Mild hypothermia protects liver against ischemia and reperfusion injury

AIM: To determine whether mild hypothermia could protect liver against ischemia and reperfusion injury in pigs. METHODS: Twenty-four healthy pigs were randomly divided into normothermia, mild hypothermia and normal control groups. The experimental procedure consisted of temporary interruption of blood flow to total hepatic lobe for different lengths of time and subsequent reperfusion. Hepatic tissue oxygen pressure (PtiO2) and aspartate aminotransferase (AST) values were evaluated, and ultrastructural analysis was carried out for all samples. RESULTS: Serum AST was significantly lower, and hepatic PtiO2 values were significantly higher in the mild hypothermia group than in the normothermia group during liver ischemia-reperfusion periods (P= 0.032, P= 0.028). Meanwhile, the histopathologic injury of liver induced by ischemia-reperfusion was significantly improved in the mild hypothermia group, compared with that in the normothermia group. CONCLUSION: Mild hypothermia can protect the liver from ischemia-reperfusion injury in pigs.     

槲皮素对缺血再灌注大鼠肝脏损伤的保护作用

目的评估槲皮素对缺血再灌注大鼠肝脏损伤大鼠的保护作用。方法健康大鼠80只被随机分为对照组5只、假手术组25只、缺血再灌注组25只和槲皮素组25只,后3组再按不同灌注时限(0、6、12、24、72 h)分为5个亚组,每个亚组5只。分别于实验结束后留取大鼠血液及肝脏组织,用全自动生化分析仪检测血清中ALT、AST水平,竞争抑制ELISA法检测血清中IL-1β、IL-6、IL-8、TNF-α水平;HE染色法观察肝脏的形态学改变,并用原位末端标记法检测肝脏细胞的凋亡情况。结果肝脏缺血再灌注组各时点血清中ALT、AST、IL-1β、IL-6、IL-8、TNF-α均有所升高,且以12 h最高,与对照组、假手术组相比差异有统计学意义(P均<0.05);除IL-6外,槲皮素组各时点以上检测指标与缺血再灌注组比较有统计学差异(P均<0.05)。光镜下,对照组、假手术组肝脏无明显改变;而在缺血再灌注后12 h,肝细胞变性明显,肝索结构消失,部分肝细胞灶状坏死;槲皮素组的肝脏仅部分细胞变性,部分肝索结构存在,该形态学变化与细胞凋亡指数的检测结果一致。结论槲皮素能够有效地改善大鼠肝脏缺血再灌注损伤。     

Melatonin protects steatotic and nonsteatotic liver grafts against cold ischemia and reperfusion injury

Abstract: Chronic organ‐donor shortage has required the acceptance of steatotic livers for transplantation purposes despite the higher risk of graft dysfunction or nonfunction associated with the cold ischemia–reperfusion injury. This study evaluated the use of melatonin as an additive to Institute Georges Lopez (IGL‐1) solution for protecting nonsteatotic and steatotic liver grafts against cold ischemia–reperfusion injury. In the current investigation, we used an ex vivo isolated perfused rat liver model. Steatotic and nonsteatotic livers were preserved for 24 hr (4°C) in University of Wisconsin or IGL‐1 solutions with or without melatonin, as well as in University of Wisconsin solution alone. Thereafter, livers were subjected to 2‐hr reperfusion (37°C). We assessed hepatic injury (transaminases) and function [bile production and sulfobromophthalein (BSP) clearance, vascular resistance], as well as other factors potentially implicated in the high vulnerability of steatotic livers against ischemia–reperfusion injury (oxidative stress and related inflammatory mediators including nitric oxide and cytokines). We also evaluated well‐known cytoprotective factors as hemeoxygenase 1 (HO‐1). Fatty livers preserved in IGL‐1 solution enriched with melatonin showed lower transaminase levels and higher bile production and BSP clearance when compared to those obtained for livers maintained in IGL‐1 solution alone. A significant diminution of vascular resistance was also observed when melatonin was added to the IGL‐1 solution. The melatonin benefits correlated with the generation of nitric oxide (through constitutive e‐NOS activation) and the prevention of oxidative stress and inflammatory cytokine release including tumor necrosis factor and adiponectin, respectively. The addition of melatonin to IGL‐1 solution improved nonsteatotic and steatotic liver graft preservation, limiting their risk against cold ischemia–reperfusion injury.     

New preservation strategies for preventing liver grafts against cold ischemia reperfusion injury

BACKGROUND: In spite of improvements in University of Wisconsin (UW) preservation solution, the injury from grafts during cold storage is an unresolved problem in liver transplantation. The aim of the present study was to evaluate the beneficial effect on ischemia-reperfusion injury associated with liver transplantation of the inversion of K(+) and Na(+) concentrations and the replacement of hydroxyethyl starch (HES) by polyethylene glycol (PEG) in UW preservation solution. METHODS: Using an orthotopic liver transplantation model, the effects on rat liver preservation of a modified preservation solution (UW-PEG) were evaluated, based on the inversion of K(+) and Na(+) concentration and the replacement of HES by PEG 35 kDa (0.03 mmol/L) in UW preservation solution. RESULTS: The use of UW-PEG preservation solution ameliorated the biochemical and histological parameters of hepatic damage. Thus, at 24 h after transplantation, transaminase levels were reduced significantly when livers were preserved during 8 h in UW-PEG preservation solution compared with the original UW solution. In addition, histological findings revealed fewer and smaller areas of hepatocyte necrosis. The benefits of UW-PEG solution cannot be explained by modifications in oxidative stress or neutrophil accumulation associated with liver transplantation. However, the results of hepatic and portal blood flow indicated that the benefits of this modified preservation solution, UW-PEG were associated with improvements in the microcirculatory disorders after reperfusion. CONCLUSIONS: The UW-PEG solution, while retaining all the advantages of UW solution, improved hepatic ischemia-reperfusion injury associated with liver transplantation.     

Ring-Box 1基因负调控抗病毒天然免疫的初步研究

目的研究Ring-Box 1基因(RBX1/ROC1)对视黄酸诱导基因-I(retinoic acid inducible gene I,DDX58/RIG-I)抗病毒感染信号通路功能的影响,寻找抗病毒治疗的潜在靶标。方法利用PCR扩增RBX1/ROC1基因,将其插入p CDNA3载体,并且通过免疫印迹检测质粒在细胞中的表达;利用荧光素酶报告基因检测RBX1/ROC1基因对DDX58/RIG-I信号通路的影响。结果本研究成功构建Flag-RBXI/ROC1真核表达载体并在HEK293细胞中得到了表达;通过荧光素酶报告基因分析发现RBX1/ROC1基因抑制DDX58/RIG-I信号通路。结论 RBX1/ROC1是宿主天然免疫DDX58/RIG-I抗病毒感染信号通路的负调控分子,为寻找抗病毒靶点打下基础。     

苦参碱对大鼠小体积肝移植缺血再灌注损伤的保护作用

目的: 探讨苦参碱对大鼠小体积肝移植缺血再灌注损伤的保护作用及机制.方法:采用大鼠30%小体积肝移植模型, ♂SD大鼠322只随机分为假手术组、小体积肝移植对照组和高、低剂量苦参碱治疗组(60、40 mg/kg). 观察术后1 wk生存率, 检测移植术2h、4 h、1 d、2 d、3d、7 d后ALT、AST及LDH值. 光镜及电镜下评估移植肝病理形态学改变, ELISA法检测肝脏IL-6, TNF-α表达.结果: 与对照组比较, 苦参碱高、低剂量治疗组术后1 wk生存率显著增加(80%, 70% vs 50%, 均P<0.05), 术后2h、4h、1d ALT、AST及LDH明显降低(P<0.01). 苦参碱治疗组中肝细胞和肝窦内皮细胞凋亡减少、细胞形态明显改善, 苦参碱治疗组术后2 h、4 h、1 d肝脏组织中IL-6, TNF-α水平明显降低(P<0.01). 结论:苦参碱可减轻肝细胞及肝窦内皮细胞的损伤, 改善小体积肝移植术后缺血再灌注损伤, 其机制可能与苦参碱抑制肝移植术后IL-6、TNF-α等炎症因子的释放有关.     

环孢菌素A拮抗心肌缺血/再灌注损伤的作用

目的:研究环孢菌素A(CsA)拮抗小型猪心肌缺血/再灌注损伤(MI/RI)的作用及可能的机制。方法:经皮球囊封堵冠状动脉左前降支制备小型猪MI/RI模型。将存活的动物随机分为3组:即对照组(n=4)、CsA组(n=6)及他可英司(FK-506)组(n=6),分别静滴生理盐水100 ml、25 mg/kg CsA及1 mg/kg FK-506。所有动物均经90 min缺血和3 h再灌注。通过病理检查评估心肌梗死(MI)面积。用免疫组化染色法检测心肌细胞凋亡。用透射电子显微镜观察各组心肌细胞线粒体的形态。结果:CsA组MI的面积比对照组[(7.5±0.6)cm2vs.(10.5±2.6)cm2]和FK-506组[(7.5±0.6)cm2vs.(9.6±2.7)cm2]明显减少(P0.01);CsA组心肌细胞的凋亡率(%)比对照组[(11.9±1.88)%vs.(22.3±1.66)%]和FK-506组[(11.9±1.88)%vs.(19.2±1.82)%]明显下降(P0.01)。透射电子显微镜检查显示,CsA组能维持线粒体的形态,线粒体坍塌的百分率为(20%±7%),比对照组(53%±12%)和FK-506组(47%±9%)明显减少(P0.01)。结论:CsA可能对MI/RI具有拮抗作用,其机制可能是通过抑制线粒体膜通透性转换孔(mPTP),保持线粒体形态完整而实现,此种效应不依赖于钙调磷酸酶抑制途径。     

环孢菌素A拮抗心肌缺血／再灌注损伤的作用

目的：研究环孢菌素A（CsA）拮抗小型猪心肌缺血／再灌注损伤（MI／RI）的作用及可能的机制。方法：经皮球囊封堵冠状动脉左前降支制备小型猪MI／RI模型。将存活的动物随机分为3组：即对照组（n=4）、CsA组（n=6）及他可英司（FK-506）组（n=6），分别静滴生理盐水100ml、25mg／kgCsA及1mg／kgFK-506。所有动物均经90rain缺血和3h再灌注。通过病理检查评估心肌梗死（MI）面积。用免疫组化染色法检测心肌细胞凋亡。用透射电子显微镜观察各组心肌细胞线粒体的形态。结果：CsA组MI的面积比对照组[（7．5±0．6）cm。粥．（10．5±2．6）cm。]和FK-506组[（7．5±0．6）cm。掷．（9．6±2．7）cm。]明显减少（P〈0．01）；CsA组心肌细胞的凋亡率（％）比对照组[（11．9±1．88）％郴．（22．3±1．66）％]和FK-506组[（11．9±1．88）％郴．（19．2±1．82）％]明显下降（JP〈0．01）。透射电子显微镜检查显示，CsA组能维持线粒体的形态，线粒体坍塌的百分率为（20％±7％），比对照组（53％±12％）和FK-506组（47％±9％）明显减少（P〈0．01）。结论：CsA可能对MI／RI具有拮抗作用，其机制可能是通过抑制线粒体膜通透性转换孔（mPTP），保持线粒体形态完整而实现，此种效应不依赖于钙调磷酸酶抑制途径。     

Cyclin-dependent kinase 2 signaling regulates myocardial ischemia/reperfusion injury

Ischemia/reperfusion (I/R) injury to the heart is accompanied by the upregulation and posttranslational modification of a number of proteins normally involved in regulating cell cycle progression. Two such proteins, cyclin-dependent kinase-2 (Cdk2) and its downstream target, the retinoblastoma gene product (Rb), also play a critical role in the control of apoptosis. Myocardial ischemia activates Cdk2, resulting in the phosphorylation and inactivation of Rb. Blocking Cdk2 activity reduces apoptosis in cultured cardiac myocytes. Genetic or pharmacological inhibition of Cdk2 activity in vivo during I/R injury led to a 36% reduction in infarct size (IFS), when compared to control mice, associated with a reduction in apoptotic myocytes. To confirm that Rb was the critical target in Cdk2-mediated I/R injury, we determined the consequences of I/R injury in cardiac-specific Rb-deficient mice (CRb^L/L). IFS was increased 140% in CRb^L/L mice compared to CRb^+/+ controls. TUNEL positive nuclei and caspase-3 activity were augmented by 92% and 36%, respectively, following injury in the CRb^L/L mice demonstrating that loss of Rb in the heart significantly exacerbates I/R injury. These data suggest that Cdk2 signaling pathways are critical regulators of cardiac I/R injury in vivo and support a cardioprotective role for Rb.     

Melatonin protects liver against ischemia and reperfusion injury through inhibition of toll-like receptor signaling pathway

Abstract: This study investigated the immunomodulating effect of melatonin on toll‐like receptor (TLR)‐stimulated signal transduction. Rats were subjected to 60 min of ischemia followed by 1 or 5 hr of reperfusion. Melatonin (10 mg/kg) or the vehicle was administered intraperitoneally 15 min prior to ischemia and immediately before reperfusion. Melatonin treatment significantly reduced the level of serum alanine aminotransferase activity. Increased levels of TLR3 and TLR4 protein expression induced by ischemia/reperfusion (I/R) were attenuated by melatonin. Serum level of high‐mobility group box 1 (HMGB1), a potent alarmin of the TLR system, increased significantly in the I/R group, and melatonin inhibited this release. Melatonin suppressed the increase in myeloid differentiation factor 88 (MyD88) protein expression, extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) phosphorylation and nuclear translocation of nuclear factor κB (NF‐κB) and phosphorylated c‐Jun, a component of activator protein 1. The increased level of toll‐receptor‐associated activator of interferon (TRIF) expression, phosphorylation of interferon (IFN) regulatory factor 3 (IRF3) and serum IFN‐β was attenuated by melatonin. Melatonin attenuated the levels of tumor necrosis factor alpha (TNF‐α), interleukin (IL)‐6 and inducible nitric oxide synthase (iNOS) protein and mRNA expression, while the level of heme oxygenase‐1 (HO‐1) was augmented. Our results suggest that melatonin ameliorates I/R‐induced liver damage by modulation of TLR‐mediated inflammatory responses.     

Inhibition of glycogen synthase kinase 3 beta ameliorates liver ischemia reperfusion injury by way of an interleukin-10-mediated immune regulatory mechanism

The ubiquitous serine/threonine kinase glycogen synthase kinase 3 beta (Gsk3β) differentially regulates macrophage Toll-like receptor (TLR)-triggered pro- and anti-inflammatory cytokine programs. This study was designed to determine the in vivo role and therapeutic potential of Gsk3β modulation in tissue inflammation and injury in a murine model of liver partial warm ischemia/reperfusion injury (IRI). As a constitutively activated liver kinase, Gsk3β became quickly inactivated (phosphorylated) following IR. The active Gsk3β, however, was essential for the development of IRI pathology, as administration of its specific inhibitor, SB216763, ameliorated the hepatocellular damage, evidenced by reduced serum alanine aminotransferase (sALT) levels and well-preserved liver architecture compared with controls. The liver protective effect of Gsk3β inhibition was dependent on an immune regulatory mechanism, rather than direct cytoprotection via mitochondria permeability transition pores (MPTP). Indeed: (1) coadministration of SB216763 and atractyloside (MPTP opener) failed to abrogate a local cytoprotective Gsk3β inhibition effect; (2) SB216763 selectively inhibited IR-triggered liver pro-inflammatory, but spared interleukin (IL)-10, gene induction programs; and (3) IL-10 neutralization restored liver inflammation and IRI in SB216763-treated mice. Gsk3β inactivation by IR was a self-regulatory mechanism in liver homeostasis, critically dependent on phosphoinositide 3 (PI3)-kinase activation, as administration of a PI3 kinase inhibitor, wortmannin, reduced Gsk3 phosphorylation and augmented liver damage. In vitro, IL-10 was critical for the suppression of pro-inflammatory gene programs by Gsk3 inhibition in bone marrow-derived macrophages in response to TLR4 stimulation. CONCLUSION: Our novel findings document the key immune regulatory function of Gsk3β signaling in the pathophysiology of liver IRI, and provide a rationale to target Gsk3β as a refined therapeutic strategy to ameliorate liver IRI.     

CXC chemokine receptor-1 is expressed by hepatocytes and regulates liver recovery after hepatic ischemia/reperfusion injury

CXC chemokines mediate hepatic inflammation and injury following ischemia/reperfusion (I/R). More recently, signaling through CXC chemokine receptor-2 (CXCR2) was shown to delay liver recovery and repair after I/R injury. The chemokine receptor CXCR1 shares ligands with CXCR2, yet nothing is known about its potential role in liver pathology. In the present study, we examined the role of CXCR1 in the injury and recovery responses to I/R using a murine model. CXCR1 expression was undetectable in livers of sham-operated mice. However, after ischemia CXCR1 expression increased 24 hours after reperfusion and was maximal after 96 hours of reperfusion. CXCR1 expression was localized largely to hepatocytes. In order to assess the function of CXCR1, CXCR2(-/-) mice were treated with the CXCR1/CXCR2 antagonist, repertaxin. Prophylactic treatment with repertaxin had no effect on acute inflammation or liver injury. However, when repertaxin was administered 24 hours postreperfusion there was a significant increase in hepatocellular injury and a delay in recovery compared to control-treated mice. CXCR1(-/-) mice also demonstrated delayed recovery and regeneration after I/R when compared to wild-type mice. In vitro, hepatocytes from CXCR2(-/-) mice that were stimulated to express CXCR1 showed increased proliferation in response to ligand. Hepatocyte proliferation was decreased in CXCR1(-/-) mice in vivo. CONCLUSION: This is the first report to show that CXCR1 expression is induced in hepatocytes after injury. Furthermore, the data suggest that CXCR1 has divergent effects from CXCR2 and appears to facilitate repair and regenerative responses after I/R injury.     

卡维地洛和庚醇对心肌缺血再灌注损伤的保护作用

目的研究卡维地洛（CVD）、庚醇（HT）在缺血再灌注心肌损伤中作用。方法通过建立的心脏缺血再灌注模型，采用2％氯化三苯四唑（TTC）染色测量心肌梗死的重量，监测心肌型肌酸激酶同功酶（CK—MB）、乳酸脱氢酶（LDH）、丙二醛（MDA）和一氧化氮（NO）等的变化。观察CVD、HT对心肌梗死的影响。结果在缺血30min，再灌注6h后，CVD减少CK—MB、LDH及MDA的释放，稳定NO的分泌。HT组在一定程度上也减少了LDH、CK—MB的释放，但对NO、MDA的释放似乎没有太大影响。CVD组的大鼠梗死心肌仅占左心室心肌重量的（6．2±1.1）％，比对照组减少了51.6％。HT组梗死心肌占左心室心肌重量的（7．4±1.0）％，比对照组减少了42．2％。HE常规染色发现CVD组心肌细胞损伤程度明显减轻，粒细胞浸润也明显减少，大部分仅表现肌纤维肿胀和断裂。CVD可以防止内皮型一氧化氮合酶的减少。电镜下，CVD组和HT组心肌润盘肌丝、线粒体等损伤较轻。结论CVD通过B受体拮抗、抗氧化等方面的作用来保护心肌。HT可能是使缝隙连接可逆性解藕联产生心肌保护作用。     

Melatonin protects against ischemia/reperfusion injury in skeletal muscle

Abstract:  Melatonin has been shown to diminish ischemia-reperfusion (I/R) injury in many tissues. The main aim of this study was to evaluate the protective antioxidant effect of melatonin in skeletal muscle during I/R injury. Wistar albino rats were randomly divided into three groups. Hindlimb ischemia was achieved by clamping the common femoral artery in two groups but not in control group. Limbs were rendered ischemic for 1.5 hr; at the end of the reperfusion period of 1.5 hr muscle tissue samples were taken for the histological evaluation and biochemical analysis. Melatonin (10 mg/kg) was injected i.p. in the I/R + Mel group at the onset of ischemia whereas the vehicle solution was injected in the I/R group. In I/R + Mel group histological damage was significantly less than in the I/R group ( P  < 0.001). In the I/R + Mel group, the mean malonedialdehyde level was lower than in the I/R group ( P  < 0.01) and was quite near to the levels in the control group ( P  > 0.05). Glutathione levels were found to be reduced in the I/R group compared with the control ( P  < 0.01) and I/R + Mel group ( P  < 0.01). Melatonin has a protective effect against I/R injury in skeletal muscle and may reduce the incidence of compartment syndrome, especially after acute or chronic peripheral arterial occlusions.     

丙酮酸对缺血/再灌注大鼠心肌的保护作用

目的:探讨丙酮酸(Pyr)对缺血/再灌注(I/R)大鼠心肌的影响及其可能的机制。方法:30只SD成年大鼠随机分为假手术(Sham)组、I/R组及I/R+Pyr组,每组10只。I/R+Pyr组大鼠于再灌前5 min,开始持续性静脉灌注Pyr 2 h[25 mg/(kg·h)]。再灌注2 h后,利用多道生理记录仪检测大鼠在体血流动力学指标:平均动脉压(MABP)、左室收缩压(LVSP)及左室最大收缩、舒张末压微分(±LV dP/dt max)。采用Western blot方法检测磷酸化-JNK(p-JNK)和总的JNK(t-JNK)表达。用原位缺口末端标记法(TUNEL)评价心肌细胞的凋亡。结果:I/R组的MABP、LVSP、±LV dP/dt max显著低于Sham组(P0.01),Pyr干预可增加I/R后MABP、LVSP及±LV dP/dt max的水平(P0.05)。与Sham组相比,I/R组大鼠左心室p-JNK的水平明显增高(P0.01);而Pyr可降低大鼠左心室p-JNK的水平(P0.01),并抑制心肌细胞凋亡(P0.05)。结论:Pyr可改善I/R大鼠心肌的功能,抑制心肌细胞凋亡,其机制可能与抑制JNK信号的激活有关。     

Cytoprotective effects of melatonin against necrosis and apoptosis induced by ischemia/reperfusion injury in rat liver

Abstract:  Melatonin protects against organ ischemia; this effect has mainly been attributed to the antioxidant properties of the indoleamine. This study examined the cytoprotective properties of melatonin against injury to the liver caused by ischemia/reperfusion (I/R). Rats were subjected to 60 min of ischemia followed by 5 hr of reperfusion. Melatonin (10 mg/kg) or the vehicle was administered intraperitoneally 15 min before ischemia and immediately before reperfusion. The serum aminotransferase activity and lipid peroxidation levels were increased markedly by hepatic I/R, which were suppressed significantly by melatonin. In contrast, the glutathione content, which is an index of the cellular redox state, and mitochondrial glutamate dehydrogenase activity, which is a maker of the mitochondrial membrane integrity, were lower in the I/R rats. These decreases were attenuated by melatonin. The rate of mitochondrial swelling, which reflects the extent of the mitochondrial permeability transition, was higher after 5 hr of reperfusion but was attenuated by melatonin. Melatonin limited the release of cytochrome c into the cytosol and the activation of caspase-3 observed in the I/R rats. The melatonin-treated rats showed markedly fewer apoptotic (TUNEL positive) cells and DNA fragmentation than did the I/R rats. These results suggest that melatonin ameliorates I/R-induced hepatocytes damage by inhibiting the level of oxidative stress and the apoptotic pathway. Consequently, melatonin may provide a new pharmacological intervention strategy for hepatic I/R injuries.