Nitric oxide is an essential mediator of the protective effects of remote ischaemic preconditioning in a mouse model of liver ischaemia/reperfusion injury

NO (nitric oxide) may protect the liver from IR (ischaemia/reperfusion) injury. RIPC (remote ischaemic preconditioning) also protects against liver IR injury; however, the molecular mediator(s) of RIPC are currently unknown. The aim of the present study was to assess the role of NO in hindlimb RIPC-induced protection against liver IR injury. Mice were allocated to the following groups: sham group; RIPC group (six cycles of 4×4?min IR of hindlimb); IR group [40?min lobar (70%) hepatic ischaemia and 2-h reperfusion]; RIPC+IR group (RIPC followed by IR group procedures); and C-PTIO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt]+RIPC+IR group [C-PTIO (a direct NO scavenger) was administered, followed by the RIPC+IR group procedure]. Hepatic MBF (microcirculatory blood flow) was measured throughout the experiment. Circulating NOx (nitrite and nitrate) levels, plasma liver transaminases, hepatic histopathological and TEM (transmission electron microscopy) studies were performed at the end of the experiment. NOx concentrations were significantly elevated (P<0.05) in the RIPC and RIPC+IR groups. Compared with liver IR alone, RIPC+IR preserved hepatic MBF during liver reperfusion (P<0.05). In contrast, C-PTIO+RIPC+IR reduced MBF compared with RIPC+IR (P<0.05). RIPC+IR reduced plasma transaminases (P<0.05), and histopathological and ultrastructural features of injury compared with IR alone. The protective effects of RIPC+IR in reducing liver IR injury were abrogated in the group that received antecedent C-PTIO (C-PTIO+RIPC+IR). In conclusion, NO is an essential mediator of the protection afforded by hindlimb RIPC against liver IR injury. The mechanisms underlying this protection involve preservation of the sinusoidal structure and maintenance of blood flow through the hepatic microcirculation.     

缺血预处理减轻胰腺移植受体大鼠小肠肠黏膜屏障的损害

背景:胰腺移植过程中的缺血再灌注损伤可以引起术后众多的并发症,其中继发性胰腺炎可以导致受体小肠黏膜的损伤,造成严重的后果。目的:观察缺血预处理对大鼠胰腺移植受体肠黏膜屏障的保护作用。设计:随机对照动物实验。单位:解放军第四军医大学西京医院胃肠外科。材料:实验于2001-09/2004-04在解放军第四军医大学西京医院胃肠外科实验室完成。动物为SD雄性大鼠83只。方法:①取47只大鼠,自阴茎静脉注射脲链霉素65mg/kg制备糖尿病大鼠模型。将造模成功的36只大鼠随机分为缺血再灌注组,供体缺血预处理组(DIPC组),受体双后肢缺血预处理组(RIPC组)3组,每组12只。剩余36只正常大鼠中随机取12只为对照组,另外24只为供体。②对照组仅行开腹术,其他3组行胰腺移植。DIPC组于获取供胰前阻断供体脾血管5min再灌注5min2次;RIPC组于供胰再灌注前阻断受体双后肢血流5min再灌注5min,重复3次;缺血再灌注组不作处理。主要观察指标:①手术后5d各组随机取6只大鼠检测小肠通透性(以血浆中FITC-dextran浓度表示)和吸收功能(以血浆中木糖浓度表示)。②各组其余6只大鼠于术后5d取血检测血清肿瘤坏死因子α、NO、超氧化物歧化酶和淀粉酶活性,取回肠黏膜组织检测小肠黏膜黏膜湿重、微绒毛高度及宽度、丙二醛含量及髓过氧化物酶活性,同时取肠系膜淋巴结、肝及脾组织进行细菌培养,观察细菌易位情况。结果:经补充后72只大鼠进入结果分析。①血浆中FITC-dextran浓度:缺血再灌注组高于对照组、DIPC组和RIPC组(P<0.01)。②血浆中木糖浓度:缺血再灌注组低于对照组、DIPC组和RIPC组(P<0.01)。③细菌易位率:缺血再灌注组高于对照组、DIPC组和RIPC组(P<0.01)。④小肠黏膜损伤程度:缺血再灌注组低于其他3组(P<0.01)。⑤缺血再灌注组小肠髓过氧化物酶活性和丙二醛含量显著高于其他3组(P<0.01),血清超氧化物歧化酶活性和NO水平低于其他3组,淀粉酶活性和肿瘤坏死因子α水平高于其他3组(P<0.01)。结论:供体和受体双后肢缺血预处理均可保护大鼠胰腺移植受体小肠肠黏膜屏障,降低细菌易位率     

Cardioprotection by remote ischemic preconditioning exhibits a signaling pattern different from local ischemic preconditioning

Remote ischemic preconditioning (RIPC) and local ischemic preconditioning (IPC) protect the myocardium from subsequent ischemia/reperfusion (I/R) injury. In this study, the protective effects of early RIPC, IPC, and the combination of both (RIPC-IPC) were characterized. Furthermore, the hypothesis was tested that protein kinase C (PKC) and mitogen-activated protein kinases (MAPKs), important mediators of IPC, are activated in RIPC. Infarct size, serum troponin T, and creatine kinase levels were assessed after 4 × 5-min noninvasive RIPC, local IPC, or a combination of both and 35 min of regional ischemia and 120 min of reperfusion. Protein kinase C ε and the MAPKs extracellular signal-regulated MAPK (ERK), c-jun N-terminal kinase (JNK), and p38 MAPK were analyzed by Western blot analysis and activity assays in the myocardium and skeletal muscle immediately after the preconditioning protocol. Remote ischemic preconditioning, IPC, and RIPC-IPC significantly reduced myocardial infarct size (RIPC-I/R: 54% ± 15%; IPC-I/R: 33% ± 15%; RIPC-IPC-I/R: 33% ± 15%; P < 0.05 vs. I/R [76% ± 14%]) and troponin T release (RIPC-I/R: 15.4 ± 6.4 ng/mL; IPC-I/R: 10.9 ± 7.0 ng/mL; RIPC-IPC-I/R: 9.8 ± 5.6 ng/mL; P < 0.05 vs. I/R [27.1 ± 12.0 ng/mL]) after myocardial I/R. Ischemic preconditioning led to an activation of PKCε and ERK 1/2, whereas RIPC did not lead to a translocation of PKCε to the mitochondria or phosphorylation of the MAPKs ERK 1/2, JNK 1/2, and p38 MAPK. Remote ischemic preconditioning did not induce translocation of PKCε to the mitochondria or phosphorylation of MAPKs in the preconditioned muscle tissue. Remote ischemic preconditioning, IPC, and RIPC-IPC exert early protection against myocardial I/R injury. Remote ischemic preconditioning and local IPC exhibit different activation dynamics of signal transducers in the myocardium. The studied PKC-MAPK pathway is likely not involved in the protective effects of RIPC.     

缺血预处理抑制大鼠胰腺移植缺血再灌注胰腺细胞凋亡:活性氧与线粒体DNA修复酶的作用

背景:缺血预处理可诱发机体内源性保护机制,可全面有效地防治器官移植缺血再灌注损伤.在胰腺移植过程中冷、热缺血均可导致移植胰腺缺血再灌注损伤,线粒体结构及功能与胰腺病变密切相关,近些年研究发现,线粒体DNA存在修复体系,其与线粒体DNA损伤之间的平衡决定了疾病的发生和转归.目的:观察缺血预处理对大鼠胰腺移植缺血再灌注损伤时的细胞凋亡的影响,分析线粒体DNA修复酶8-氧鸟嘌呤DNA糖基化酶和氧化应激在其中的变化规律及可能途径.方法:纳入健康雄性SD大鼠50只,其中20只为供体,10只为假手术组,另20只糖尿病造模后分为缺血再灌注组和缺血预处理组,每组10只.假手术组只行开、关腹手术,缺血再灌注组和缺血预处理组行异位全胰十二指肠移植.缺血再灌注组对应供体大鼠于获取供胰前以4℃ UW液灌洗20 min:缺血预处理组对应供体大鼠于获取供胰前阻断腹上动脉5 min,再灌注5 min,共2次.供胰均控制热缺血时间为15 min,冷缺血时间为180 min.再灌注后12 h检测血浆淀粉酶活性、血糖浓度及Caspase-3,9活化水平,流式细胞法检测腺泡细胞凋亡率,罗丹明123法检测线粒体膜电位,二氯荧光素法检测线粒体过氧化氢产生速率,高效液相色谱法检测线粒体DNA中8-氧鸟嘌呤质量浓度,荧光定量聚合酶链反应法检测8-氧鸟嘌呤DNA糖基化酶mRNA的表达,Westenn-blotting法检测细胞色素C释放、磷酸化Akt及线粒体8-氧鸟嘌呤DNA糖基化酶蛋白表达水平.结果与结论:缺血预处理可降低线粒体氧化应激,提高Akt磷酸化水平,从而上调8-氧鸟嘌呤DNA糖基化酶表达,减少线粒体DNA氧化损伤,抑制腺泡细胞凋亡,减轻移植胰缺血再灌注损伤.     

缺血预处理对大鼠移植胰细胞凋亡的影响

目的 观察缺血预处理对大鼠移植胰细胞凋亡的影响。方法 6只正常大鼠为对照组，18只糖尿病SD大鼠随机分为缺血再灌注组（I／R组，n=6）、供胰缺血预处理组（DIPC组，n=6）和受体双后肢缺血预处理组（RIPC组，n=6），均行单纯胰腺移植（18只正常SD大鼠为供体）；检测各组大鼠再灌注前、后血糖；再灌注后2h移植胰组织中超氧化物歧化酶（SOD）和髓过氧化物酶（MPO）含量；用TUNEL法观察移植胰组织细胞凋亡情况。结果 与I／R组比较，DIPC组和RIPC组大鼠再灌注后的血糖、MPO活性和移植胰组织细胞调亡指数明显降低，移植胰组织中SOD活性明显增高（均为P〈0．01）。结论 供胰和受体大鼠双后肢缺血预处理能减轻大鼠移植胰细胞的凋亡现象。     

缺血预处理减轻大鼠减体积肝移植损伤并促进肝再生

背景:近年来,肝移植技术迅速发展,如何预防缺血再灌注损伤并有效保护肝再生成为研究的热点.缺血预处理是保护肝缺血损伤的有效方法,但其确切机制尚存争议.目的:研究缺血预处理在大鼠减体积肝移植肝损伤和肝再生中的作用及机制.方法:动物随机分为3组,肝移植组建立大鼠减体积肝移植模型.缺血预处理+肝移植组在供肝灌注前阻断第1肝门行缺血预处理10 min,再灌注15 min.假手术组在开腹后游离肝周韧带,然后关腹.分别于术后0.5,2,6,24 h取材.通过血清谷丙转氨酶水平和移植肝组织病理检查评估肝损伤.半定量免疫组织化学和western blot法测定氧化还原蛋白1表达水平,检测移植肝细胞增殖细胞核抗原评估肝再生情况.结果与结论:与肝移植组相比,缺血预处理+肝移植组术后6,24 h受体血清谷丙转氨酶明显降低(P<0.05;P<0.01).病理学分析显示肝移植组术后24 h可见到门脉周围大量炎细胞浸润,肝窦扩张明显,肝组织损伤较重;而缺血预处理+肝移植组则损伤较轻.半定量免疫组织化学显示缺血预处理+肝移植组移植肝中Ref-1蛋白表达明显增加,这一结果同样在westernblot检测中得到验证:缺血预处理+肝移植组移植肝术后24 h Ref-1蛋白表达较肝移植组明显增强(P<0.05).同时,术后2,6和24 h缺血预处理+肝移植组增殖细胞核抗原阳性细胞数较肝移植组明显增加(P<0.05).结果提示缺血预处理可减轻大鼠减体积肝移植术后早期移植物肝损伤并促进肝再生,这与Ref-1蛋白高表达密切相关.     

Ischemic preconditioning protects against gut dysfunction and mucosal injury after ischemia/reperfusion injury

Mesenteric ischemia/reperfusion (IR) damages the gastrointestinal epithelia and impairs gut function. Ischemic preconditioning (IPC) has been shown to protect organs against IR injury. We hypothesized that IPC protects the gut from IR injury. Rats were randomized to a sham group, a sham early IPC + IR group (sham IPC + SMA occlusion for 30 min and 6 h of reperfusion), an early IPC + IR group (IPC, three cycles of SMA occlusion for 4 min and reperfusion for 10 min) followed immediately by SMA occlusion for 30 min and 6 h of reperfusion), a sham 24-h group, a sham late IPC + IR group (sham IPC followed by additional reperfusion for 24 h + SMA occlusion for 30 min and 6 h of reperfusion), and a late IPC + IR group (IPC protocol followed by additional reperfusion for 24 h, and then SMA occlusion for 30 min followed by 6 h of reperfusion). At 6 h, transit was determined and expressed as the mean geometric center. Ileum was harvested for assessment of mucosal injury and myeloperoxidase (MPO) activity. Tissue water was determined using the wet-to-dry weight ratio to assess gut edema. Early IPC + IR significantly improved transit (3.9 +/- 0.2), decreased MPO levels (3 +/- 2), and lessened mucosal injury (1.2 +/- 0.3) compared with animals subjected to sham early IPC + IR (transit, 2.9 +/- 0.2; MPO levels, 9 +/- 1; mucosal injury, 3.0 +/- 0.6). Late IPC + IR also improved transit (6.0 +/- 0.4) and decreased MPO levels (1 +/- 1) compared with sham late IPC + IR (transit, 4.4 +/- 0.2; MPO levels, 8 +/- 1), however, there was no difference in the mucosal protection between late IPC + IR (1 +/- 0.3) and sham late IPC + IR (1 +/- 1). Our results suggest that early and late IPC improves intestinal dysfunction, decreases inflammation, and provides mucosal protection in the intestine after IR. Our results show that IR-induced gut dysfunction can be improved by IPC. Both phases of IPC can potentially be useful in the clinical setting of surgical patient care.     

Ischaemic and pharmacological preconditionings protect liver via adenosine and redox status following hepatic ischaemia/reperfusion in rats

Although IPC (ischaemic preconditioning) is considered as a protective strategy in HI/R (hepatic ischaemia/reperfusion), the mechanisms for this effect have not been fully elucidated. In the present study we investigate whether PPC (pharmacological preconditioning) by transient activation of A(1)R (adenosine A(1) receptor) protects against long-term HI/R and whether the protective effects of IPC depend on A(1)R activation and whether both preconditionings affect remote organs. Wistar rats underwent IPC and long-term HI/R. Another set of animals were pharmacologically preconditioned with the A(1)R-agonist CCPA [2-chloro-N(6)-cyclopentyladenosine; 0.1 mg/kg of body weight, i.p. (intraperitoneally)] 24 h before HI/R. In other groups, rats received an A(1)R-antagonist, DPCPX (1,3-dipropyl-8-cyclopentylxanthine; 0.1 mg/kg of body weight, i.p.) 24 h before HI/R. Hepatic damage was evaluated by transaminase [AST (aspartate transaminase), ALT (alanine transaminase)] release; inflammation was assessed by hepatic MPO (myeloperoxidase) and serum TNFalpha (tumour necrosis factor alpha) and NO; oxidative stress was estimated by MDA (malondialdehyde) and 4-HDA (4-hydroxyalkenals), SOD (superoxide dismutase) activity, GSH and ADA (adenosine deaminase) as adenosine metabolism. Both preconditionings protected liver and lung against HI/R as indicated by the reduction in transaminases, MPO, MDA+4-HDA, NO, TNFalpha and ADA activity as compared with HI/R (P<0.05). However, pre-treatment with DPCPX abolished the protective effects of IPC and PPC. Preconditionings induced a significant increase in hepatic MnSOD (manganese SOD) activity and NO generation compared with the sham group, and this activity was abolished by DPCPX pre-treatment. A(1)R activation induced hepatic delayed preconditioning and blockade of A(1)R abolished hepatic IPC. IPC, as well as PPC, were able to prevent lung damage. These protective effects are associated with a reduction in oxidative stress, inflammation and endogenous antioxidant preservation.     

Effect of ischaemic preconditioning on hepatic oxygenation, microcirculation and function in a rat model of moderate hepatic steatosis

IPC (ischaemic preconditioning) may protect the steatotic liver, which is particularly susceptible to I/R (ischaemia/reperfusion) injury. Hepatic steatosis was induced in Sprague-Dawley rats with a high-cholesterol (2%) diet for 12 weeks after which rats were subjected to I/R (ischaemia/reperfusion; 45 min of lobar ischaemia followed by 2 h of reperfusion). Rats were divided into three study groups (n=6 each) receiving: (i) sham laparotomy alone, (ii) I/R, and (iii) IPC (5 min of ischaemia, followed by 10 min of reperfusion) before I/R. Hepatic extra- and intra-cellular oxygenation and HM (hepatic microcirculation) were measured with near-infrared spectroscopy and laser Doppler flowmetry respectively. Plasma liver enzymes and hepatic tissue ATP were measured as markers of liver injury. Histology showed moderate-grade steatosis in the livers. At the end of 2 h of reperfusion, I/R significantly decreased extra- and intra-cellular oxygenation concomitant with a failure of recovery of HM (21.1+/-14.4% of baseline; P<0.001 compared with sham animals). IPC increased intracellular oxygenation (redox state of the copper centre of cytochrome oxidase; P<0.05 compared with rats receiving I/R alone) and flow in HM (70.9+/-17.1% of baseline; P<0.001 compared with rats receiving I/R alone). Hepatocellular injury was significantly reduced with IPC compared with I/R injury alone (alanine aminotransferase, 474.8+/-122.3 compared with 5436.3+/-984.7 units/l respectively; P<0.01; aspartate aminotransferase, 630.8+/-76.9 compared with 3166.3+/-379.6 units/l respectively; P<0.01]. In conclusion, IPC has a hepatoprotective effect against I/R injury in livers with moderate steatosis. These data may have important clinical implications in liver surgery and transplantation.     

Inhaled NO accelerates restoration of liver function in adults following orthotopic liver transplantation

Ischemia/reperfusion (IR) injury in transplanted livers contributes to organ dysfunction and failure and is characterized in part by loss of NO bioavailability. Inhalation of NO is nontoxic and at high concentrations (80 ppm) inhibits IR injury in extrapulmonary tissues. In this prospective, blinded, placebo-controlled study, we evaluated the hypothesis that administration of inhaled NO (iNO; 80 ppm) to patients undergoing orthotopic liver transplantation inhibits hepatic IR injury, resulting in improved liver function. Patients were randomized to receive either placebo or iNO (n = 10 per group) during the operative period only. When results were adjusted for cold ischemia time and sex, iNO significantly decreased hospital length of stay, and evaluation of serum transaminases (alanine transaminase, aspartate aminotransferase) and coagulation times (prothrombin time, partial thromboplastin time) indicated that iNO improved the rate at which liver function was restored after transplantation. iNO did not significantly affect changes in inflammatory markers in liver tissue 1 hour after reperfusion but significantly lowered hepatocyte apoptosis. Evaluation of circulating NO metabolites indicated that the most likely candidate transducer of extrapulmonary effects of iNO was nitrite. In summary, this study supports the clinical use of iNO as an extrapulmonary therapeutic to improve organ function following transplantation.     

远程缺血预处理对大鼠局灶性脑缺血/再灌注损伤的保护作用

目的探讨远程缺血预处理(RIPC)对大鼠局灶性脑缺血/再灌注(I/R)损伤的影响。方法SD雄性大鼠70只,随机分组,每组10只。对照组仅行单纯缺血后再灌注;RIPC组按RIPC与脑缺血间隔时间不同分为30min及1、2、12、24和48h组,即反复3次夹闭双侧股动脉造成肢体缺血5min、再灌注5min后,分别间隔30min及1、2、12、24和48h后,行大脑中动脉栓塞(MCAO)120min、再灌注24h。对各组动物进行神经功能缺损评分,然后行氯化三苯四唑(TTC)染色,计算脑梗死容积。结果与对照组比较,RIPC1、2和24h组神经功能缺损评分显著下降,差异有显著性(P均<0.05);而RIPC30min、12h和48h组与对照组比较差异均无显著性(P均>0.05)。脑梗死容积百分比RIPC1h组〔(17.9±7.5)%,P=0.016〕、2h组〔(18.3±11.2)%,P=0.019〕和24h组〔(20.2±11.9)%,P=0.047〕均明显小于对照组〔(30.5±9.8)%〕;而RIPC30min、12h和48h组与对照组比较差异无显著性(P均>0.05)。结论RIPC对大鼠局灶性脑I/R损伤有保护作用,其保护时程为预处理后1～2h,24h后再次出现。     

缺血预处理预防肝细胞分离过程中的缺血再灌注损伤

背景：缺血预处理能否减轻肝细胞分离过程中的缺血再灌注损伤及改善供体残留肝脏功能？经检索国内外罕见这方面的研究。目的：探讨缺血预处理对分离肝细胞及供鼠残肝缺血再灌注损伤的防治作用。方法：12只SD大鼠随机分为2组：单纯肝部分切除组和缺血预处理组,各6只。采用改良四步胶原酶灌注法分离上述切除肝脏肝细胞,同时收集术前和术后1d大鼠的血清。结果与结论：缺血预处理组切除肝脏分离肝细胞成活率、增殖活性及白蛋白合成、超氧化物歧化酶水平显著高于单纯肝部分切除组（P〈0.05）,而乳酸脱氢酶、谷丙转氨酶、丙二醛水平显著减少（P〈0.05）;与单纯肝部分切除组比较,缺血预处理组大鼠血清白蛋白、乳酸脱氢酶、谷丙转氨酶、超氧化物歧化酶、丙二醛水平差异无显著性意义（P均〉0.05）。结果表明缺血预处理能减轻肝细胞分离过程中的缺血再灌注损伤,其机制可能与其自身缺血性预适应、抗氧化、清除氧自由基的能力相关,但对供鼠肝部分切除后残肝功能的影响不明显。     

Amiodarone offsets the cardioprotective effects of ischaemic preconditioning against ischaemia/reperfusion injury

Both ischaemic preconditioning (IPC) and amiodarone protect against myocardial ischaemia. We examined whether a combination of IPC and amiodarone demonstrated an additive protective effect in isolated rat hearts (n = 40). The controls (group I) were subjected to ischaemia/ reperfusion injury; group II was subjected to cycles of IPC prior to ischaemia/ reperfusion injury; group III was subjected to ischaemia in the presence of amiodarone (10(-10) mol/1); and group IV was subjected to IPC followed by ischaemia in the presence of amiodarone (10(-10) mol/l). Amiodarone produced the best preserved left ventricular end-systolic pressure and dP/dtmax, less developed ventricular stiffness, the shortest arrhythmia duration, and the smallest infarct size among the groups. All of the myocardial protective effects against ischaemia/reperfusion injury were diminished or abolished when IPC and amiodarone were applied sequentially.     

Fructose-fed rats are protected against ischemia/reperfusion injury

This study examines the relationship between insulin resistance (IR) induced by fructose feeding (FF) and susceptibility to myocardial ischemia/reperfusion injury (MI/R). Six-week-old male Sprague-Dawley rats were randomized into control (CON; n = 59) or FF (n = 58) groups. After 4 weeks, rats were further randomized into one of the following groups: placebo, ischemic preconditioning (IPC), 5-hydroxydecanoic acid (5-HD) (10 mg/kg), or 5-HD + IPC. Moreover, to determine the role of fructose, a second model of IR (Zucker obese) and rats fed fructose diet for 3 days (FF-3) were also subjected to MI/R. In all experiments, rats were subjected to 30 min of myocardial ischemia and 4 h of reperfusion. In rats randomized to placebo, infarct size was significantly reduced by FF (24 +/- 5%) compared with CON (54 +/- 1%, p < 0.05). Pretreatment with 5-HD did not alter the infarct size in CON (45 +/- 5%) but inhibited the protection afforded by FF (53 +/- 7%). IPC reduced the infarct size to an equivalent level in both groups, whereas 5-HD administration prior to IPC blunted the IPC effect. In Zucker obese rats, infarct size was significantly larger (57 +/- 4%) compared with lean controls (37 +/- 4%, p < 0.05). In FF-3 rats, infarct size was also decreased (20 +/- 2%, p < 0.01) compared with CON. This study suggests that fructose feeding affords protection against MI/R that is related to or mimics preconditioning. This protection is not consistent with other models of IR and is likely related to the fructose diet itself.     

Intestinal ischemic preconditioning protects the intestine and reduces bacterial translocation

Ischemic preconditioning (IPC) was first demonstrated in the heart, but this protective effect has been also recently described in the intestine. The aim of this study was to determine the effects of intestinal ischemic preconditioning on the morphology of intestine and bacterial translocation. Twenty-four male Wistar rats weighting 250 to 300 g were randomized into three groups. A control group of rats (n = 8) were subjected laparotomy. In an ischemic group (n = 8), laparotomy was performed and the superior mesenteric artery was occluded by an atraumatic clamp for 30 min. In the preconditioned group (n = 8), before the ischemia-reperfusion (I/R) period (as in ischemic group), rats were subjected to an initial 10 min of intestinal ischemia and 10 min of reperfusion. Twenty-four hours later, to evaluate whether the I/R induced intestinal injury and bacterial translocation (BT), tissue and blood samples were collected, and liver, spleen, and mesenteric lymph node specimens were obtained under sterile conditions for microbiological analysis. Samples of ileum were removed for both biochemical and histopathological evaluation. In the I/R group, the incidence of bacteria-isolated mesenteric lymph nodes, spleen, liver, and blood was significantly higher than other groups (P < 0.05). IPC prevented I/R-induced BT and it significantly reduced the I/R-induced intestinal injury (P < 0.05). Increased inducible nitric oxide (NO) synthase (iNOS) expression observed on the ileal specimens of the I/R group was found to be prevented by IPC. Our data suggest IPC as a key factor that reduces BT and iNOS activation in intestinal I/R. This is the first study showing that intestinal IPC blocks the cascade of events that causes BT and intestinal injury that may lead to sepsis.     

运动预处理对心肌缺血再灌注损伤老龄大鼠心肌的影响

目的:观察运动预处理对心肌缺血再灌注损伤后老龄大鼠心功能、心肌梗死面积、心肌细胞超微结构及抗氧化能力的影响。方法:选择60只SPF级雄性SD老龄大鼠按照随机数字表法分为对照组(Con组)、缺血再灌注模型组(IR组)、运动预处理+缺血再灌注组(EP+IR组)、缺血预适应组(IPC组)、运动预处理+缺血预适应组(EP+IPC组),每组12只。Con组、IR组、IPC组不做特殊运动干预;EP+IR组、EP+IPC组接受运动预处理干预(采用电动动物实验跑台进行梯度运动训练,1次/d,5 d/周,共训练6周)。利用Langendorff装置制备老龄大鼠离体心肌缺血/再灌注模型,具体如下:Con组仅进行心肌离体灌流,持续平衡灌注180 min,不进行缺血操作;IR组平衡预灌注20 min,然后保持心脏温度恒定在37℃,通过控制灌流设备的三通阀,使全心缺血40 min,再复灌120 min;EP+IR组大鼠心脏离体后,模型制备方法同IR组;IPC组大鼠心脏离体后平衡灌注20 min,给予3次缺血预处理(短暂缺血5 min,再灌注10 min),之后缺血40 min,再复灌120 min;EP+IPC组大鼠心脏离体后,模型制备方法同IPC组。于再灌注前、再灌注30、60、120 min分别采用多导生理记录仪记录心脏功能变化;于再灌注结束后,采用TTC染色法测定心肌梗死面积,比色法检测冠脉流出液中LDH活性、心肌组织中MDA含量及SOD活力。结果:(1)心脏功能指标:与Con组同一时间点比较,IR组再灌注前、再灌注后30、60、120 min心功能各项指标[心率(CR)、左心室舒张压(LVDP)、左心室压力最大变化速率(±dp/dtmax)、冠脉流量(CF)]均明显降低(P<0.05)。与IR组同一时间点比较,EP+IR、IPC、EP+IPC组再灌注30、60、120 min心功能各项指标均明显升高(P<0.05)。与IPC组同一时间点比较,EP+IPC组再灌注30、60、120 min心功能HR、±dp/dt_max、CF均明显更高(P<0.05)。(2)心肌梗死面积:与Con组比较,IR组心肌梗死面积明显增大(P<0.05);与IR组比较,EP+IR、IPC、EP+IPC组心肌梗死面积明显缩小(P<0.05)。(3)LDH活性、MDA含量、SOD活力:与Con组比较,IR组LDH活性水平明显升高(P<0.05);与IR组比较,EP+IR、IPC、EP+IPC组LDH活性水平降低(P<0.05);与EP+IR、IPC组比较,EP+IPC组LDH活性水平明显更低(P<0.05)。与Con组比较,IR组MDA含量明显升高,SOD活力明显降低(P<0.05);与EP+IR、IPC组比较,EP+IPC组MDA含量明显更低,SOD活力明显更高(P<0.05)。结论:运动预处理可诱导老龄大鼠心肌IPC保护作用,能有效改善心肌缺血再灌注损伤老龄大鼠心脏功能,减小心肌梗死面积,减轻心肌细胞损伤,这可能与其降低心肌缺血/再灌注时心肌LDH活性、MDA含量,提高SOD活力,增强心肌抗氧化能力有关。     

Pretreatment with bone morphogenetic protein-7 (BMP-7) mimics ischemia preconditioning following intestinal ischemia/reperfusion injury in the intestine and liver

Intestinal ischemia/reperfusion (I/R) injury has been shown to cause intestinal mucosal injury and adversely affect function. Ischemic preconditioning (IPC) has been shown to protect against intestinal I/R injury by reducing polymorphonuclear leukocyte infiltration, intestinal mucosal injury, and liver injury, and preserve intestinal transit. Bone morphogenetic protein 7 (BMP-7) has been shown to protect against I/R injury in the kidney and brain. Recently, microarray analysis has been used to examine the possible IPC candidate pathways. This work revealed that IPC may work through upregulation of BMP-7. The purpose of this study was to examine if pretreatment with BMP-7 would replicate the effects seen with IPC in the intestine and liver after intestinal I/R. Rats were randomized to six groups: sham, I/R (30 min of superior mesenteric artery occlusion and 6 h of R), IPC+R (three cycles of superior mesenteric artery occlusion for 4 min and R for 10 min), IPC+I/R, BMP-7+R (100 microm/kg recombinant human BMP-7), or BMP-7+I/R. A duodenal catheter was placed, and 30 min before sacrifice, fluorescein isothiocyanate-Dextran was injected. At sacrifice, dye concentrations were measured to determine intestinal transit. Ileal mucosal injury was determined by histology and myeloperoxidase activity was used as a marker of polymorphonuclear leukocyte infiltration. Serum levels of aspartate aminotransferase were measured at sacrifice to determine liver injury. Pretreatment with BMP-7 significantly improved intestinal transit and significantly decreased intestinal mucosal injury and serum aspartate aminotransferase levels, comparable to animals undergoing IPC. In conclusion, BMP-7 protected against intestinal I/R-induced intestinal and liver injury. Bone morphogenetic protein 7 may be a more logical surrogate to IPC in the prevention of injury in the setting of intestinal I/R.     

缺血预处理预防肝细胞分离过程中的缺血再灌注损伤

背景:缺血预处理能否减轻肝细胞分离过程中的缺血再灌注损伤及改善供体残留肝脏功能? 经检索国内外罕见这方面的研究.目的:探讨缺血预处理对分离肝细胞及供鼠残肝缺血再灌注损伤的防治作用.方法:12 只SD大鼠随机分为2组:单纯肝部分切除组和缺血预处理组,各6只.采用改良四步胶原酶灌注法分离上述切除肝脏肝细胞,同时收集术前和术后1 d大鼠的血清.结果与结论:缺血预处理组切除肝脏分离肝细胞成活率、增殖活性及白蛋白合成、超氧化物歧化酶水平显著高于单纯肝部分切除组(P < 0.05),而乳酸脱氢酶、谷丙转氨酶、丙二醛水平显著减少(P < 0.05);与单纯肝部分切除组比较,缺血预处理组大鼠血清白蛋白、乳酸脱氢酶、谷丙转氨酶、超氧化物歧化酶、丙二醛水平差异无显著性意义(P均> 0.05).结果表明缺血预处理能减轻肝细胞分离过程中的缺血再灌注损伤,其机制可能与其自身缺血性预适应、抗氧化、清除氧自由基的能力相关,但对供鼠肝部分切除后残肝功能的影响不明显.     

一氧化氮合酶在缺血预处理对大鼠肾脏缺血-再灌注损伤保护中的作用

目的：探讨一氧化氮合酶（ＮＯＳ）在缺血预处理对大鼠肾脏缺血－再灌注损伤保护中的作用。方法：将大鼠随机分为对照组（ＣＯＮ）、缺血－再灌注组（ＩＲ）和血预处理后血－再灌注组（ＩＰＣ）。光镜下观察并行肾小管评分,用免疫组化法检测各组中不同灌注时间的３种ＮＯＳ的变化。结果：不同缺血－再灌注时间点病理组织学肾小管评分ＩＰＣ组均低于ＩＲ组,而ＩＰＣ组和ＣＯＮ组之间无显著性差异;诱导型ＮＯＳ在ＩＲ组中的表达明显     

解偶联蛋白-2在大鼠缺血预适应心肌中的表达

目的：探讨解偶联蛋白-2（UCP2）在心肌缺血预适应（IPC）心肌保护中的作用。方法：采取结扎左冠状动脉的方法复制大鼠心肌缺血再灌注模型。IPC组行3次缺血5min，再灌注10min的预处理。缺血再灌注（IR）组与IPC组行30min缺血及120min再灌注；对照组不结扎左冠状动脉。电镜观察心肌超微结构。据Rainio评分标准进行心肌超微结构损伤程度的半定量分析，随机选取20个低倍视野，计算平均心肌细胞凋亡数。采用RT—PCR和Western印迹法检测心肌中UCP2的表达。结果：IPC组Rainio评分和心肌细胞凋亡率均低于IR组（氏0．05），IPC组的UCP2 mRNA和蛋白表达水平均较IR组明显增加（P〈0．01）。结论：IPC可减轻心肌超微结构损伤程度和减少细胞凋亡。IPC可诱导UCP2表达，提示UCP2可能参与了IPC的心肌保护作用。