Steatotic liver grafts tolerate ischemia-reperfusion (I/R) injury poorly, contributing to increased primary graft nonfunction following transplantation. Activation of nuclear factor kappa-B (NFκB) following I/R injury plays a crucial role in activation of pro-inflammatory responses leading to injury.
We evaluated the role of NFκB in steatotic liver injury by using an orthotopic liver transplant (OLT) model in Zucker rats (lean to lean or obese to lean) to define the mechanisms of steatotic liver injury. Obese donors were treated with bortezomib to assess the role of NF-κB in steatotic liver I/R injury. Hepatic levels of NF-κB and pro-inflammatory cytokines were analyzed by ELISA. Serum transaminase levels and histopathological analysis were performed to assess associated graft injury.
I/R injury in steatotic liver results in significant increases in activation of NF-κB (40%, p<0.003), specifically the p65 subunit following transplantation. Steatotic donor pretreatment with proteasome inhibitor bortezomib (0.1 mg/kg) resulted in significant reduction in levels of activated NF-κB (0.58±0.18 vs. 1.37±0.06 O.D./min/10μg protein, p<0.003). Bortezomib treatment also reduced expression of pro-inflammatory cytokines MIP-2 compared with control treated steatotic and lean liver transplants respectively (106±17.5 vs. 443.3±49.9 vs. 176±10.6 pg/mL, p=0.02), TNF-α (223.8±29.9 vs. 518.5±66.5 vs 264.5±30.1 pg/2μg protein, p=0.003) and IL-1β (6.0±0.91 vs. 19.8±5.2 vs 5±1.7 pg/10μg protein, p= 0.02) along with a significant reduction in ALT levels (715±71 vs 3712.5±437.5 vs 606±286 U/L, p=0.01).
These results suggest that I/R injury in steatotic liver transplantation are associated with exaggerated activation of NFκB subunit p65, leading to an inflammatory mechanism of reperfusion injury and necrosis. Proteasome inhibition in steatotic liver donor reduces NFκB p65 activation and inflammatory I/R injury, improving transplant outcomes of steatotic grafts in a rat model.
hepatic steatosis; I/R injury; liver transplantation; NFκB; PS-341; bortezomib; obese; marginal graft
Hepatic steatosis continues to present a major challenge in liver transplantation. These organs have been shown to have an increased susceptibility to cold ischemia and reperfusion (CIR) injury compared to otherwise comparable lean livers; the mechanisms governing this increased susceptibility to CIR injury are not fully understood. Endoplasmic reticulum (ER) stress is an important link between hepatic steatosis, insulin resistance and the metabolic syndrome. In this study, we investigated ER stress signaling and blockade in the mediation of CIR injury in severely steatotic rodent allografts. Steatotic allografts from genetically leptin-resistant rodents had increased ER stress responses and increased markers of hepatocellular injury following liver transplantation into strain-matched lean recipients. ER stress response components were decreased by the chemical chaperone, TUDCA, resulting in improvement of the allograft injury. TUDCA treatment decreased NF-κB activation, and the pro-inflammatory cytokines IL-6 and IL-1β. However, the predominant response was decreased expression of the ER stress cell death mediator, CHOP. Further, activation of the inflammation-associated caspase 11 was decreased linking ER Stress/CHOP to pro-inflammatory cytokine production following steatotic liver transplantation. These data confirm ER stress in steatotic allografts, and implicate this as a mediating mechanism of inflammation and hepatocyte death in the steatotic liver allograft.
Liver Transplantation; Endoplasmic Reticulum Stress; Hepatic Steatosis; Ischemia-Reperfusion Injury
During partial hepatectomy, ischemia–reperfusion (I/R) is commonly applied in clinical practice to reduce blood flow. Steatotic livers show impaired regenerative response and reduced tolerance to hepatic injury. We examined the effects of tauroursodeoxycholic acid (TUDCA) and 4-phenyl butyric acid (PBA) in steatotic and non-steatotic livers during partial hepatectomy under I/R (PH+I/R). Their effects on the induction of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress were also evaluated. We report that PBA, and especially TUDCA, reduced inflammation, apoptosis and necrosis, and improved liver regeneration in both liver types. Both compounds, especially TUDCA, protected both liver types against ER damage, as they reduced the activation of two of the three pathways of UPR (namely inositol-requiring enzyme and PKR-like ER kinase) and their target molecules caspase 12, c-Jun N-terminal kinase and C/EBP homologous protein-10. Only TUDCA, possibly mediated by extracellular signal-regulated kinase upregulation, inactivated glycogen synthase kinase-3β. This is turn, inactivated mitochondrial voltage-dependent anion channel, reduced cytochrome c release from the mitochondria and caspase 9 activation and protected both liver types against mitochondrial damage. These findings indicate that chemical chaperones, especially TUDCA, could protect steatotic and non-steatotic livers against injury and regeneration failure after PH+I/R.
hepatic ischemia–reperfusion; hepatic resection; steatotic liver; endoplasmic reticulum stress; 4-phenyl butyric acid; taurine-conjugated ursodeoxycholic acid
AIM: To investigate the effect of mild steatotic liver on ischemia-reperfusion injury by focusing on Kupffer cells (KCs) and platelets.
METHODS: Wistar rats were divided into a normal liver group (N group) and a mild steatotic liver group (S group) induced by feeding a choline-deficient diet for 2 wk. Both groups were subjected to 20 min of warm ischemia followed by 120 min of reperfusion. The number of labeled KCs and platelets in sinusoids and the blood perfusion in sinusoids were observed by intravital microscopy (IVM), which was performed at 30, 60 and 120 min after reperfusion. To evaluate serum alanine aminotransferase as a marker of liver deterioration, blood samples were taken at the same time as IVM.
RESULTS: In the S group, the number of platelets adhering to KCs decreased significantly compared with the N group (120 after reperfusion; 2.9 ± 1.1 cells/acinus vs 4.8 ± 1.2 cells/acinus, P < 0.01). The number of KCs in sinusoids was significantly less in the S group than in the N group throughout the observation periods (before ischemia, 19.6 ± 3.3 cells/acinus vs 28.2 ± 4.1 cells/acinus, P < 0.01 and 120 min after reperfusion, 29.0 ± 4.3 cells/acinus vs 40.2 ± 3.3 cells/acinus, P < 0.01). The blood perfusion of sinusoids 120 min after reperfusion was maintained in the S group more than in the N group. Furthermore, elevation of serum alanine aminotransferase was lower in the S group than in the N group 120 min after reperfusion (99.7 ± 19.8 IU/L vs 166.3 ± 61.1 IU/L, P = 0.041), and histological impairment of hepatocyte structure was prevented in the S group.
CONCLUSION: Ischemia-reperfusion injury in mild steatotic liver was attenuated compared with normal liver due to the decreased number of KCs and the reduction of the KC-platelet interaction.
Steatotic liver; Mild steatotic liver; Kupffer cell; Platelet; Ischemia-reperfusion; Intravital microscopy
AIM: To examine the relevance of hypoxia inducible factor (HIF-1) and nitric oxide (NO) on the preservation of fatty liver against cold ischemia-reperfusion injury (IRI).
METHODS: We used an isolated perfused rat liver model and we evaluated HIF-1α in steatotic and non-steatotic livers preserved for 24 h at 4°C in University of Wisconsin and IGL-1 solutions, and then subjected to 2 h of normothermic reperfusion. After normoxic reperfusion, liver enzymes, bile production, bromosulfophthalein clearance, as well as HIF-1α and NO [endothelial NO synthase (eNOS) activity and nitrites/nitrates] were also measured. Other factors associated with the higher susceptibility of steatotic livers to IRI, such as mitochondrial damage and vascular resistance were evaluated.
RESULTS: A significant increase in HIF-1α was found in steatotic and non-steatotic livers preserved in IGL-1 after cold storage. Livers preserved in IGL-1 showed a significant attenuation of liver injury and improvement in liver function parameters. These benefits were enhanced by the addition of trimetazidine (an anti-ischemic drug), which induces NO and eNOS activation, to IGL-1 solution. In normoxic reperfusion, the presence of NO favors HIF-1α accumulation, promoting also the activation of other cytoprotective genes, such as heme-oxygenase-1.
CONCLUSION: We found evidence for the role of the HIF-1α/NO system in fatty liver preservation, especially when IGL-1 solution is used.
Fatty liver; Tissue preservation; Hypoxia inducible factor-1α; IGL-1; Nitric oxide; Trimetazidine
Steatosis is currently the most common chronic liver disease and it can aggravate ischemia-reperfusion (IR) lesions. We hypothesized that S-nitroso-N-acetylcysteine (SNAC), an NO donor component, can ameliorate cell damage from IR injury. In this paper, we report the effect of SNAC on liver IR in rats with normal livers compared to those with steatotic livers.
Thirty-four rats were divided into five groups: I (n=8), IR in normal liver; II (n=8), IR in normal liver with SNAC; III (n=9), IR in steatotic liver; IV (n=9), IR in steatotic liver with SNAC; and V (n=10), SHAN. Liver steatosis was achieved by administration of a protein-free diet. A SNAC solution was infused intraperitoneally for one hour, beginning 30 min. after partial (70%) liver ischemia. The volume of solution infused was 1 ml/100 g body weight. The animals were sacrificed four hours after reperfusion, and the liver and lung were removed for analysis. We assessed hepatic histology, mitochondrial respiration, oxidative stress (MDA), and pulmonary myeloperoxidase.
All groups showed significant alterations compared with the group that received SHAN. The results from the steatotic SNAC group revealed a significant improvement in liver mitochondrial respiration and oxidative stress compared to the steatotic group without SNAC. No difference in myeloperoxidase was observed. Histological analysis revealed no difference between the non-steatotic groups. However, the SNAC groups showed less intraparenchymal hemorrhage than groups without SNAC (p=0.02).
This study suggests that SNAC effectively protects against IR injury in the steatotic liver but not in the normal liver.
Fatty liver; S-nitrosothiol; S-nitroso-N-acetylcysteine; reperfusion injury; oxidative stress
We examined the effects of upregulation of heme oxygenase-1 (HO-1) in steatotic rat liver models of ex vivo cold ischemia/reperfusion (I/R) injury. In the model of ischemia/isolated perfusion, treatment of genetically obese Zucker rats with the HO-1 inducer cobalt protoporphyrin (CoPP) or with adenoviral HO-1 (Ad-HO-1) significantly improved portal venous blood flow, increased bile production, and decreased hepatocyte injury. Unlike in untreated rats or those pretreated with the HO-1 inhibitor zinc protoporphyrin (ZnPP), upregulation of HO-1 by Western blots correlated with amelioration of histologic features of I/R injury. Adjunctive infusion of ZnPP abrogated the beneficial effects of Ad-HO-1 gene transfer, documenting the direct involvement of HO-1 in protection against I/R injury. Following cold ischemia/isotransplantation, HO-1 overexpression extended animal survival from 40% in untreated controls to about 80% after CoPP or Ad-HO-1 therapy. This effect correlated with preserved hepatic architecture, improved liver function, and depressed infiltration by T cells and macrophages. Hence, CoPP- or gene therapy–induced HO-1 prevented I/R injury in steatotic rat livers. These findings provide the rationale for refined new treatments that should increase the supply of usable donor livers and ultimately improve the overall success of liver transplantation.
J. Clin. Invest. 104:1631–1639 (1999).
AIM: To investigate the benefits of insulin like growth factor-1 (IGF-1) supplementation to serum-free institut georges lopez-1 (IGL-1)® solution to protect fatty liver against cold ischemia reperfusion injury.
METHODS: Steatotic livers were preserved for 24 h in IGL-1® solution supplemented with or without IGF-1 and then perfused “ex vivo” for 2 h at 37°C. We examined the effects of IGF-1 on hepatic damage and function (transaminases, percentage of sulfobromophthalein clearance in bile and vascular resistance). We also studied other factors associated with the poor tolerance of fatty livers to cold ischemia reperfusion injury such as mitochondrial damage, oxidative stress, nitric oxide, tumor necrosis factor-α (TNF-α) and mitogen-activated protein kinases.
RESULTS: Steatotic livers preserved in IGL-1® solution supplemented with IGF-1 showed lower transaminase levels, increased bile clearance and a reduction in vascular resistance when compared to those preserved in IGL-1® solution alone. These benefits are mediated by activation of AKT and constitutive endothelial nitric oxide synthase (eNOS), as well as the inhibition of inflammatory cytokines such as TNF-α. Mitochondrial damage and oxidative stress were also prevented.
CONCLUSION: IGL-1® enrichment with IGF-1 increased fatty liver graft preservation through AKT and eNOS activation, and prevented TNF-α release during normothermic reperfusion.
AKT; Institut georges lopez-1® solution; Insulin like growth factor-1; Ischemia reperfusion injury; Nitric oxide; Oxidative stress; Steatotic graft preservation
The serious need for expanding the donor population has attracted attention to the use of steatotic donor livers in orthotopic liver transplantation (OLT). However, steatotic livers are highly susceptible to hepatic ischemia-reperfusion injury (IRI). Expression of fibronectin (FN) by endothelial cells is an important feature of hepatic response to injury. We report the effect of a cyclic RGD peptide with high affinity for the α5β1, the FN integrin receptor, in a rat model of steatotic liver cold ischemia, followed by transplantation. RGD peptide therapy ameliorated steatotic IRI and improved recipient survival rate. It significantly inhibited the recruitment of monocyte/macrophages and neutrophils, and depressed the expression of pro-inflammatory mediators, such as inducible nitric oxide synthase (iNOS) and interferon (IFN)-γ. Moreover, it resulted in profound inhibition of metalloproteinase-9 (MMP-9) expression, a gelatinase implied in leukocyte migration in damaged livers. Finally, we show that RGD peptide therapy reduced the expression of the 17-kDa active caspase-3 and the number of apoptotic cells in steatotic OLTs. The observed protection against steatotic liver IRI by the cyclic RGD peptides with high affinity for the α5β1 integrin suggests that this integrin is a potential therapeutic target to allow the successful utilization of marginal steatotic livers in transplantation.
Although endoplasmic reticulum (ER) stress has been implicated in the pathophysiology of organ ischemia and reperfusion injury (IRI), the underlying mechanisms have yet to be fully elucidated. In particular, as tissue pro-inflammatory immune response is the key mediator of local IRI, how ER stress impacts liver immune cell activation cascade remains to be determined.
In vitro, ER stress in macrophages and hepatocytes were induced by pharmacological agents. Macrophage TLR4 and hepatocyte TNF-α responses were studied. In vivo, the induction of ER stress by IR and the impact of ER stress amelioration by a small molecule chaperon 4-phenyl butyric acid (PBA) on liver immune response were studied in a murine partial liver warm ischemia model.
ER stressed macrophages generated a significantly enhanced pro-inflammatory immune response against TLR4 stimulation; while ER stressed hepatocyte became more susceptible to TNF-α induced cell death. IR resulted in upregulations of sXBP-1 and ATF6 levels in affected livers. Mice pre-treated with PBA were protected from liver IRI, in parallel with diminished local pro-inflammatory gene induction program.
Our study documents a potential immune regulatory role of ER stress in the mechanism of liver IRI, and provides a rationale for targeting stress response as a new therapeutic means to ameliorate tissue inflammation in organ transplant recipients.
ER stress; Liver ischemia; TLR4; Inflammation
Liver steatosis is associated with organ dysfunction after hepatic resection and transplantation which may be caused by hepatic ischemia/reperfusion injury. The aim of the current study was to determine the precise mechanism leading to hepatocyte apoptosis after steatotic liver ischemia/reperfusion. Using a murine model of partial hepatic ischemia for 90 min, we examined the levels and pathway of apoptosis, and the peroxynitrite expression, serum alanine aminotransferase levels, and liver histology 1 and 4 h after reperfusion. In the steatotic liver, the peroxynitrite expression increased after ischemia/reperfusion. Significant hepatocyte apoptosis in the steatotic liver was seen after reperfusion, caused by upregulation of cleaved caspases 9 and 3, but not caspase 8. Serum alanine aminotransferase levels were elevated and histological examination revealed severe liver injury in the steatotic liver 4 h after reperfusion. In mice treated with aminoguanidine, ischemia/reperfusion-induced increases in serum alanine aminotransferase levels and apoptosis were significantly reduced in steatotic liver compared with mice treated with phosphate buffered saline. Survival of mice with steatotic livers significantly improved by treatment with aminoguanidine. Our data suggested that the steatotic liver is vulnerable to hepatic ischemia/reperfusion, leading to significant hepatocyte apoptosis by the mitochondrial permeability transition, and thereby resulting in organ dysfunction.
steatosis; apoptosis; peroxynitrite; hepatic resection
Hepatic ischemia-reperfusion can be associated with acute lung injury. Alveolar epithelial type II cells (ATII) play an important role in maintaining lung homeostasis in acute lung injury.
To study potentially new mechanisms of hepatic ischemia-reperfusion-induced lung injury, we examined how liver ischemia-reperfusion altered the proteome of ATII.
Spontaneously breathing male Zucker rats.
Rats were anesthetized with isoflurane. The vascular supply to the left and medial lobe of the liver was clamped for 75 mins and then reperfused. Sham-operated rats were used as controls. After 8 hrs, rats were killed.
Measurements and Main Results
Bronchoalveolar lavage and differential cell counts were performed, and tumor necrosis factor-α and cytokine-induced neutrophil chemotactic factor-1 in plasma were determined by enzyme-linked immunosorbent assay. ATII were isolated, lysed, tryptically digested, and labeled using isobaric tags (iTRAQ). The samples were fractionated by cation exchange chromatography, separated by high-performance liquid-chromatography, and identified using electrospray tandem mass spectrometry. Spectra were interrogated and quantified using ProteinProspector. Quantitative proteomics provided quantitative data for 94 and 97 proteins in the two groups. Significant changes in ATII protein content included 30% to 40% increases in adenosine triphosphate synthases, adenosine triphosphate/adenosine diphosphate translocase, and catalase (all p < .001). Following liver ischemia-reperfusion, there was also a significant increase in the percentage of neutrophils in bronchoalveolar lavage (48% ± 26%) compared with sham-operated controls (5% ± 3%) (p < .01), and plasma tumor necrosis factor-α levels were also significantly increased.
The proteins identified by quantitative proteomics indicated significant changes in moderators of cell metabolism and host defense in ATII. These findings provide new insights into possible mechanisms responsible for hepatic ischemia-reperfusion-related acute lung injury and suggest that ATII cells in the lung sense and respond to hepatic injury.
liver; lung; quantitative proteomics; isotope ratio mass spectrometry; ischemia; reperfusion injury; Zucker rats; iTRAQ
We have previously shown that treatment of steatotic livers with vitamin E succinate decreases liver injury and increases survival after ischemia/reperfusion (I/R). It is now understood that compromised energy status is associated with increased injury following liver ischemia in the setting of hepatic steatosis at least partially as a result of increased reactive oxygen species (ROS) and induction of mitochondrial uncoupling protein-2 (UCP2). Given the association between ROS, mitochondrial function, and UCP2, it was our goal to determine whether the protective effects of vitamin E succinate were associated with decreased ROS injury, down-regulation of UCP2, or improvement of ATP levels following I/R. To test this, leptin deficient (ob/ob) mice with steatotic livers that had received other 50 IU of vitamin E succinate supplement per day or control chow for 7 days were subjected to total hepatic ischemia (15 minutes) followed by reperfusion. We measured liver expressions of ATP, glutathione (GSH), and UCP2 as well as mitochondrial DNA damage. Vitamin E treatment decreased hepatic UCP2 expression and increased ATP and GSH levels prior to I/R. These levels were maintained at 1 hour after I/R. At 24 hours, while hepatic UCP2 expression, ATP, and GSH levels were similar to those of mice not receiving vitamin E, mitochondrial DNA damage was blocked. These results revealed that vitamin E succinate decreased hepatic UCP2 expression, reduced oxidative stress, and improved mitochondrial function in mice with steatotic livers before and after I/R, identifying mechanisms of protection in this setting.
Although recent studies indicate that renal ischemic preconditioning (IPC) protects the kidney from ischemia-reperfusion (I/R) injury, the precise protective mechanism remains unclear. In the current study, we investigated whether early IPC could upregulate hypoxia inducible transcription factor-1α (HIF-1α) expression and could reduce endoplasmic reticulum (ER) stress after renal I/R and whether pharmacological inhibition of nitric oxide (NO) production would abolish these protective effects.
Kidneys of Wistar rats were subjected to 60 min of warm ischemia followed by 120 min of reperfusion (I/R group), or to 2 preceding cycles of 5 min ischemia and 5 min reperfusion (IPC group), or to intravenously injection of NG-nitro-L-arginine methylester (L-NAME, 5 mg/kg) 5 min before IPC (L-NAME+IPC group). The results of these experimental groups were compared to those of a sham-operated group. Sodium reabsorption rate, creatinine clearance, plasma lactate dehydrogenase (LDH) activity, tissues concentrations of malonedialdehyde (MDA), HIF-1α and nitrite/nitrate were determined. In addition, Western blot analyses were performed to identify the amounts of Akt, endothelial nitric oxide synthase (eNOS) and ER stress parameters.
IPC decreased cytolysis, lipid peroxidation and improved renal function. Parallely, IPC enhanced Akt phosphorylation, eNOS, nitrite/nitrate and HIF-1α levels as compared to I/R group. Moreover, our results showed that IPC increased the relative amounts of glucose-regulated protein 78 (GRP78) and decreased those of RNA activated protein kinase (PKR)-like ER kinase (PERK), activating transcription factor 4 (ATF4) and TNF-receptor-associated factor 2 (TRAF2) as judged to I/R group. However, pre treatment with L-NAME abolished these beneficial effects of IPC against renal I/R insults.
These findings suggest that early IPC protects kidney against renal I/R injury via reducing oxidative and ER stresses. These effects are associated with phosphorylation of Akt, eNOS activation and NO production contributing thus to HIF-1α stabilization. The beneficial impact of IPC was abolished when NO production is inhibited before IPC application.
kidney; ischemia-reperfusion; ischemic preconditioning; Akt; eNOS, HIF1-α; ER stress
Endoplasmic reticulum (ER) and mitochondria have been implicated in the pathology of renal ischemia/reperfusion (I/R). In the present study, we investigated whether the use of ischemic postconditioning (IPostC) and trimetazidine (TMZ) separately or combined could reduce ER stress and mitochondria damage after renal ischemia.
Kidneys of Wistar rats were subjected to 60-min of warm ischemia followed by 120-min of reperfusion (I/R group, n = 6), or to 6 cycles of ischemia/reperfusion (10-s each cycle) just after 60-min of warm ischemia (IPostC group, n = 6), or to i.p. injection of TMZ (3 mg/kg) 30-min before ischemia (TMZ group, n = 6), or to the combination of both treatments (IPostC+TMZ group, n = 6). The results of these experimental groups were compared to those of a sham-operated group in which rat renal pedicles were only dissected. Sodium reabsorption rate, creatinine clearance lactate deshydrogenase (LDH) activity in plasma, and concentration of malonedialdehyde (MDA) in tissue were determined. In addition, Western blot analysis was performed to identify the amounts of cytochrome c, c-JunNH2-terminal kinase (JNK), voltage-dependent anion channel (VDAC), glycogen synthase kinase 3-beta (GSK3-β), and ER stress parameters.
IPostC or/and TMZ significantly decreased cytolysis, oxidative stress and improved renal function in comparison to I/R group. IPostC but not TMZ significantly attenuated ER stress parameters versus I/R group. Indeed, it down-regulated the glucose-regulated protein 78 (GRP78), the activating transcription factor 4 (ATF4), the RNA activated protein kinase (PKR)-like ER kinas (PERK), the X box binding protein-1 (XBP-1) and the caspase12 protein levels. TMZ treatment significantly augmented GSK3-β phosphorylation and reduced levels of cytochrome c and VDAC phosphorylation in comparison to IPostC application. The combination of both treatments gave a synergetic effect. It significantly improved the survival rate, attenuated cytolysis, oxidative stress and improved renal function.
This study revealed that IPostC protects kidney from I/R injury by suppressing ER stress while the beneficial effects of TMZ are mediated by mitochondria protection. The combination of both treatments ameliorated functional recovery.
Kidney; Ischemia-reperfusion; Ischemic postconditioning; Trimetazidine; Endoplasmic reticulum stress; Mitochondria
Whether ischemic preconditioning (IP) reduces ischemia/reperfusion (I/R) injury in human normal and fatty livers remains controversial. We compared two independent groups of liver donor transplants with versus without steatosis to evaluate IP consequences. Liver donors with (n=22) or without (n=28) steatosis either did or did not undergo IP before graft retrieval. Clinical data from the recipients, as well as histological and immunohistological characteristics of post-reperfusion biopsies were analyzed. Incidence of post-reperfusion necrosis was increased (10/10 versus 9/14, respectively; P<0.05) and the clinical outcome of recipients was worse for non-IP steatotic liver grafts compared with non-IP non-steatotic grafts. IP significantly lowered the transaminase values only in patients receiving a non-steatotic liver. An increased expression of beclin-1 and LC3, two pro-autophagic proteins, tended to decrease the incidence of necrosis (P=0.067) in IP steatotic livers compared with non-IP steatotic group. IP decreased the incidence of acute and chronic rejection episodes in steatotic livers (2/12 versus 6/10; P=0.07 and 2/12 versus 7/10; P<0.05, respectively), but not in non-steatotic livers. Thus, IP may induce autophagy in human steatotic liver grafts and reduce rejection in their recipients.
steatosis; liver transplantation; ischemia/reperfusion; ischemic preconditioning; autophagy; liver
The 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.
A 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).
Fat 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.
AIM: To investigate the effect of different secondary warm ischemia time (SWIT) on bile duct injury in liver-transplanted rats.
METHODS: Forty-eight male inbred Sprague-Dawley rats were randomly assigned into four groups: a sham-operation group and three groups with secondary biliary warm ischemia time of 0 min, 10 min and 20 min. A rat model of autologous liver transplantation under ether anesthesia was established, and six rats were killed in each group and blood samples and the median lobe of the liver were collected for assay at 6 h and 24 h after hepatic arterial reperfusion.
RESULTS: With prolongation of biliary warm ischemia time, the level of vascular endothelial growth factor-A was significantly decreased, and the value at 24 h was higher than that at 6 h after hepatic arterial reperfusion, but with no significant difference. The extended biliary SWIT led to a significant increase in bile duct epithelial cell apoptosis, and a decrease in the number of blood vessels, the bile duct surrounding the blood vessels and bile duct epithelial cell proliferation in the early postoperative portal area. Pathologic examinations showed that inflammation of the rat portal area was aggravated, and biliary epithelial cell injury was significantly worsened.
CONCLUSION: A prolonged biliary warm ischemia time results in aggravated injury of the bile duct and the surrounding vascular plexus in rat autologous orthotopic liver transplantation.
Bile duct; Liver; Transplantation; Warm ischemia; Rat
The role of uncoupling protein-2 (UCP2) in the liver is currently unclear. Emerging evidence suggests a relationship between UCP2 and oxidative stress. In the present study, we tested the hypothesis that UCP2 expression in the liver might change during warm ischemia-reperfusion (I/R) according to oxidative stress.
Wistar rats were subjected to 40 (short ischemia) or 90 (long ischemia) minutes of partial lobar ischemia followed by 4 hours of reperfusion. UCP2 expression in the ischemic and nonischemic lobes was assessed using reverse transcription-polymerase chain reaction and immunohistochemistry. Malondialdehyde concentrations in the liver tissue were also compared.
Malondialdehyde concentrations in the ischemic lobes were significantly higher in the long ischemia group. In the ischemic lobes of the short ischemia group, UCP2 protein expression was induced in hepatocytes, which did not express the protein prior to treatment, and the expression levels were higher than in the long ischemia group. The intralobular distribution of UCP2 seemed to correlate inversely with that of the necrotic area. UCP2 expression was observed, even in nonischemic lobes with similar intralobular heterogeneity.
UCP2 was induced in hepatocytes after warm I/R. Although the primitive role of UCP2 expression may be cytoprotective in nature, its actual protective effect in hepatic I/R may be minimal
Oxidative stress; Ischemia-reperfusion; Uncoupling protein; Liver; Surgery
Steatotic donors are routinely rejected for transplantation because of their increased rate of primary nonfunction. These grafts are more sensitive to ischemia/reperfusion (I/R) during transplantation. Removal of endotoxin before reperfusion improves liver performance post-I/R. We hypothesize that the main modality of injury in steatotic livers is toll-like receptor 4 (TLR4) signaling. We fed 4-week-old control and TLR4-deficient (TLR4KO) mice a normal diet (ND) or a 60% high-fat diet (HFD) for 4 weeks to induce steatosis. Mice were subjected to total hepatic ischemia (35 minutes) and reperfusion (1 or 24 hours). Survival improved and liver pathology decreased at 24 hours in TLR4KO HFD animals compared to control HFD animals. An investigation of infiltrates showed that neutrophils and CD4+ cells were increased at 24 hours in control HFD animals, whereas TLR4KO HFD animals were similar to ND controls. Messenger RNA levels of interleukin 6 (IL-6), IL-12, and interferon gamma were elevated at 1 hour in control HFD animals, whereas TLR4KO HFD animals were similar to ND controls. IL-10 levels at 1 hour of reperfusion in control HFD and TLR4KO animals were decreased versus control ND animals. In conclusion, these improvements in liver function in TLR4KO HFD animals implicate TLR4 as a mediator of steatotic graft failure after I/R.
Warm ischemia-reperfusion injury remains a crucial issue in transplantation following the cardiac death of donors. Previously, we showed that surfactant inhalation during warm ischemia mitigated ischemia-reperfusion injury. This study investigated the mechanisms of surfactant inhalation protection of the warm ischemic lung after reoxygenation with ventilation alone. In an isolated rat lung ventilation model, cardiac arrest was induced in the CTRL (control) and SURF (surfactant treatment) groups by ventricular fibrillation. Ventilation was restarted 110 min later; the lungs were flushed, and a heart and lung block was procured. In the SURF group, a natural bovine surfactant (Surfacten®) was inhaled for 3 min at the end of warm ischemia. In the Sham (no ischemia) group, lungs were flushed, procured, and ventilated in the same way. Afterwards, the lungs were ventilated with room air without reperfusion for 60 min. Surfactant inhalation significantly improved dynamic compliance and airway resistance. Moreover, surfactant inhalation significantly decreased inducible nitric oxide synthase and caspase-3 transcript levels, and increased those of Bcl-2 and surfactant protein-C. Immunohistochemically, lungs in the SURF group showed weaker staining for 8-hydroxy-2′-deoxyguanosine, inducible nitric oxide synthase, and apoptosis, and stronger staining for Bcl-2 and surfactant protein-C. Our results indicate that surfactant inhalation in the last phase of warm ischemia mitigated the injury resulting from reoxygenation after warm ischemia. The reduction in oxidative damage and the inhibition of apoptosis might contribute to the protection of the warm ischemic lungs.
Background: In hepatic ischaemia/reperfusion injury, activated liver macrophages (Kupffer cells) are dominantly regulated by a transcription factor, nuclear factor κB (NFκB), with respect to expression of inflammatory cytokines, acute phase response proteins, and cell adhesion molecules.
Aims: We assessed whether inactivation of NFκB in the liver could attenuate total hepatic warm ischaemia/reperfusion injury.
Methods: We studied rats with hepatic overexpression of inhibitor κBα super-repressor (IκBα SR) caused by a transgene introduced using an adenoviral vector. Hepatic ischaemia/reperfusion injury was induced under warm conditions by total occlusion of hepatoduodenal ligament structures for 20 minutes, followed by reperfusion. Controls included uninfected and control virus (AdLacZ) infected rats.
Results: IκBα SR was overexpressed in Kupffer cells as well as in hepatocytes, blocking nuclear translocation of NFκB (p65) into the nucleus after reperfusion. Gene transfection with IκBα SR, but not with LacZ, markedly attenuated ischaemia/reperfusion injury, suppressing inducible nitric oxide synthase and nitrotyrosine expression in the liver. Moreover, no remarkable hepatocyte apoptosis was detected under IκBα SR overexpression.
Conclusions: Adenoviral transfer of the IκBα SR gene in the liver ameliorates short term warm ischaemia/reperfusion injury, possibly through attenuation of hepatic macrophage activation.
Kupffer cells; rats; ischaemia/reperfusion injury; nuclear factor κB; repressor gene transfection
Beside lung transplantation, cardiopulmonary bypass, isolated lung perfusion and sleeve resection result in serious pulmonary ischemia–reperfusion injury, clinically known as acute respiratory distress syndrome. Very little is known about cells infiltrating the lung during ischemia–reperfusion. Therefore, a model of warm ischemia–reperfusion injury was applied to differentiate cellular infiltrates and to quantify tissue damage.
Fifty rats were randomized into eight groups. Five groups underwent warm ischemia for 60 min followed by 30 min and 1–4 hours of warm reperfusion. An additional group was flushed with the use of isolated lung perfusion after 4 hours of reperfusion. One of two sham groups was also flushed. Neutrophils and oedema were investigated by using samples processed with hematoxylin/eosin stain at a magnification of ×500. Immunohistochemistry with antibody ED-1 (magnification ×250) and antibody 1F4 (magnification ×400) was applied to visualize macrophages and T cells. TdT-mediated dUTP nick end labelling was used for detecting apoptosis. Statistical significance was accepted at P < 0.05.
Neutrophils were increased after 30 min until 4 hours of reperfusion as well as after flushing. A doubling in number of macrophages and a fourfold increase in T cells were observed after 30 min until 1 and 2 hours of reperfusion, respectively. Apoptosis with significant oedema in the absence of necrosis was seen after 30 min to 4 hours of reperfusion.
After warm ischemia–reperfusion a significant increase in infiltration of neutrophils, T cells and macrophages was observed. This study showed apoptosis with serious oedema in the absence of necrosis after all periods of reperfusion.
acute lung injury; acute respiratory distress syndrome; neutrophils; T cells; warm pulmonary ischemia–reperfusion injury
Fatty liver or hepatic steatosis is a common health problem associated with abnormal liver function and increased susceptibility to ischemia/reperfusion injury. The objective of this study was to investigate the effect of the fatty acid synthase inhibitor cerulenin on hepatic function in steatotic ob/ob mice. Different dosages of cerulenin were administered intraperitoneally to ob/ob mice for 2 to 7 days. Body weight, serum AST/ALT, hepatic energy state, and gene expression patterns in ob/ob mice were examined. We found that cerulenin treatment markedly improved hepatic function in ob/ob mice. Serum AST/ALT levels were significantly decreased and hepatic ATP levels increased in treated obese mice compared to obese controls, accompanied by fat depletion in the hepatocyte. Expression of peroxisome proliferator-activated receptors α and γ and uncoupling protein 2 were suppressed with cerulenin treatment and paralleled changes in AST/ALT levels. Hepatic glutathione content were increased in some cases and apoptotic activity in the steatotic livers was minimally changed with cerulenin treatment. In conclusion, these results demonstrate that fatty acid synthase blockade constitutes a novel therapeutic strategy for altering hepatic steatosis at non-stressed states in obese livers.
The anti-malaria drug chloroquine is well known as autophagy inhibitor. Chloroquine has also been used as anti-inflammatory drugs to treat inflammatory diseases. We hypothesized that chloroquine could have a dual effect in liver ischemia/reperfusion (I/R) injury: chloroquine on the one hand could protect the liver against I/R injury via inhibition of inflammatory response, but on the other hand could aggravate liver I/R injury through inhibition of autophagy. Rats (n=6 per group) were pre-treated with chloroquine (60 mg/kg, i.p.) 1 h before warm ischemia, and they were continuously subjected to a daily chloroquine injection for up to 2 days. Rats were killed 0.5, 6, 24 and 48 h after reperfusion. At the early phase (i.e., 0–6 h after reperfusion), chloroquine treatment ameliorated liver I/R injury, as indicated by lower serum aminotransferase levels, lower hepatic inflammatory cytokines and fewer histopathologic changes. In contrast, chloroquine worsened liver injury at the late phase of reperfusion (i.e., 24–48 h after reperfusion). The mechanism of protective action of chloroquine appeared to involve its ability to modulate mitogen-activated protein kinase activation, reduce high-mobility group box 1 release and inflammatory cytokines production, whereas chloroquine worsened liver injury via inhibition of autophagy and induction of hepatic apoptosis at the late phase. In conclusion, chloroquine prevents ischemic liver damage at the early phase, but aggravates liver damage at the late phase in liver I/R injury. This dual role of chloroquine should be considered when using chloroquine as an inhibitor of inflammation or autophagy in I/R injury.
chloroquine; liver I/R injury; autophagy; apoptosis; HMGB1