Role of NADPH oxidase-derived superoxide in reduced size liver ischemia and reperfusion injury

Hepatic resection with concomitant periods of ischemia and reperfusion (I/R) is required to perform reduced size liver transplantation such as split liver or liver donor transplantation. Although great progress has been made using these types of surgeries, there remains substantial risk to both donors and recipients, with a significant number of patients developing liver injury and failure. The objective of this study was to assess the roles of superoxide (O(2)(-)) and tumor necrosis factor-alpha (TNF-alpha) in the pathophysiology of a mouse model of reduced size liver combined with ischemia and reperfusion (RSL+I/R). We found that all male mice subjected to RSL+I/R died within 3-5 days following surgery. Mortality was always preceded by dramatic increases in liver injury and TNF-alpha expression in the absence of neutrophil infiltration. Using a long-lived, polycationic form of human manganese superoxide dismutase (pcMnSOD), NADPH oxidase-deficient mice (gp91(-/-)) or a monoclonal antibody directed against mouse TNF-alpha, we demonstrated that hepatocellular injury (and mortality) were significantly attenuated. In addition, we found that pcMnSOD administration or NADPH deficiency reduced expression of TNF-alpha. Taken together, our data suggest that NADPH oxidase-derived O(2)(-) plays an important role in the pathophysiology of RSL+I/R-induced liver injury via its ability to enhance expression of TNF-alpha. We propose that therapies directed toward scavenging of O(2)(-), inhibiting NADPH oxidase, and/or immuno-neutralizing TNF-alpha may prove useful in limiting the liver injury induced by surgical procedures that require resection and I/R such as split liver or living donor liver transplantation.     

ARRB1 ameliorates liver ischaemia/reperfusion injury via antagonizing TRAF6‐mediated Lysine 6‐linked polyubiquitination of ASK1 in hepatocytes

Hepatic ischaemia/reperfusion (I/R) injury is a major clinical problem during liver surgical procedures, which usually lead to early transplantation failure and higher organ rejection rate, and current effective therapeutic strategies are still limited. Therefore, in‐depth exploring of the molecular mechanisms underlying liver I/R injury is key to the development of new therapeutic methods. β‐arrestins are multifunctional proteins serving as important signalling scaffolds in numerous physiopathological processes, including liver‐specific diseases. However, the role and underlying mechanism of β‐arrestins in hepatic I/R injury remain largely unknown. Here, we showed that only ARRB1, but not ARRB2, was down‐regulated during liver I/R injury. Hepatocyte‐specific overexpression of ARRB1 significantly ameliorated liver damage, as demonstrated by decreases in serum aminotransferases, hepatocellular necrosis and apoptosis, infiltrating inflammatory cells and secretion of pro‐inflammatory cytokines relative to control mice, whereas experiments with ARRB1 knockout mice gotten opposite effects. Mechanistically, ARRB1 directly interacts with ASK1 in hepatocytes and inhibits its TRAF6‐mediated Lysine 6‐linked polyubiquitination, which then prevents the activation of ASK1 and its downstream signalling pathway during hepatic I/R injury. In addition, inhibition of ASK1 remarkably abolished the disruptive effect result from ARRB1 deficiency in liver I/R injury in vivo, indicating that ASK1 was required for ARRB1 function in hepatic I/R injury. In conclusion, we proposed that ARRB1 is a novel protective regulator during liver I/R injury, and modulation of the regulatory axis between ARRB1 and ASK1 could be a novel therapeutic strategy to prevent this pathological process.     

FTY720 suppresses liver tumor metastasis by reducing the population of circulating endothelial progenitor cells

Background

Surgical procedures such as liver resection and liver transplantation are the first-line treatments for hepatocellular carcinoma (HCC) patients. However, the high incidence of tumor recurrence and metastasis after liver surgery remains a major problem. Recent studies have shown that hepatic ischemia-reperfusion (I/R) injury and endothelial progenitor cells (EPCs) contribute to tumor growth and metastasis. We aim to investigate the mechanism of FTY720, which was originally applied as an immunomodulator, on suppression of liver tumor metastasis after liver resection and partial hepatic I/R injury.

Methodology/Principal Findings

An orthotopic liver tumor model in Buffalo rat was established using the hepatocellular carcinoma cell line McA-RH7777. Two weeks after orthotopic liver tumor implantation, the rats underwent liver resection for tumor-bearing lobe and partial hepatic I/R injury. FTY720 (2 mg/kg) was administered through the inferior caval vein before and after I/R injury. Blood samples were taken at days 0, 1, 3, 7, 14, 21 and 28 for detection of circulating EPCs (CD133+CD34+). Our results showed that intrahepatic and lung metastases were significantly inhibited together with less tumor angiogenesis by FTY720 treatment. The number of circulating EPCs was also significantly decreased by FTY720 treatment from day 7 to day 28. Hepatic gene expressions of CXCL10, VEGF, CXCR3, CXCR4 induced by hepatic I/R injury were down-regulated in the treatment group.

Conclusions/Significance

FTY720 suppressed liver tumor metastasis after liver resection marred by hepatic I/R injury in a rat liver tumor model by attenuating hepatic I/R injury and reducing circulating EPCs.     

Cannabidiol protects against hepatic ischemia/reperfusion injury by attenuating inflammatory signaling and response, oxidative/nitrative stress, and cell death

Ischemia/reperfusion (I/R) is a pivotal mechanism of liver damage after liver transplantation or hepatic surgery. We have investigated the effects of cannabidiol (CBD), the nonpsychotropic constituent of marijuana, in a mouse model of hepatic I/R injury. I/R triggered time-dependent increases/changes in markers of liver injury (serum transaminases), hepatic oxidative/nitrative stress (4-hydroxy-2-nonenal, nitrotyrosine content/staining, and gp91phox and inducible nitric oxide synthase mRNA), mitochondrial dysfunction (decreased complex I activity), inflammation (tumor necrosis factor α (TNF-α), cyclooxygenase 2, macrophage inflammatory protein-1α/2, intercellular adhesion molecule 1 mRNA levels; tissue neutrophil infiltration; nuclear factor κB (NF-κB) activation), stress signaling (p38MAPK and JNK), and cell death (DNA fragmentation, PARP activity, and TUNEL). CBD significantly reduced the extent of liver inflammation, oxidative/nitrative stress, and cell death and also attenuated the bacterial endotoxin-triggered NF-κB activation and TNF-α production in isolated Kupffer cells, likewise the adhesion molecule expression in primary human liver sinusoidal endothelial cells stimulated with TNF-α and attachment of human neutrophils to the activated endothelium. These protective effects were preserved in CB2 knockout mice and were not prevented by CB1/2 antagonists in vitro. Thus, CBD may represent a novel, protective strategy against I/R injury by attenuating key inflammatory pathways and oxidative/nitrative tissue injury, independent of classical CB1/2 receptors.     

A causal analysis of intra-abdominal hemorrhage after reduced-size liver transplantation in rat

In this study, we analyzed causes of abdominal hemorrhage after reduced-size liver transplantation in rat. Healthy SD rats (weight range of 260–280 g) underwent liver transplantation, the donors were female and the recipients were male rats. The recipients were, on average, by <10 g heavier than the donors. All operations on donor rats were performed by the same person using unaided eyesight. Operations on recipient rats were performed by two persons using unaided eyesight. About 270 rats received reduced-size liver transplantation of which 44 died because of intra-abdominal hemorrhages. The distribution of abdominal hemorrhage sites was as follows: 28 cases with anastomotic hemorrhages of the inferior vena cava of the superior liver, 9 cases with subcapsular hemorrhage, 9 cases with tied hemorrhages from the left lateral lobe, 7 cases with hemorrhages from the papillary lobe, 7 hemorrhages from the triangular lobe, 5 hemorrhages of the right suprarenal vein and lumbar veins, 4 hemorrhages caused by mechanical injury, 4 cuff hemorrhages of the portal vein and the inferior vein cava of the inferior liver, 8 anastomotic hemorrhages of the inferior vena cava of the superior liver and the tied hemorrhage of the left outboard lobe, and 5 hemorrhages of the two tied points of the reduced-size liver. Ten hemorrhages were stopped by suture or/and ligature, and 6 were stopped by washing with or soaking in hot water. Results of this study will be helpful to raise success rates of reduced-size liver transplantation.     

Ischemic preconditioning: tolerance to hepatic ischemia-reperfusion injury

Hepatic ischemia-reperfusion (I/R) injury still remains an unresolved problem in both liver resectional surgery and liver transplantation and may be responsible for liver failure, lung injury and death. The current review summarizes the findings reported to date on the effectiveness of ischemic preconditioning against liver and lung damage associated with hepatic I/R injury and the underlying protective mechanisms. The effect of ischemic preconditioning on the mechanisms potentially involved in hepatic I/R injury, including alterations in energy metabolism, neutrophil accumulation, microcirculatory disturbances, formation of proinflammatory mediators, such as endothelin and tumor necrosis factor-alpha, and reactive oxygen species generation have been evaluated. In this review, we address the role of preconditioning in the increased vulnerability of fatty livers to hepatic I/R injury. The effectiveness of ischemic preconditioning versus pharmacological strategies that could simulate the benefits of liver preconditioning has been also discussed.     

Hepatic microvascular responses to ischemia-reperfusion in low-density lipoprotein receptor knockout mice

The overall objective of this study was to determine whether genetically induced hypercholesterolemia alters the inflammatory and microvascular responses of mouse liver to ischemia-reperfusion (I/R). The accumulation of rhodamine 6G-labeled leukocytes and the number of nonperfused sinusoids (NPS) were monitored (by intravital microscopy) in the liver of wild-type (WT) and low-density lipoprotein receptor-deficient (LDLr(-/-)) mice for 1 h after a 30-min period of normothermic ischemia. Plasma alanine transaminase (ALT) levels were used to monitor hepatocellular injury. Microvascular leukostasis as well as increases in NPS and plasma ALT were observed at 60 min after hepatic I/R in both WT and in LDLr(-/-) mice; however, these responses were greatly exaggerated in LDLr(-/-) mice. Pretreatment of LDLr(-/-) mice with gadolinium chloride, which reduces Kupffer cell function, attenuated the hepatic leukostasis, NPS, and hepatocellular injury elicited by I/R. Similar protection against I/R was observed in LDLr(-/-) mice pretreated with antibodies directed against tumor necrosis factor-alpha, intercellular adhesion molecule-1 (ICAM-1), or P-selectin. These findings indicate that chronic hypercholesterolemia predisposes the hepatic microvasculature to the deleterious effects of I/R. Kupffer cell activation and the leukocyte adhesion receptors ICAM-1 and P-selectin appear to contribute to the exaggerated inflammatory responses observed in the postischemic liver of LDLr(-/-) mice.     

Impaired autophagy contributes to hepatocellular damage during ischemia/reperfusion: Heme oxygenase-1 as a possible regulator

Liver ischemia and reperfusion (I/R) injury is characterized by oxidative stress that is accompanied by alterations of the endogenous defensive system. Emerging evidence suggests a protective role for autophagy induced by multiple stressors including reactive oxygen species. Meanwhile, heme oxygenase-1 (HO-1) has long been implicated in cytoprotection against oxidative stress in vitro and in vivo. Therefore, we investigated the impact of autophagy in the pathogenesis of liver I/R and its molecular mechanisms, particularly its linkage to HO-1. By using transmission electron microscopic analysis and biochemical autophagic flux assays with microtubule-associated protein 1 light chain 3-II, and beclin-1, representative autophagy markers, and p62, a selective substrate for autophagy, we found that reperfusion reduced autophagy both in the rat liver and in primary cultured hepatocytes. When autophagy was further inhibited with chloroquine or wortmannin, I/R-induced hepatocellular injury was aggravated. While livers that underwent I/R showed increased levels of mammalian target of rapamaycin and calpain 1 and 2, inhibition of calpain 1 and 2 induced an autophagic response in hepatocytes subjected to hypoxia/reoxygenation. HO-1 increased autophagy, and HO-1 reduced I/R-induced calcium overload in hepatocytes and prevented calpain 2 activation both in vivo and in vitro. Taken together, these findings suggest that the impaired autophagy during liver I/R, which is mediated by calcium overload and calpain activation, contributes to hepatocellular damage and the HO-1 system protects the liver from I/R injury through enhancing autophagy.     

Role of IL-17A in neutrophil recruitment and hepatic injury after warm ischemia-reperfusion mice

Recent evidence suggests that IL-17A regulates neutrophil-dependent organ injury. Accordingly, the purpose of this study was to determine the role of IL-17A in neutrophil recruitment after ischemia-reperfusion (I/R) and in subsequent liver injury. Two mouse models including wild-type and IL-17A knockout mice were evaluated for I/R injury. The medial largest lobe of the liver was clamped for 90 min. In another set of experiments, recombinant mouse (rm)IL-17A homodimer or rmIL-17A/F heterodimer were administered to knockout mice before I/R, and liver injury was investigated. Isolated Kupffer cells were incubated with rmIL-17A or rmIL-17F, and production of TNF-α was measured. Studies evaluating the extent of liver injury as measured by serum transaminase levels demonstrated similar levels in the acute phase (6 h) in these two models. In contrast, in the subacute phase (20 h) after I/R, both serum transaminase levels and percent of hepatic necrosis were significantly reduced in the knockout mice compared with the wild-type mice. This reduction in liver injury seen in the knockout mice was associated with suppression of chemokine and adhesion molecule expression and reduction in infiltration of neutrophils into the liver. Administration of rmIL-17A homodimer, but not IL-17A/F heterodimer, increased liver injury in the subacute phase of I/R in KO mice. TNF-α production by isolated Kupffer cells increased significantly in the cells incubated with rmIL-17A compared with rmIL-17F. These results indicate that IL-17A is a key regulator in initiating neutrophil-induced inflammatory responses and hepatic injury in the subacute phase after reperfusion.     

Protective effect of Urtica dioica L. on renal ischemia/reperfusion injury in rat

Renal ischemia–reperfusion (I/R) injury may occur after renal transplantation, thoracoabdominal aortic surgery, and renal artery interventions. This study was designed to investigate the effect of Urtica dioica L. (UD), in I/R induced renal injury. A total of 32 male Sprague–Dawley rats were divided into four groups: control, UD alone, I/R and I/R?+?UD; each group contain 8 animals. A rat model of renal I/R injury was induced by 45-min occlusion of the bilateral renal pedicles and 24-h reperfusion. In the UD group, 3?days before I/R, UD (2?ml/kg/day intraperitoneal) was administered by gastric gavage. All animals were sacrificed at the end of reperfusion and kidney tissues samples were obtained for histopathological investigation in all groups. To date, no more histopathological changes on intestinal I/R injury in rats by UD treatment have been reported. Renal I/R caused severe histopathological injury including tubular damage, atrophy dilatation, loss of brush border and hydropic epithelial cell degenerations, renal corpuscle atrophy, glomerular shrinkage, markedly focal mononuclear cell infiltrations in the kidney. UD treatment significantly attenuated the severity of intestinal I/R injury and significantly lowered tubulointerstitial damage score than the I/R group. The number of PCNA and TUNEL positive cells in the control and UD alone groups was negligible. When kidney sections were PCNA and TUNEL stained, there was a clear increase in the number of positive cells in the I/R group rats in the renal cortical tissues. However, there is a significant reduction in the activity of PCNA and TUNEL in kidney tissue of renal injury induced by renal I/R with UD therapy. Our results suggest that administration of UD attenuates renal I/R injury. These results suggest that UD treatment has a protective effect against renal damage induced by renal I/R. This protective effect is possibly due to its ability to inhibit I/R induced renal damage, apoptosis and cell proliferation.     

The role of excessive versus acute administration of erythropoietin in attenuating hepatic ischemia-reperfusion injury

Ischemia-reperfusion injury (I/R) is the main cause of primary graft nonfunction. Our aim was to evaluate the effect of excessive versus acute administration of erythropoietin (EPO) in attenuating the hepatic injury induced by I/R in mice. The effect of segmental (70%) hepatic ischemia was evaluated in a transgenic mouse line with constitutive overexpression of human EPO cDNA and in wild-type (WT) mice. Mice were randomly allocated to 5 main experimental groups: (i) WT-sham, (ii) WT ischemia, (iii) WT ischemia + recombinant human erythropoietin (rhEPO), (iv) transgenic-sham, and (v) transgenic ischemia. The EPO-pretreated mice showed a significant reduction in liver enzyme levels and intrahepatic caspase-3 activity and fewer apoptotic hepatocytes (p < 0.05 for all) compared with the WT untreated I/R group. EPO decreased c-Jun N-terminal kinase (JNK) phosphorylation and nuclear factor-κB (NF-κB) expression during I/R. In transgenic I/R livers, baseline histology showed diffused hepatic injury, and no significant beneficial effect was noted between the WT untreated and the transgenic I/R mice. In conclusion, acute pretreatment with EPO in WT mice attenuated in vivo I/R liver injury. However, in excessive EPO overexpression, the initial liver injury abolished the beneficial effect of EPO. These findings have important implications for the potential use of acute EPO in I/R injury during liver transplantation.     

The damage-associated molecular pattern HMGB1 is released early after clinical hepatic ischemia/reperfusion

Objective and backgroundActivation of sterile inflammation after hepatic ischemia/reperfusion (I/R) culminates in liver injury. The route to liver damage starts with mitochondrial oxidative stress and cell death during early reperfusion. The link between mitochondrial oxidative stress, damage-associate molecular pattern (DAMP) release, and sterile immune signaling is incompletely understood and lacks clinical validation. The aim of the study was to validate this relation in a clinical liver I/R cohort and to limit DAMP release using a mitochondria-targeted antioxidant in I/R-subjected mice.MethodsPlasma levels of the DAMPs high-mobility group box 1 (HMGB1), mitochondrial DNA, and nucleosomes were measured in 39 patients enrolled in an observational study who underwent a major liver resection with (N = 29) or without (N = 13) intraoperative liver ischemia. Circulating cytokine and neutrophil activation markers were also determined. In mice, the mitochondria-targeted antioxidant MitoQ was intravenously infused in an attempt to limit DAMP release, reduce sterile inflammation, and suppress I/R injury.ResultsIn patients, HMGB1 was elevated following liver resection with I/R compared to liver resection without I/R. HMGB1 levels correlated positively with ischemia duration and peak post-operative transaminase (ALT) levels. There were no differences in mitochondrial DNA, nucleosome, or cytokine levels between the two groups. In mice, MitoQ neutralized hepatic oxidative stress and decreased HMGB1 release by ±50%. MitoQ suppressed transaminase release, hepatocellular necrosis, and cytokine production. Reconstituting disulfide HMGB1 during reperfusion reversed these protective effects.ConclusionHMGB1 seems the most pertinent DAMP in clinical hepatic I/R injury. Neutralizing mitochondrial oxidative stress may limit DAMP release after hepatic I/R and reduce liver damage.     

The Role of N-Acetyltransferase 8 in Mesenchymal Stem Cell-Based Therapy for Liver Ischemia/Reperfusion Injury in Rats

Objective

To evaluate the impact of mesenchymal stem cells (MSCs) against hepatic I/R injury and explore the role of N-acetyltransferase 8 (NAT8) in the process.

Methods

We investigated the potential of injected MSCs systemically via the tail vein in healing injuried liver of the SD rat model of 70% hepatic I/R injury by measuring the biochemical and pathologic alterations. Subsequently, we evaluated the expression levels of NAT8 by western blotting in vivo. Concurrently, hydrogen peroxide (H₂O₂)-induced apoptosis in the human normal liver cell line L02 was performed in vitro to evaluate the protective effects of MSC conditioned medium (MSC-CM) on L02 cells. In addition, we downregulated and upregulated NAT8 expression in L02 cells and induced apoptosis by using H₂O₂ to study the protective role of NAT8.

Results

MSCs implantation led to a significant reduced liver enzyme levels, an advanced protection in the histopathological findings of the acutely injured liver and a significantly lower percentage of TUNEL-positive cells, which were increased after I/R injury. In vitro assays, MSC-CM inhibited hepatocyte apoptosis induced by H₂O_2. Moreover, overexpression or downregulation of NAT8 prevented or aggravated hepatocyte apoptosis induced by H₂O₂, respectively.

Conclusions

MSC transplantation provides support to the I/R-injured liver by inhibiting hepatocellular apoptosis and stimulating NAT8 regeneration.     

Hepatocellular protection by nitric oxide or nitrite in ischemia and reperfusion injury

Ischemia and reperfusion (I/R)-induced liver injury occurs in several pathophysiological disorders including hemorrhagic shock and burn as well as resectional and transplantation surgery. One of the earliest events associated with reperfusion of ischemic liver is endothelial dysfunction characterized by the decreased production of endothelial cell-derived nitric oxide (NO). This rapid post-ischemic decrease in NO bioavailability appears to be due to decreased synthesis of NO, enhanced inactivation of NO by the overproduction of superoxide or both. This review presents the most current evidence supporting the concept that decreased bioavailability of NO concomitant with enhanced production of reactive oxygen species initiates hepatocellular injury and that endogenous NO or exogenous NO produced from nitrite play important roles in limiting post-ischemic tissue injury.     

Targeting p21-activated kinase 4 (PAK4) with pyrazolo[3,4-d]pyrimidine derivative SPA7012 attenuates hepatic ischaemia-reperfusion injury in mice

p21-Activated kinase 4 (PAK4), one of the serine/threonine kinases activated by Rho-family GTPases, has been widely studied as an oncogenic protein that is overexpressed in many types of cancers. In our recent study, PAK4 upregulation was observed in mice exhibiting hepatic ischaemia-reperfusion (I/R) and in liver transplantation patients. Liver I/R injury was also attenuated in Pak4 KO mice. Herein, we report a novel series of pyrazolo[3,4-d]pyrimidine derivatives of type I ½ PAK4 inhibitors. The most potent compound SPA7012 was evaluated to determine the pharmacological potential of PAK4 inhibitor in I/R injury in mice. Mice with I/R injury showed typical patterns of liver damage, as demonstrated by increases in serum levels of aminotransferases and proinflammatory cytokines, hepatocellular necrosis and apoptosis, and inflammatory cell infiltration, relative to sham mice. Conversely, intraperitoneal administration of SPA7012 dramatically attenuated biochemical and histopathologic changes. Mechanistically, stabilisation of nuclear factor-erythroid 2-related factor 2 (Nrf2), a master regulator of anti-oxidative response, was observed following SPA7012 treatment. SPA7012 treatment in primary hepatocytes also attenuated hypoxia-reoxygenation-induced apoptotic cell death and inflammation. Together, these results provide experimental evidence supporting the use of PAK4 inhibitors for alleviation of I/R-induced liver damage.     

Regulation of postischemic liver injury following different durations of ischemia

The objective of this study was to define the relationship among Kupffer cells, O(2)(-) production, and TNF-alpha expression in the pathophysiology of postischemic liver injury following short and long periods of ischemia. Using different forms of superoxide dismutase with varying circulating half-lives, a monoclonal antibody directed against mouse TNF-alpha, and NADPH oxidase-deficient mice, we found that 45 or 90 min of partial (70%) liver ischemia and 6 h of reperfusion (I/R) produced time-dependent increases in liver injury and TNF-alpha expression in the absence of neutrophil infiltration. Furthermore, we observed that hepatocellular injury induced by short periods of ischemia were not dependent on formation of TNF-alpha but were dependent on Kupffer cells and NADPH oxidase-independent production of O(2)(-). However, liver injury induced by extended periods of ischemia appeared to require the presence of Kupffer cells, NADPH oxidase-derived O(2)(-), and TNF-alpha expression. We conclude that the sources for O(2)(-) formation and the relative importance of TNF-alpha in the pathophysiology of I/R-induced hepatocellular injury differ depending on the duration of ischemia.     

Involvement of GPX4 in irisin's protection against ischemia reperfusion‐induced acute kidney injury

Ischemia reperfusion (I/R)‐induced acute kidney injury (AKI) is a common and serious condition. Irisin, an exercise‐induced hormone, improves mitochondrial function and reduces reactive oxygen species (ROS) production. Glutathione peroxidase 4 (GPX4) is a key regulator of ferroptosis and its inactivation aggravates renal I/R injury by inducing ROS production. However, the effect of irisin on GPX4 and I/R‐induced AKI is still unknown. To study this, male adult mice were subjected to renal I/R by occluding bilateral renal hilum for 30 min, which was followed by 24 hr reperfusion. Our results showed serum irisin levels were decreased in renal I/R mice. Irisin (250 μg/kg) treatment alleviated renal injury, downregulated inflammatory response, improved mitochondrial function, and reduced ER stress and oxidative stress after renal I/R, which were associated with upregulation of GPX4. Treated with RSL3 (a GPX4 inhibitor) abolished irisin's protective effect. Thus, irisin attenuates I/R‐induced AKI through upregulating GPX4.