Effects of Quercetin on Gene and Protein Expression of NOX and NOS after Myocardial Ischemia and Reperfusion in Rabbit

Previous studies have suggested that reactive oxygen species (ROS), endothelial nitricoxide synthase (eNOS), and inducible nitric oxide synthase (iNOS) are involved in the pathophysiology of myocardial ischemia‐reperfusion injury (MIRI). The NOX family of NADPH oxidases share the capacity to generate superoxide and ROS. Several studies have demonstrated that quercetin possesses a protective effect against MIRI. Our aim is to investigate the effects of quercetin on NOX2, eNOS, and iNOS after MIRI in rabbits. New Zealand rabbits were subjected to 30 min of myocardial ischemia followed by 12 h of reperfusion. They were then randomly assigned to four experimental groups: control, I/R (ischemia/reperfusion), quercetin (Que), I/R + Que. Gene and protein expression of NOX2, eNOS, and iNOS were compared. Both in real‐time PCR and in the Western blotting studies, myocardial ischemia‐reperfusion‐induced NOX2 and iNOS expression were enhanced (P < 0.01) but eNOS mRNA and protein expression in I/R hearts were not significantly different from those in control (P < 0.01). Administration of quercetin reduced NOX2, eNOS, and iNOS mRNA and protein expression both in control and in I/R heart (P < 0.01). Gene and protein expression of NOX2 and iNOS were increased after MIRI. Quercetin not only inhibited myocardial ischemia‐reperfusion‐induced NOX2 and iNOS mRNA and protein expression but also inhibited eNOS mRNA and protein expression.     

Fundamental Efforts toward the Development of a Therapeutic Cocktail with a Manifold Ameliorative Effect on Hepatic Ischemia/Reperfusion Injury

Objective: Ischemia/reperfusion injury is mediated by various mechanisms. The present study was designed to evaluate the effect of a multiple pharmacological approach toward ischemia/reperfusion injury reduction. Methods : The left liver lobe of Sprague‐Dawley rats underwent normothermic ischemia for 90 minutes after a cocktail (glycine, taurine, alanine, arginine, and prednisolon) intravenous administration. Controls received normal saline. Liver injury (transaminases, histology) and cellular activation [Kupffer cell phagocytosis, production of tumor necrosis factor‐alpha (TNFα), and prostaglandin E‐2 (PGE₂)], as well as microcirculation and leukocyte‐endothelial interaction (in vivo microscopy), were assessed. Results : Whereas in controls a substantial increase of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase to 2,341±477, 1,442±262, and 1,973±73 U/L, respectively, was measured eight hours after reperfusion, the cocktail significantly reduced the increase of enzymes by 33–80% (P<0.05). Further, necrosis, index of liver damage, and index of leukocyte infiltration significantly decreased after the cocktail (P<0.05). Moreover, the cocktail improved acinar and sinusoidal perfusion, while the sinusoidal diameters, leukocyte‐endothelial interaction, Kupffer cell phagocytic activity, and TNFα/PGE₂ serum levels were significantly reduced, the latter by 86% and to 1.64‐fold, respectively. Conclusions : This study depicts that multifaceted pharmacological tackling of ischemia/reperfusion injury is feasible and protects rat liver tissue from warm ischemia/reperfusion injury.     

Deletion of macrophage migration inhibitory factor protects the heart from severe ischemia-reperfusion injury: a predominant role of anti-inflammation

Inflammation plays an important role in mediating and exacerbating myocardial ischemia–reperfusion (I/R) injury. Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, facilitates inflammation and modulates metabolism. However, the role of MIF in mediating local inflammation subsequent to ischemic myocardial injury has not been established. We hypothesized that genetic deletion of MIF protects the heart against severe I/R injury by suppressing inflammation and/or modulating energy metabolism. We showed in the mouse I/R model that duration of both ischemia and reperfusion is a determinant for the degree of regional inflammation and tissue damage. Following a prolonged cardiac I/R (60 min/24 h) MIF KO mice had a significant reduction in both infarct size (26 ± 3% vs. 45 ± 4%, P < 0.05) and cardiomyocyte apoptosis (1.4 ± 0.2% vs. 5.4 ± 0.4%, P < 0.05) and preserved contractile function compared with WT. MIF KO mice with I/R had reduced expression of various inflammatory cytokines and mediators (P < 0.05), suppressed infiltration of neutrophils (− 40%) and macrophages (− 33%, both P < 0.05), and increased macrophage apoptosis (14.4 ± 1.4% vs. 5.2 ± 0.6%, P < 0.05). Expression of toll-like receptor-4 (TLR-4), phosphorylation of c-Jun N-terminal kinase (JNK), and nuclear fraction of NF-κB p65 were also significantly lower in MIF KO hearts with I/R. Further, MIF KO mice exhibited a lower glucose uptake but higher fatty acid oxidation rate than that in WT (both P < 0.05). In conclusion, MIF deficiency protected the heart from prolonged/severe I/R injury by suppressing inflammatory responses. We have identified a critical role of MIF in mediating severe I/R injury. Thus, MIF inhibitory therapy may be a novel strategy to protect the heart against severe I/R injury.     

Effect of adrenomedullin on hepatic damage in hepatic ischaemia/reperfusion injury in rats

Aims: Adrenomedullin (AM) is a multifunctional peptide with a putative beneficial role after an ischaemic insult. The aim of this study was to evaluate the effect of AM on partial hepatic ischaemia reperfusion (I/R) injury. Methods: Rats were subjected to 1 h of 70% hepatic ischaemia, followed by reperfusion or sham. At the end of ischaemia, vehicle (phosphate‐buffered saline solution), N‐nitro‐l ‐arginine methyl ester (l ‐NAME) and AM with or without l ‐NAME were infused via the portal vein. Analysis was performed at pre‐ischaemia, ischaemia onset and 1, 2 and 4 h after reperfusion. Hepatic tissue blood flow (HTBF) was evaluated by laser Doppler. Results: Plasma AM levels in the I/R groups were significantly lower than the levels in the sham group. AM treatment significantly reduced levels of aspartate transaminase and tissue arginase (P<0.05). Significant decreases of tumour necrosis factor‐α, interleukin‐1β and endothelin‐1 levels were also found in the serum. Endothelin‐1, malondialdehyde and necrosis were observed more frequently in liver tissue in the AM group than the control (P<0.05). Tissue nitric oxide, energy charge and HTBF were significantly increased in AM treatment experiments (P<0.05). Conclusion: The improved HTBF, energy charge and nitric oxide and the reduction of hepatic necrosis, oxidative stress, liver enzymes, endotelin‐1 and pro‐inflammatory cytokines demonstrate that treatment with AM attenuates liver I/R injury.     

Glycine maintains mitochondrial activity and bile composition following warm liver ischemia-reperfusion injury

Background and Aim: Experimental studies have shown protective effect by the non‐essential amino acid glycine to liver ischemia‐reperfusion (I/R) injury but the mechanism of action is unknown. Methods: A rabbit model of hepatic lobar I/R was used. Three groups of animals (n = 6) were studied: Sham group (laparotomy alone), ischemia reperfusion (I/R) group (1 h of liver lobar ischemia and 6 h of reperfusion), and a glycine I/R group (intravenous glycine 5 mg/kg prior to the I/R protocol). Systemic and hepatic hemodynamics, degree of liver injury (bile flow, transaminases), hepatic microcirculation, mitochondrial activity (redox state of cytochrome oxidase), bile composition and cytokines (tumor necrosis factor‐α and interleukin‐8) were measured during the experiment. Results: Glycine administration increased portal blood flow, bile production, hepatic microcirculation and maintained cytochrome oxidase activity as compared with the I/R group during reperfusion. Glycine also reduced bile lactate surge and stimulated acetoacetate release in bile during reperfusion versus the I/R group. Cytokine levels (tumor necrosis factor‐α, interleukin‐8) and hepatocellular injury (aspartate aminotransferase and alanine aminotransferase) were significantly reduced by glycine administration. Conclusion: Intravenous glycine administration reduces liver warm I/R injury by reducing the systemic inflammatory response, and maintaining cellular energy production.     

Dihydrolipoyl histidinate zinc complex, a new antioxidant, attenuates hepatic ischemia-reperfusion injury in rats

Background and Aims: Ischemia/reperfusion (I/R) injury is characterized by significant oxidative stress, which induces characteristic changes in the antioxidant system and organ injury leading to significant morbidity and mortality. The aim of this study was to evaluate the protective effect of dihydrolipoyl histidinate zinc complex (DHLHZn) on oxidative damage after severe hepatic I/R injury. Methods: Thirty male Wistar rats were subjected to 45 min of hepatic ischemia by clamping of the hepatic artery and portal vein, followed by a 6‐h reperfusion period. DHLHZn (10 mg/kg) (I/R + DHLHZn group) or saline (I/R group) was administered intraperitoneally twice, 30 min before ischemia and at the beginning of the reperfusion. Sham‐operated animals (sham group) received equal amounts of saline. The rats were killed at the end of the reperfusion period. Serum levels of aspartate aminotransferase and alanine aminotransferase were determined, and histological examination and oxidative stress were evaluated in liver tissues. In addition, antimycin A‐stimulated RAW264.7 cells (murine macrophage‐like cells) were treated with DHLHZn to estimate its antioxidant effect. Results: Serum aspartate aminotransferase and alanine aminotransferase levels were increased in the I/R group, but these increases were significantly inhibited in the I/R + DHLHZn group. Similarly, liver tissue damage observed in the I/R group was attenuated in the I/R + DHLHZn group. Cells treated in vitro with both DHLHZn and antimycin A showed reduced reactive oxygen species activity compared to cells treated with antimycin A alone. Conclusion: The new antioxidant DHLHZn may have potential for therapeutic application in liver I/R injury, although this is a limited animal study.     

Melatonin attenuates hepatic ischemia-reperfusion injury in rats by inhibiting NF-κB signaling pathway

BackgroundThe sterile inflammatory response is one of the key mechanisms leading to hepatic ischemia-reperfusion injury. Melatonin has been shown to prevent organ injuries, but its roles in the inflammatory response after hepatic ischemia-reperfusion injury have not been fully explored, especially in late ischemia-reperfusion injury. The present study aimed to investigate the roles and possible mechanisms of melatonin in the inflammatory response after hepatic ischemia-reperfusion injury.MethodsSixty Sprague-Dawley rats were randomly divided into a sham group, ischemia-reperfusion injury group (I/R group), and melatonin-treated group (M + I/R group). The rats in the I/R group were subjected to 70% hepatic ischemia for 45 min, followed by 5 or 24 h of reperfusion. The rats in the M + I/R group were injected with melatonin (10 mg/kg, intravenous injection) 15 min prior to ischemia and immediately before reperfusion. Serum and samples of ischemic liver lobes were harvested for future analysis, and the 7-day survival rate was assessed after hepatic ischemia-reperfusion surgery.ResultsIn comparison with the I/R group, the M + I/R group showed markedly decreased expression levels of inflammatory cytokines (IL-6 and TNF-α) and numbers of apoptotic hepatocytes (P < 0.05). Immunoblotting showed that the expression levels of IL-6, p-NF-κBp65/t-NF-κBp65 and p-IκB-α/t-IκB-α in the M + I/R group were significantly lower than those in the I/R group, and immunofluorescence staining showed that the expression level of p-NF-κBp65 in the M + I/R group was lower than that in the I/R group (P < 0.05). The 7-day survival rates were 20% in the I/R group and 50% in the M + I/R group (P < 0.05).ConclusionsMelatonin downregulated the activity of the NF-κB signaling pathway in the early and late stages of hepatic ischemia-reperfusion injury, alleviated the inflammatory response, protected the liver from ischemia-reperfusion injury, and increased the survival rate.     

Inhibition of nitric oxide synthesis increases apoptotic cardiomyocyte death and myocardial angiotensin-converting enzyme gene expression in ischemia/reperfusion-injured myocardium of rats

Cardiomyocyte apoptosis is an important pathogenic mechanism in myocardial ischemia/reperfusion (I/R) injury. It has been shown that nitric oxide (NO) and the renin–angiotensin system (RAS) are closely related, and both systems regulate apoptotic cell death. However, the effects of NO modulation on myocardial apoptotic cell death and changes in the RAS in the I/R-injured myocardium have not been studied. Female Sprague–Dawley rats were randomized into three groups: NO synthesis inhibitor, N^G-nitro-l-arginine-methyl ester (l-NAME, 10 mg/kg); NO precursor, l-arginine (540 mg/kg); and vehicle. The rats were then subjected to 45 min coronary occlusion followed by 4 h reperfusion. The TdT-mediated in situ nick and labeling (TUNEL) indices were 39.9% ± 0.8% at the border and 30.9% ± 1.2% at the center of the I/R area in the vehicle group. L-NAME administration significantly increased these TUNEL-positive cells to 45.3% ± 1.9% and 37.9% ± 1.3%, respectively (P < 0.05 each). L-arginine administration reduced the TUNEL index at the border zone with marginal significance (P = 0.08 vs vehicle group). I/R injury significantly reduced the angiotensin-converting enzyme (ACE) mRNA expression in the left (ventricular) free wall of vehicle group rats. However, ACE mRNA expression was 1.9 times greater in the L-NAME group than that in the vehicle group (P < 0.05). This study showed that the inhibition of NO synthesis increased apoptotic cardiomyocyte death and local ACE mRNA expression in the I/R-injured myocardium. Our observations indicate that NO, ACE, and apoptotic cardiomyocyte death are related to each other during I/R injury. Received: July 9, 2001 / Accepted: August 17, 2001     

Opioid system blockade decreases collagenase activity and improves liver injury in a rat model of cholestasis

BACKGROUND: Following bile duct ligation (BDL) endogenous opioids accumulate in plasma and play a role in the pathophysiology and manifestation of cholestasis. Evidence of centrally mediated induction of liver injury by exogenous opioid agonist administration, prompts the question of whether opioid receptor blockade by naltrexone can affect cholestasis-induced liver injury. METHODS: Cholestasis was induced by BDL and cholestatic and sham-operated rats received either naltrexone or saline for 7 consecutive days. On the 7th day, liver samples were collected for determining matrix metalloproteinase-2 (MMP-2) activity, S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) content and blood samples were obtained for measuring plasma nitrite/nitrate and liver enzyme activities. RESULTS: Naltrexone-treated BDL animals had a significant reduction in plasma enzyme activity and nitrite/nitrate level. Liver SAM : SAH ratio and SAM level improved by naltrexone treatment in cholestatic animals compared to saline-treated BDL ones. Naltrexone treatment in BDL rats led to a decrease in the level of liver MMP-2 activity, which had already increased during cholestasis. CONCLUSION: Opioid receptor blockade improved the degree of liver injury in cholestasis, as assessed by plasma enzyme and liver MMP-2 activities. The beneficial effect of naltrexone may be due to its ability to increase liver SAM level and restore the SAM : SAH ratio.     

Nuclear factor-kappaB decoy oligodeoxynucleotides attenuates ischemia/reperfusion injury in rat liver graft

AIM: To evaluate the protective effect of NF-kappaB decoy oligodeoxynucleotides (ODNs) on ischemia/reperfusion (I/R) injury in rat liver graft. METHODS: Orthotopic syngeneic rat liver transplantation was performed with 3 h of cold preservation of liver graft in University of Wisconsin solution containing phosphorothioated double-stranded NF-kappaB decoy ODNs or scrambled ODNs. NF-kappaB decoy ODNs or scrambled ODNs were injected intravenously into donor and recipient rats 6 and 1 h before operation, respectively. Recipients were killed 0 to 16 h after liver graft reperfusion. NF-kappaB activity in the liver graft was analyzed by electrophoretic mobility shift assay (EMSA). Hepatic mRNA expression of TNF-alpha, IFN-gamma and intercellular adhesion molecule-1 (ICAM-1) were determined by semiquantitative RT-PCR. Serum levels of TNF-alpha and IFN-gamma were measured by enzyme-linked immunosorbent assays (ELISA). Serum level of alanine transaminase (ALT) was measured using a diagnostic kit. Liver graft myeloperoxidase (MPO) content was assessed. RESULTS: NF-kappaB activation in liver graft was induced in a time-dependent manner, and NF-kappaB remained activated for 16 h after graft reperfusion. NF-kappaB activation in liver graft was significant at 2 to 8 h and slightly decreased at 16 h after graft reperfusion. Administration of NF-kappaB decoy ODNs significantly suppressed NF-kappaB activation as well as mRNA expression of TNF-alpha, IFN-gamma and ICAM-1 in the liver graft. The hepatic NF-kappaB DNA binding activity [presented as integral optical density (IOD) value] in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (2.16+/-0.78 vs 36.78+/-6.35 and 3.06+/-0.84 vs 47.62+/- 8.71 for IOD value after 4 and 8 h of reperfusion, respectively, P<0.001). The hepatic mRNA expression level of TNF-alpha, IFN-gamma and ICAM-1 [presented as percent of beta-actin mRNA (%)] in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (8.31+/-3.48 vs 46.37+/-10.65 and 7.46+/- 3.72 vs 74.82+/-12.25 for hepatic TNF-alpha mRNA, 5.58+/-2.16 vs 50.46+/-9.35 and 6.47+/-2.53 vs 69.72+/-13.41 for hepatic IFN-gamma mRNA, 6.79+/-2.83 vs 46.23+/-8.74 and 5.28+/-2.46 vs 67.44+/-10.12 for hepatic ICAM-1 mRNA expression after 4 and 8 h of reperfusion, respectively, P<0.001). Administration of NF-kappaB decoy ODNs almost completely abolished the increase of serum level of TNF-alpha and IFN-gamma induced by hepatic ischemia/reperfusion, the serum level (pg/mL) of TNF-alpha and IFN-gamma in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (42.7+/-13.6 vs 176.7+/-15.8 and 48.4+/-15.1 vs 216.8+/-17.6 for TNF-alpha level, 31.5+/-12.1 vs 102.1+/-14.5 and 40.2+/-13.5 vs 118.6+/-16.7 for IFN-gamma level after 4 and 8 h of reperfusion, respectively, P<0.001). Liver graft neutrophil recruitment indicated by MPO content and hepatocellular injury indicated by serum ALT level were significantly reduced by NF-kappaB decoy ODNs, the hepatic MPO content (A655) and serum ALT level (IU/L) in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (0.17+/-0.07 vs 1.12+/-0.25 and 0.46+/-0.17 vs 1.46+/-0.32 for hepatic MPO content, 71.7+/-33.2 vs 286.1+/-49.6 and 84.3+/-39.7 vs 467.8+/-62.3 for ALT level after 4 and 8 h of reperfusion, respectively, P<0.001). CONCLUSION: The data suggest that NF-kappaB decoy ODNs protects against I/R injury in liver graft by suppressing NF-kappaB activation and subsequent expression of proinflammatory mediators.     

Protection by Glycyrrhizin against Warm Ischemia‐Reperfusion–Induced Cellular Injury and Derangement of the Microcirculatory Blood Flow in the Rat Liver

Background/Aim: The mechanism by which ischemia‐reperfusion (I/R)‐induced derangement of the hepatic microcirculation leads to tissue injury is not fully understood. We postulated that alterations to the hepatic microcirculation, including hemodynamic derangement and increased leukocyte‐endothelium interaction, play a role, and that glycyrrhizin exerts its hepatoprotective effects, in part, by reducing these microcirculatory changes. Materials and Methods: Wistar rats were subjected to 30–60 minutes segmental hepatic ischemia, followed by 120 minutes of reperfusion. Glycyrrhizin was administered prior to ischemia. Using intravital fluorescence microscopy, the administration of fluorescein isothiocyanate–conjugated erythrocytes allowed the measurement of erythrocyte‐velocity (RBC_vel), lobular, and sinusoidal perfusion. Bleb formation was observed by electron microscopy. Blood and tissue were taken for the assessment of liver injury. Results: Glycyrrhizin reduced I/R‐induced liver injury (histology, liver enzymes) and reduced hepatocyte apoptosis (TUNEL, caspase‐3 activity). Glycyrrhizin inhibited hepatocyte bleb formation and reversed the I/R‐induced reductions in lobular perfusion and RBC_vel. Leukocyte rolling and adherence in postsinusoidal venules and neutrophil infiltration were reduced by glycyrrhizin. I/R‐induced elevation in HMGB1 was prevented by glycyrrhizin. Conclusions: Early bleb formation with deranged microcirculatory flow and leukocyte‐endothelium interaction would appear to contribute to I/R‐induced hepatocellular injury. Glycyrrhizin exerts its hepatoprotective effect by preventing these changes, in addition to a direct cellular effect.     

Dexrazoxane Prevents Myocardial Ischemia/Reperfusion-Induced Oxidative Stress in the Rat Heart

Introduction Dexrazoxane (Dex), used clinically to protect against anthracycline-induced cardiotoxicity, possesses iron-chelating properties. The present study was designed to examine whether Dex could inhibit the ischemia/reperfusion (I/R) induced damage to the rat heart. Materials and methods Isolated perfused rat hearts were exposed to global ischemia (37°C) and 60 min reperfusion. Dex was perfused for 10 min prior to the ischemia, or administered intraperitoneally (150 mg) 30 min prior to anesthesia of the rats. I/R caused a significant hemodynamic function decline in control hearts during the reperfusion (e.g., the work index LVDP X HR declined to 42.7 ± 10%). Dex (200 μM) applied during the preischemia significantly increased the hemodynamic recovery following reperfusion (LVDP X HR recovered to 55.7 ± 8.8%, p < 0.05 vs. control). Intraperitoneal Dex, too, significantly increased the hemodynamic recovery of the reperfused hearts. I/R caused an increase in oxidation of cytosolic proteins, while Dex decreased this oxidation. Discussion The decrease in proteins carbonylation and correlative hemodynamic improvement suggests that Dex decreases I/R free radical formation and reperfusion injury. Eyal Ramu and Amit Korach contributed equally to this study.     

缺血预处理对大鼠脑缺血再灌注损伤神经细胞的保护作用

目的探讨大鼠局灶性脑缺血预处理对脑缺血再灌注损伤后神经元的保护作用。方法健康雄性SD大鼠60只,随机分为3组:假手术组、大脑中动脉缺血再灌注(MCAO)组、预处理(BIP)组,每组按照再灌注后12 h、1、2、3 d四个时间点平均分为4个亚组,制备缺血预处理模型,分别用流式细胞术和ELISA法观察脑缺血预处理对缺血再灌注大鼠缺血半暗带神经细胞凋亡率及血清神经元特异性烯醇化酶(NSE)含量的影响。结果大鼠脑缺血再灌注后12 h,MCAO组细胞凋亡发生率及血清中NSE的含量较假手术组显著增加(P<0.01),1 d时达到高峰,以后时间点逐渐下降,但仍高于假手术组(P<0.01);BIP组各个时间点神经元凋亡发生率及血清NSE较MCAO组显著降低(P<0.05,P<0.01)。结论大鼠局灶性脑缺血预处理对脑缺血再灌注神经元损伤有保护作用。     

Endotoxin tolerance protects against local hepatic ischemia/reperfusion injury in the rat

Liver surgery and liver transplantation as well as circulatory shock are often associated with hepatic ischemia/reperfusion (I/R) injury. Recent evidence suggests that TNF-alpha plays a central role in I/R injury and, therefore, down-regulation of TNF-alpha seems to be a promising way to protect against the deleterious consequences of I/R. Endotoxin tolerance represents a state of unresponsiveness to endotoxin and is associated with diminished TNF-alpha production. Thus, the effect of endotoxin tolerance on hepatic I/R injury of the liver was investigated in a rat model. I/R injury was induced by temporary ischemia of the left lateral liver lobe for 90 min followed by a 3 h observation period of reperfusion. I/R injury resulted in functional hepatic disorder characterized by a decrease both in bile flow and bile acid concentration and 50% mortality. This was prevented by induction of endotoxin tolerance. Hepatic TNF-alpha mRNA expression after I/R of the liver was determined by RT-PCR. In untreated rats, TNF-alpha mRNA was induced in the liver 60 min after reperfusion and further increased until 3 h after reperfusion. In contrast, in endotoxin-tolerant rats, no increases in TNF-alpha mRNA expression were detected. This suggests that induction of endotoxin tolerance protects against hepatic I/R injury possibly via down-regulation of intra-organ TNF-alpha expression.     

Melatonin pretreatment enhances the therapeutic effects of exogenous mitochondria against hepatic ischemia–reperfusion injury in rats through suppression of mitochondrial permeability transition

We tested the hypothesis that melatonin (Mel) enhances exogenous mitochondria (Mito) treatment against rodent hepatic ischemia–reperfusion (IR) injury. In vitro study utilized three groups of hepatocytes (i.e. nontreatment, menadione, and menadione–melatonin treatment, 4.0 × 10⁵ each), while in vivo study used adult male Sprague Dawley rats (n = 40) equally divided into sham‐control (SC), IR (60‐min left‐lobe ischemia + 72‐hr reperfusion), IR‐Mel (melatonin at 30 min/6/8 hr after reperfusion), IR‐Mito (mitochondria 15,000 μg/rat 30 min after reperfusion), and IR‐Mel‐Mito. Following menadione treatment in vitro, oxidative stress (NOX‐1/NOX‐2/oxidized protein), apoptotic (cleaved caspase‐3/PARP), DNA damage (γ‐H2AX/CD90/XRCC1), mitochondria damage (cytosolic cytochrome c) biomarkers, and mitochondrial permeability transition were found to be lower, whereas mitochondrial cytochrome c were found to be higher in hepatocytes with melatonin treatment compared to those without (all P < 0.001). In vivo study demonstrated highest liver injury score and serum AST in IR group, but lowest in SC group and higher in IR‐Mito group than that in groups IR‐Mel and IR‐Mel‐Mito, and higher in IR‐Mel group than that in IR‐Mel‐Mito group after 72‐hr reperfusion (all P < 0.003). Protein expressions of inflammatory (TNF‐α/NF‐κB/IL‐1β/MMP‐9), oxidative stress (NOX‐1/NOX‐2/oxidized protein), apoptotic (caspase‐3/PARP/Bax), and mitochondria damage (cytosolic cytochrome c) biomarkers displayed an identical pattern, whereas mitochondria integrity marker (mitochondrial cytochrome c) showed an opposite pattern compared to that of liver injury score (all P < 0.001) among five groups. Microscopically, expressions of apoptotic nuclei, inflammatory (MPO ⁺ /CD68 ⁺ /CD14 ⁺ cells), and DNA damage (γ‐H2AX ⁺ cells) biomarkers exhibited an identical pattern compared to that of liver injury score (all P < 0.001) among five groups. Melatonin‐supported mitochondria treatment offered an additional benefit of alleviating hepatic IR injury.     

Gender differences in hepatic ischemic reperfusion injury in rats are associated with endothelial cell nitric oxide synthase-derived nitric oxide

AIM: This study was designed to examine the hypothesis that gender differences in I/R injury are associated with endothelial cell nitric oxide synthase (eNOS)-derived nitric oxide (NO). METHODS: Wistar rats were randomized into seven experimental groups (12 animals per group). Except for the sham operated groups, all rats were subjected to total liver ischemia for 40 min followed by reperfusion. All experimental groups received different treatments 45 min before the laparotomy. For each group, half of the animals (six) were used to investigate the survival; blood samples and liver tissues were obtained in the remaining six animals after 3 h of reperfusion to assess serum NO, alanine aminotransferase (ALT) and TNF-α levels, liver tissue malondialdehyde (MDA) content, and severity of hepatic I/R injury. RESULTS: Basal serum NO levels in female sham operated (FS) group were nearly 1.5-fold of male sham operated (MS) group (66.7±11.0 μmol/L vs45.3μ10.1 μmol/L, P<0.01). Although serum NO levels decreased significantly after hepatic I/R (P<0.01, vs sham operated groups), they were still significantly higher in female rat (F) group than in male rat (M) group (47.8±8.6 μmol/L vs 23.8±4.7 μmol/L, P<0.01). Serum ALT and TNF-α levels, and liver tissue MDA content were significantly lower in F group than in M group (370.5±46.4 U/L, 0.99±0.11 μg/L and 0.57±0.10 μmol/g vs668.7±78.7 U/L, 1.71±0.18μg/L and 0.86±0.11 μmol/g, respectively, P<0.01). I/R induced significant injury to the liver both in M and F groups (P<0.01 vs sham operated groups). But the degree of hepatocyte injury was significantly milder in F group than in M group (P<0.05 and P<0.01). The median survival time was six days in F group and one day in M group. The overall survival rate was significantly higher in F group than in M group (P<0.05). When compared with male rats pretreated with saline (M group), pretreatment of male rats with 17-β-estradiol (E2) (M+E2 group) significantly increased serum NO levels and significantly decreased serum ALT and TNF-α levels, and liver tissue MDA content after I/R (P<0.01). The degree of hepatocyte injury was significantly decreased and the overall survival rate was significantly improved in M+E2 group than in M group (P<0.01 and P<0.05). The NOS inhibitor Nw-nitro-L-arginine methyl ester (L-NAME) treatment could completely abolish the protective effects of estrogen in both male and female rats. CONCLUSION: The protective effects afforded to female rats subjected to hepatic I/R are associated with eNOS-derived NO.     

Bucillamine improves hepatic microcirculation and reduces hepatocellular injury after liver warm ischaemia-reperfusion injury

Background:

Liver transplantation and resection surgery involve a period of ischaemia and reperfusion to the liver which initiates an inflammatory cascade resulting in liver and remote organ injury. Bucillamine is a low-molecular-weight thiol antioxidant that is capable of rapidly entering cells.

Methods:

The effect of bucillamine was studied in a rat model of liver ischaemia–reperfusion injury with 45 min of partial (70%) liver ischaemia and at 3 and 24 h of reperfusion. Controls included ischaemia-reperfusion (I/R) only, sham and bucillamine alone (without ischaemia reperfusion). Liver injury was assessed by serum transaminases (AST and ALT). Sinusoidal blood flow and hepatocyte apoptosis were measured using intravital microscopy (IVM).

Results:

The hepatocellular injury of I/R produced a markedly elevated serum AST which was reduced with bucillamine (2072.5 ± 511.79 vs. 932 ± 200.8, P < 0.05) at 3 h reperfusion. Bucillamine treatment with I/R also increased parenchymal blood flow [red blood cell (RBC) velocity 242.66 ± 16.86 vs. 181.11 ± 17.59, at the end of 3 h of reperfusion) and reduced hepatocyte necrosis/apoptosis at 3 h as well as 24 h (P > 0.001).

Conclusion:

Bucillamine reduces the hepatocellular injury of liver ischaemia reperfusion and improves parenchymal perfusion.     

Amelioration of hepatic ischemia/reperfusion injury in the remnant liver after partial hepatectomy in rats

BACKGROUND AND AIMS: Reactive oxygen species have been implicated in the development of hepatic ischemia/reperfusion (I/R) injury. I/R injury remains an important problem in massive hepatectomy and organ transplantation. The aim of this study was to examine the effect of edaravone, a newly synthesized free radical scavenger, on I/R injury in the remnant liver after partial hepatectomy in rats. METHODS: Partial (70%) hepatic ischemia was induced in rats by occluding the hepatic artery, portal vein, and bile duct to left and median lobes of liver. Total hepatic ischemia (Pringle maneuver) was induced by occluding the hepatoduodenal ligament. Edaravone was intravenously administered to rats just before reperfusion and partial (70%) hepatectomy was performed just after reperfusion. RESULTS: Edaravone significantly reduced the increases in the levels of serum alanine aminotransferase and aspartate aminotransferase in rats with liver injury induced by 90-min of partial ischemia followed by 120-min of reperfusion. Histopathological analysis showed that edaravone prevented inflammatory changes in the livers with I/R injury. Edaravone also decreased the levels of myeloperoxidase activity, which is an index of neutrophil infiltration, and interleukin-6 mRNA, which is a proinflammatory cytokine. Additionally, edaravone improved the survival rate in partial hepatectomy rats with I/R injury induced by the Pringle maneuver. CONCLUSIONS: Edaravone administration prior to reperfusion protected the liver against I/R injury. Edaravone also improved the function of the remnant liver with I/R injury after partial hepatectomy. Therefore, edaravone may have applicability for major hepatectomy and liver transplantation in the clinical setting.     

Antiapoptotic and protective effects of roscovitine on ischemia–reperfusion injury of the rat liver

Objective: This study was designed to investigate the role of apoptosis on liver IR injury and to determine the effects of roscovitine on this process. Materials and methods: Rat livers pretreated with roscovitine received 60 min right lobe ischemia followed by 4 h (n=8) and 24 h (n=8) reperfusion. Tissue injury was evaluated by serum alanine aminotransferase, aspartate aminotransferase, tissue malondialdehyde, myeloperoxidase measurements and histological examination. Dead (apoptotic and necrotic) hepatocytes were determined by trypan blue dye and apoptosis was evaluated with M30 monoclonal antibody. Results: Administration of roscovitine significantly decreased both apoptotic and dead hepatocyte counts compared to controls (P<0.01). Liver transaminase levels were decreased significantly in the roscovitine‐pretreated groups (P<0.05). In the groups evaluated 24 h after reperfusion, MPO levels (P<0.02) and leukocyte infiltration on histologic sections were decreased significantly in the roscovitine‐administered group compared to its control. Conclusions: This study indicates that hepatocyte apoptosis may play a role in the development of IR injury of the liver. Administration of roscovitine may be beneficial in preventing this injury.