzyme content, lipid peroxidation, and oxidative DNA adducts. Nonetheless, ischemic preconditioning partially protected from toxic liver injury. Similarly, primary hepatocytes from donor livers preconditioned with ischemia exhibited undesirably altered antioxidant enzyme content and lipid peroxidation, but better withstood insults. However, donor hepatocytes from livers preconditioned with ischemia did not engraft better than hepatocytes from control livers. Moreover, proliferation of hepatocytes from donor livers preconditioned with ischemia decreased under liver repopulation conditions. Hepatocytes from donor livers preconditioned with ischemia showed oxidative DNA damage with expression of genes involved in MAPK signaling that impose G1/S and G2/M checkpoint restrictions, including p38 MAPK-regulated or ERK-1/2-regulated cell-cycle genes such as FOS, MAPK8, MYC, various cyclins, CDKN2A, CDKN2B, TP53, and RB1. Thus, although ischemic preconditioning allowed hepatocytes to better withstand secondary insults, accompanying DNA damage and molecular events simultaneously impaired their proliferation capacity over the long term. Mitigation of ischemic preconditioning-induced DNA damage and deleterious molecular perturbations holds promise for advancing clinical applications.

Differential consequences of protein kinase C activation during early and late hepatic ischemic preconditioning

Activation of protein kinase C (PKC) has been implicated in the protection of ischemic preconditioning (IPC), but the exact role of PKC in early and late hepatic IPC is still unclear. The present study was conducted in order to investigate the differential role of PKC during early and late hepatic IPC. Rats were subjected to 90 min of partial hepatic ischemia followed by 3 (early IPC) and 24 h (late IPC) of reperfusion. IPC was induced by 10 min of ischemia following 10 min of reperfusion prior to sustained ischemia, and chelerythrine, a PKC inhibitor, was injected 10 min before IPC (5 mg/kg, i.v.). Chelerythrine abrogated the protection of early IPC, as indicated by increased serum aminotransferase activities and decreased hepatic glutathione content. While the IPC-treated group showed a few apoptotic cell deaths during both phases, chelerythrine attenuated these changes only at late IPC and limited IPC-induced inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) overexpression. Membrane translocation of PKC-δ and -ε during IPC was blocked by chelerythrine. Our results suggest that PKC might play a differential role in early and late IPC; activation of PKC-δ and -ε prevents necrosis in early IPC through preservation of redox state and prevents apoptosis in late IPC with iNOS and HO-1 induction. Therefore, PKC represents a promising target for hepatocyte tolerance to ischemic injury, and understanding the differential role of PKC in early and late IPC is important for clinical application of IPC.

Hepatic Ischemic Preconditioning Provides Protection Against Distant Renal Ischemia and Reperfusion Injury in Mice

We previously demonstrated that there are acute and delayed phases of renal protection against renal ischemia and reperfusion (IR) injury with renal ischemic preconditioning (IPC). This study assessed whether hepatic IPC could also reduce distant renal IR injury through the blood stream-mediated supply of reactive oxygen species (ROS). Male C57BL/6 mice were randomly divided into four groups: group I, sham operated including right nephrectomy; group II (IR), left renal ischemia for 30 min and reperfusion injury; group III (IPC-IR), hepatic ischemia for 10 min followed by 10 min of reperfusion before left renal IR injury; group IV (MPG - IPC + IR), pretreated with 100 mg/kg N-(2-mercaptopropionyl)-glycine (MPG) 15 min before hepatic IPC and left renal IR injury. Renal function, histopathologic findings, proinflammatory cytokines, and cytoprotective proteins were evaluated 15 min or 24 hr after reperfusion. Hepatic IPC attenuated the expression of proinflammatory cytokines, tumor necrosis factor α, intercellular adhesion molecule 1, and induced inducible nitric-oxide synthase, and the phosphorylation of Akt in the murine kidney. Renal function was better preserved in mice with hepatic IPC (group III) than groups II or IV. Hepatic IPC protects against distant renal IR injury through the blood stream-delivery of hepatic IPC-induced ROS, by inducing cytoprotective proteins, and by inhibiting inflammatory reactions.

Topical hepatic hypothermia plus ischemic preconditioning: analysis of bile flow and ischemic injuries after initial reperfusion in rats

To evaluate the effects of the topical liver hypothermia and IPC combination against I/R injury after initial reperfusion. In 32 Wistar rats, partial liver ischemia was induced for 90 minutes in normothermia (IN), ischemic preconditioning (IPC), 26ºC topical hypothermia (H) and 26ºC topical hypothermia plus IPC (H+IPC). MAP, body temperature and bile flow were recorded each 15 minutes. Plasmatic injury markers and tissue antioxidant defenses were assessed after 120 minutes of reperfusion. MAP and body temperature remained constant during all experiment. Bile flow returned to levels similar to controls after 45 minutes of reperfusion in the H and H+IPC groups and increased significantly in comparison to the NI and IPC groups after 105 and 120 minutes. AST and ALT increased significantly in the normothermic groups in comparison to controls. TBARS levels decreased significantly in the H+IPC group in comparison to the other groups whereas Catalase levels increased significantly in the IPC group. SOD levels were significantly higher in the H group in comparison to all groups. The induction of 26ºC topical hypothermia associated or not to IPC protected the ischemic liver against ischemia/reperfusion injuries and allowed an early recovery of the hepatic function.

SP1-Dependent Induction of CD39 Facilitates Hepatic Ischemic Preconditioning

Ischemia/reperfusion injury (IRI) of the liver is an important cause of hepatic dysfunction. Ischemic preconditioning (IP) is associated with adenosine-mediated tissue protection from subsequent IRI. Extracellular nucleotides (e.g., ATP) represent the main source for extracellular adenosine. Therefore, we hypothesized that phosphohydrolysis of ATP/ADP via the ectonucleoside triphosphate diphosphohydrolase-1 (CD39), conversion of ATP/ADP to AMP, mediates IP-dependent liver protection. We found that hepatic IP was associated with significant induction of CD39 transcript, heightened protein expression, and improved outcomes after IRI. Targeted gene deletion or pharmacological inhibition of CD39 abolished hepatoprotection by IP as measured by serum markers of liver injury or histology. Therapeutic studies to mimic IP with i.p. apyrase (a soluble ectonucleoside triphosphate diphosphohydrolase, NTPDase) in the absence of IP attenuated hepatic injury after IRI. In additional in vivo studies, small interfering RNA treatment was used to achieve repression of the transcription factor Sp1, known to be implicated in CD39 transcriptional regulation. In fact, Sp1 small interfering RNA treatment was associated with attenuated CD39 induction and increased hepatic injury in vivo. Our data suggest a Sp1-dependent regulatory pathway for CD39 during hepatic IP. These studies reveal a novel role of CD39 in hepatic protection and suggest soluble apyrase for the treatment of liver ischemia.

The value of hepatic ischemic preconditioning in hepatectomy with a prospective randomized controlled study

To evaluate the value of ischemic preconditioning in clinical hepatectomy. A total of 48 unselected patients undergoing liver resection were analyzed by randomized controlled trial from December 2004 to June 2006. Forty-eight unselected patients were randomized into two groups: IP group (5 minutes of ischemia followed by 5 minutes of reperfusion) and control group (received Pringle's maneuver no and no IP was given). Postoperative days 1, 3 and 7, the liver function were checked. Perioperative mortality, morbidity and hospitalized days were compared. In IP group, ischemic times were 5 - 80 min, mean 31 min, hospitalized days were 13 - 50 days, mean 20 days. In control group, ischemic times were 10 - 60 min, mean 27 min, hospitalized days were 10 - 33 days, mean 17 days. Forty-seven patients were satisfactory with postoperative recovery, except one patient died of chronic liver dysfunction after 3 months postoperatively. Postoperative days 1, 3 and 7, the ALT, AST, TBIL, ALB levels in two groups were not statistically significant (P > 0.05). The clinical use of IP through 5 minutes of warm ischemia in this technique of hepatectomy does not protect the liver from hepatic injury induced by the IRI.

Ischemic preconditioning of the liver: A few perspectives from the bench to bedside translation

Utilization of ischemic preconditioning to ameliorate ischemia/reperfusion injury has been extensively studied in various organs and species for the past two decades. While hepatic ischemic preconditioning in animals has been largely beneficial, translational efforts in the two clinical contexts--liver resection and decreased donor liver transplantation--have yielded mixed results. This review is intended to critically examine the translational data and identify some potential reasons for the disparate clinical results, and highlight some issues for further studies.

Extracellular Adenosine Production by Ecto-5′-Nucleotidase Protects During Murine Hepatic Ischemic Preconditioning

The liver tolerates ischemia/reperfusion (IR) poorly. The discovery of ischemic preconditioning (IP) has raised hopes that natural pathways could be activated to increase hepatic resistance to ischemia. However, mechanisms of hepatic IP remain largely unknown. Extracellular adenosine has been implicated as an innate anti-inflammatory metabolite, particularly during ischemia. We investigated whether ecto-5'-nucleotidase (CD73), the "pacemaker" enzyme of extracellular adenosine production, is critical for hepatic protection by IP. Mice were subjected to 4 cycles of portal triad occlusion and reperfusion (3 minutes of ischemia/3 minutes of reperfusion) prior to IR or IR alone. Hepatic IP was associated with a significant induction of CD73 transcript and protein. Targeted gene deletion or pharmacologic inhibition of CD73 abolished hepatic protection by IP as measured by lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase serum levels or histologic injury. Increases in extracellular adenosine with IP were significantly attenuated in cd73-deficient (cd73(-/-)) mice. Reconstitution of cd73(-/-) mice with soluble 5'-nucleotidase resulted in complete restoration of hepatoprotection by IP, and hepatic injury following ischemia was attenuated by treatment of WT mice with soluble 5'-nucleotidase. Mice deficient in CD73 did not demonstrate the same degree of IP-dependent inhibition of acute phase complement gene expression/activation as did wild-type mice suggesting that extracellular adenosine attenuates hepatic IR via complement regulation. Extracellular adenosine production by CD73 mediates protection during murine hepatic IP. Use of soluble 5'-nucleotidase may be a potential therapeutic for hepatic ischemia.

Use of a hanging-weight system for liver ischemic preconditioning in mice

Ischemic preconditioning (IP) represents a powerful experimental strategy to identify novel molecular targets to attenuate hepatic injury during ischemia. As a result, murine studies of hepatic IP have become an important field of research. However, murine IP is technically challenging, and experimental details can alter the results. Therefore, we systematically tested a novel model of hepatic IP by using a hanging-weight system for portal triad occlusion. This system has the benefit of applying intermittent hepatic ischemia and reperfusion without manipulation of a surgical clamp or suture, thus minimizing surgical trauma. Systematic evaluation of this model revealed a close correlation of hepatic ischemia time with liver damage as measured by alanine (ALT) and aspartate (AST) aminotransferase serum levels. Using different numbers of IP cycles and times intervals, we found optimal liver protection with four cycles of 3 min ischemia/3 min reperfusion as measured by ALT, AST, lactate dehydrogenase, and interleukin-6. Similarly, ischemia-associated increases in hepatic infarct size, neutrophil infiltration, and histological injury were maximally attenuated with the above regimen. To demonstrate transcriptional consequences of liver IP, we isolated RNA from preconditioned liver and confirmed transcriptional modulation of known target genes (equilibrative nucleoside transporters, acute-phase complement genes). Taken together, these studies confirm highly reproducible liver injury and protection by IP when using the hanging-weight system for hepatic ischemia and intermittent reperfusion. Further studies of murine IP may consider this technique.

Mediators of rat ischemic hepatic preconditioning after cold preservation identified by microarray analysis

Hepatic ischemia-reperfusion injury associated with liver transplantation is an as yet unresolved problem in clinical practice. Preconditioning protects the liver against the deleterious effects of ischemia, although the mechanism underlying this preconditioning is still unclear. To profile gene expression patterns involved in hepatic ischemic preconditioning, we analyzed the changes in gene expression in rat livers by DNA microarray analysis. Approximately 116 genes were found to have altered gene expression after 8 hours of cold ischemia. Moreover, the expression of 218 genes was modified by classic preconditioning followed by the same ischemia process. Given the importance of the effects of ischemic preconditioning (IP) in minimizing the liver damage induced by sustained ischemia before reperfusion, this study analyzed the putative genes involved in the beneficial role of IP in liver grafts undergoing cold ischemia before its implantation in the recipient (IP+I). Great differences were found in the gene expression pattern of ischemic preconditioning + long cold ischemia (IP+I) group when compared with the long cold ischemia alone condition (I), which could explain the protective regulatory mechanisms that take place after preconditioning. Twenty-six genes that were downregulated in cold ischemia were found upregulated after preconditioning preceding a long cold ischemia period. These would be genes activated or maintained by preconditioning. Heat shock protein genes and 3-hydroxy-3-methylglutaryl-coenzyme A reductase are among the most markedly induced transcripts.

Ischemic preconditioning of the murine liver protects through the Akt kinase pathway

Hepatic ischemia-reperfusion (I/R) injury occurs in the settings of transplantation, trauma, and elective liver resection. Ischemic preconditioning has been used as a strategy to reduce inflammation and organ damage from I/R of the liver. However, the mechanisms involved in this process are poorly understood. We examined the role of the phosphatidylinositol 3 (PI3) kinase/Akt-signaling pathway during hepatic ischemic preconditioning (IPC). Prior to a prolonged warm ischemic insult, BALB/c mice were subjected to a 20-minute IPC period consisting of 10 minutes of ischemia and 10 minutes of reperfusion. Mice undergoing IPC demonstrated a significantly greater level and earlier activation of Akt in the liver compared with control animals. IPC also resulted in markedly less hepatocellular injury and improved survival compared with control animals. Akt activation associated with hepatic IPC suppressed the activity of several modulators of apoptosis, including Bad, glycogen synthase kinase beta, and caspase-3. In addition, IPC also inhibited the activities of c-Jun N-terminal kinase and nuclear factor kappaB after I/R. Pretreatment of mice with PI3 kinase inhibitors completely abolished Akt phosphorylation and the protective effects seen with IPC. In conclusion, these results indicate that the PI3 kinase/Akt pathway plays an essential role in the protective effects of IPC in hepatic I/R injury. Modulation of this pathway may be a potential strategy in clinical settings of ischemic liver injury to decrease organ damage.

Mecanismos moleculares en el daño por isquemia-reperfusión hepática y en el preacondicionamiento isquémico

Ischemia-reperfusion (IR) liver injury is associated with temporary clamping of hepatoduodenal ligament during liver surgery, hypoperfusion shock and graft failure after liver transplantation. Mechanisms of IR liver injury include: i) loss of calcium homeostasis, ii) reactive oxygen and nitrogen species generation, iii) changes in microcirculation, iv) Kupffer cell activation, and (v) complement activation. Pre-exposure of the liver to transient ischemia increases the tolerance to IR injury, a phenomenon known as hepatic ischemic preconditioning (IP). IP involves: i) recovery of the energy supply and calcium, sodium and pH homeostasis, ii) enhancement in the antioxidant potential, and iii) expression of multiple stress-response proteins, including acute phase proteins, heat shock proteins, and heme oxygenase. These observations and preliminary studies in humans give a rationale for the assessment of IP in minimizing or preventing IR injury during surgery and non surgical conditions of tissue hypoperfusion.

Beneficial Effects of Ischemic Preconditioning in Patients Undergoing Hepatectomy

Temporary vascular clampage (Pringle maneuver) during liver surgery can cause ischemia-reperfusion injury. In this process, activation of polymorphonuclear leukocytes (PMNLs) might play a major role. Thus, we investigated the effects of hepatic ischemic preconditioning on PMNL functions. Prospective randomized study. Patients who underwent partial liver resection were randomly assigned to 3 groups: group 1 without Pringle maneuver; group 2 with Pringle maneuver, and group 3 with ischemic preconditioning using 10 minutes of ischemia and 10 minutes of reperfusion prior to Pringle maneuver for resection. University hospital, Munich, Germany. Seventy-five patients underwent hepatic surgery mostly owing to metastasis. Perioperative factors for PMNL activation, inflammation, and postoperative hepatocellular integrity. Ischemia-reperfusion of the human liver (mean +/- SD time to perform the Pringle maneuver, 35.5 +/- 2.6 minutes) caused (1) a decrease in the number of circulating PMNLs, (2) their intrahepatic sequestration, (3) their systemic activation, and (4) a significant correlation between the degree of their postischemic activation and the postoperative rise in liver enzyme serum levels. In parallel, cytokines with proinflammatory and chemotactic properties were released reaching the highest values when stimulation of PMNLs was most pronounced. When ischemic preconditioning preceded the Pringle maneuver, activation of PMNLs and cytokine plasma levels was reduced as evidenced by the attenuation of superoxide anion production, beta(2)-integrin up-regulation, and interleukin 8 serum concentrations, followed by a significant reduction in serum alanine aminotransferase levels on the first and second postoperative days. These results demonstrate in humans that ischemic preconditioning reduces activation of PMNLs elicited by the Pringle maneuver. The down-regulation of potentially cytotoxic functions of PMNLs might be one of yet unknown important pathways that altogether mediate protection by ischemic preconditioning.

Nitric oxide as mediator of hepatic ischemic preconditioning: intracellular mechanisms of hepatocyte protection against hypoxic injury: Rita Carini, Maria Grazia De Cesaris, Roberta Splendore, Emanuele Albano, Univ of East Piedmont, Novara Italy

Nitric oxide as mediator of hepatic ischemic preconditioning: intracellular mechanisms of hepatocyte protection against hypoxic injury: Rita Carini, Maria Grazia De Cesaris, Roberta Splendore, Emanuele Albano, Univ of East Piedmont, Novara Italy

Ischemic preconditioning protects hepatocytes via reactive oxygen species derived from Kupffer cells in rats

Hepatic ischemic preconditioning decreases sinusoidal endothelial cell injury and Kupffer cell activation after cold ischemia/reperfusion, leading to improved survival of liver transplant recipients in rats. Ischemic preconditioning also protects livers against warm ischemia/reperfusion injury, in which hepatocyte injury is remarkable. We aimed to determine whether ischemic preconditioning directly protects hepatocytes and to elucidate its mechanisms. Rats were injected with gadolinium chloride to deplete Kupffer cells or with N -acetyl- l -cysteine, superoxide dismutase, or catalase to scavenge reactive oxygen species. Livers were then preconditioned by 10 minutes of ischemia and 10 minutes of reperfusion. Subsequently, livers were subjected to 40 minutes of warm ischemia and 60 minutes of reperfusion in vivo or in a liver perfusion system. In other rats, livers were preconditioned by H(2)O(2) perfusion instead of ischemia. In the other experiments, livers were perfused with nitro blue tetrazolium to detect reactive oxygen species formation. Ischemic preconditioning decreased injury in hepatocytes, but not in sinusoidal endothelial cells. Kupffer cell depletion itself did not change hepatocyte injury after ischemia/reperfusion, indicating no contribution of Kupffer cells to ischemia/reperfusion injury. However, Kupffer cell depletion reversed hepatoprotection by ischemic preconditioning. Reactive oxygen species formation occurred in Kupffer cells after ischemic preconditioning. Scavenging of reactive oxygen species reversed the effect of ischemic preconditioning, and H(2)O(2) preconditioning mimicked ischemic preconditioning. Ischemic preconditioning directly protected hepatocytes after warm ischemia/reperfusion, which is not via suppression of changes in sinusoidal cells as in cold ischemia/reperfusion injury. This hepatocyte protection was mediated by reactive oxygen species produced by Kupffer cells.

Expression of type 1 plasminogen activator inhibitor and nitric oxide generation in ischemic preconditioning of rat liver

Aims: Nitric oxide (NO) has protective effects against ischemia–reperfusion (I/R) injury and plays an important role in ischemic preconditioning. Type 1 plasminogen activator inhibitor (PAI-1) regulates plasminogen activators, which may have cytotoxic effects in I/R injury. I/R injury is reduced by the inhibition of fibrinolytic enzymes. To clarify the mechanism of ischemic preconditioning, PAI-1 induction and NO generation were studied in hepatic ischemic preconditioning. Methods: Total hepatic ischemia was achieved by Pringle's maneuver. FK409 was used as an NO donor. Plasma alanine aminotransferase (ALT) and hyaluronic acid (HA) were measured to estimate hepatic damage and serum nitrite (NO 2 −) and nitrate (NO 3 −) were also determined to assess NO generation. Reserve transcription-polymerase chain reaction (RT-PCR) and in situ hybridization were carried out to determine the quantitative changes in the expression of PAI-1 mRNA. Plasma PAI-1 concentration was determined using an enzyme-linked immunosorbent assay (ELISA) system. Results: No increase in ALT or HA was found with 5 min I/R. NO and PAI-1 in plasma and PAI-1 mRNA in liver were not increased in the ischemic period, but were increased during the reperfusion period. Infusion of FK409 stimulated induction of PAI-1 mRNA dose dependently. In situ hybridization studies indicated that hepatocytes expressed PAI-1 mRNA after I/R treatment. Conclusions: I/R increased the concentration of plasma PAI-1. Reperfusion following ischemia was needed for the induction of PAI-1 mRNA and increase of plasma NO concentration. FK409 stimulated PAI-1 mRNA induction in the liver. These results indicate that PAI-1 is a component of the ischemic preconditioning mechanisms, which is stimulated by NO generation.

The NO/ET-1 system is involved in the protection of the hepatic ischemic preconditioning

To study the relationship between the disturbance of nitric oxide/endothelin-1 (NO/ET-1) and the hepatic ischemia/reperfusion (I/R) injury as well as the regulation of the NO/ET-1 system by the hepatic ischemic preconditioning (IPC), the changes of the NO/ET-1 system and their relationship with the hepatic I/R injury were compared between the I/R group and the IPC+I/R group in a rat hepatic I/R model. 2 h after reperfusion, the liver tissues were examined for expressed inducible nitric oxide synthase (iNOS) mRNA by RT-PCR. In the acute phase of hepatic reperfusion, the ratio of NO/ET-1 was reduced, which was due to the significant reduction of NO 2 − /NO 3 − (the metabolic product of NO) and significant elevation of ET-1 in the blood plasma. The content of ALT, AST, LDH and TNF-α in blood plasma, and level of MDA in liver tissue were increased but ATP in liver tissue was reduced, and the hepatic damage was deteriorated. The protection of the hepatic IPC was associated with the elevated ratio of NO/ET-1 caused by the elevation of NO 2 − /NO 3 − , and reduction of ET-1 as well. No iNOS mRNA was detected in the liver tissues. It was concluded that hepatic I/R injury was related to the disturbance of NO/ET-1. The protection of the hepatic IPC in the acute phase might be mediated by its regulation of NO/ET-1 system. The cNOS rather than the iNOS generated the NO in this scenario.

Regulation of NFkappaB in hepatic ischemic preconditioning.

The second messengers tyrosine kinase (TK) and protein kinase C (PKC) have been implicated in mediating the cellular signaling cascade during hepatic ischemic preconditioning (IPC). We evaluated the role of TK and PKC on the modulation of the transcription factor nuclear factor kappa B (NFkappaB) and its inhibitor IkappaB alpha during IPC. Yorkshire pigs underwent routine harvest. IPC livers underwent 15 minutes of ischemia and 15 minutes of in situ perfusion before harvest, with or without pretreatment with a TK inhibitor (genistein) or a PKC inhibitor (chelerythrine). During cold storage and reperfusion, tissue extracts were analyzed for IkappaB alpha phosphorylation and NFkappaB levels and for TK and PKC activity by Western blot. Control pig livers demonstrated no change in the levels of TK, PKC, IkappaB alpha, or NFkappaB before cold ischemia. IPC grafts demonstrated activation of TK and PKC with increased IkappaB alpha phosphorylation and NFkappaB levels before cold ischemia. IPC grafts pretreated with genistein demonstrated inhibition of TK activation but not of PKC activation. Genistein-pretreated grafts also demonstrated inhibition of IkappaB alpha phosphorylation and a lack of NFkappaB translocation to the nucleus throughout the entire experiment. IPC grafts pretreated with chelerythrine demonstrated inhibition of PKC activation but not TK activation. Chelerythrine-pretreated grafts also demonstrated IkappaB alpha phosphorylation before cold ischemia and enhanced nuclear levels of NFkappaB. Data suggest that the role of TK in IPC might be mediated in part by NFkappaB, but PKC does not depend on NFkappaB for its effect. Two parallel signaling pathways might explain these data.

Hepatic ischemic preconditioning in mice is associated with activation of NF-κB, 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-κB), 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-κB within 30 minutes of the subsequent ischemic interval, paralleled by degradation of inhibitory (binding) protein for NF-κB (IκBα). 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-κB 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. (H EPATOLOGY 2002;36:94-102.)