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。     

Ischemic preconditioning reduces intestinal epithelial apoptosis in rats

Recent experimental studies have described protective effect of ischemic preconditioning (IPC) on ischemia-reperfusion (I/R) injury of the intestine. We hypothesize that to reach a new point of view on the effect of IPC in intestinal barrier function, the relationship between I/R-induced mucosal injury and apoptosis must first be clarified. The present study was undertaken to investigate the role of IPC on intestinal apoptosis and probable contributions of bcl-2 expression to this process. We also investigated the effect of intestinal IPC on ileal malondyaldihyde levels. Forty-four male Wistar rats were randomized into four groups each consisting of 11 rats: sham-operated control, I/R group (30 min of superior mesenteric artery occlusion), IPC-I/R group (10 min of temporary artery occlusion prior before an ischemic insult of 30 min), and IPC alone group (10 min of preconditioning). Twenty-four hours later, ileum samples were obtained. Ileal malondyaldihyde levels were increased in the I/R group (31.9 +/- 18.8 vs. 106.8 +/- 39.8) but not in the IPC alone and IPC-I/R groups (38.1 +/- 13.6 and 44.7 +/- 12.7; P < 0.01). The number of apoptotic cells was significantly lower in IPC-I/R group than that of I/R group, and these findings were further supported by DNA laddering and M30 findings. Diminished bcl-2 expression observed in the ileal specimens of I/R group was prevented by IPC. Our results indicate that IPC may provide a protective effect on ileal epithelium and that this effect is probably the result of a significant increase in the expression of bcl-2 after the insult. The reversal of apoptosis by IPC might help preserving the vitality of intestinal structures that have a critical function, cessation of which often leads to multiorgan dysfunction syndrome.     

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。     

Remote ischemic preconditioning: current knowledge, unresolved questions, and future priorities

Remote ischemic preconditioning (RIPC) is the phenomenon whereby brief episodes of ischemia-reperfusion applied in distant tissues or organs render the myocardium resistant to a subsequent sustained episode of ischemia. Reduction of infarct size with RIPC has been documented in response to (i) brief antecedent ischemia in a remote coronary vascular bed (intra-cardiac protection); (ii) collection and transfer of coronary effluent from perconditioning "donor" hearts to naive "receptor" hearts (inter-cardiac protection); (iii) brief ischemia applied in skeletal muscle, mesentery, and other organs (interorgan protection); and (iv) remote nociception ("remote PC of trauma"). Moreover, the paradigm has expanded to encompass temporal modifications in the application of the remote stimulus (remote perconditioning and remote postconditioning). Progress has also been made in translating the concept of RIPC to patients undergoing planned ischemic events: evidence for attenuation of cardiac enzyme release with RIPC has been reported after elective abdominal aortic aneurysm repair, angioplasty, and coronary artery bypass graft surgery. However, despite these advances in characterization and clinical application, the mechanisms of RIPC--most notably, the means by which the protective stimulus is communicated to the heart--remain poorly defined and, in all likelihood, are model dependent.     

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.     

丙酮酸乙酯对肾缺血/再灌注损伤小鼠炎症因子及丝裂素活化蛋白激酶表达的影响

目的 观察丙酮酸乙酯(EP)对小鼠缺血/再灌注(I/R)损伤肾脏的保护作用,研究其对肾组织中炎症因子及丝裂素活化蛋白激酶(MAPK)信号通路相关蛋白表达的影响. 方法 将50只雄性BABL/c小鼠按随机数字表法分为假手术组(n=8)、模型组(n=10)和EP治疗组(n=32);EP治疗组再分为EP预处理组和EP 4、6、12 h处理组,每组8只,分别于制模前30 min及制模后4、6、12 h腹腔注射EP 40 mg/kg.采用夹闭双肾动脉30 min制备肾I/R损伤模型.于I/R 24 h取肾组织,采用实时聚合酶链反应(PCR)检测白细胞介素(IL-1β、IL-6)、肿瘤坏死因子-α(TNF-α)、细胞间黏附分子-1(ICAM-1)、高迁移率族蛋白B1(HMGB1)的mRNA表达;采用蛋白质免疫印迹法(Western blotting)检测MAPK信号转导通路中细胞外信号调节激酶1/2(ERK1/2)、c-Jun氨基末端蛋白激酶(JNK)、p38MAPK等的蛋白表达. 结果 实时PCR结果显示,与假手术组比较,模型组小鼠肾组织IL-1β、IL-6、TNF-α、ICAM-1及HMGB1的mRNA表达均显著增高(IL-1β:12.05±8.08比3.18±1.13;IL-6:10.26±6.85比0.81±0.34;TNF-α:5.83±3.85比0.67±0.34;ICAM-1:3.87±2.02比0.29±0.13;HMGB1:652.82±78.50比112.31±32.50,均P＜0.05);而EP各处理组能显著抑制上述炎症因子的表达,尤其以12 h处理组最为显著,分别为0.45±0.26、 0.66±0.13、 0.21±0.11、 0.05±0.02、 212.26±3.20(均P＜0.05).Western blotting结果显示,与假手术组比较,模型组小鼠肾组织磷酸化的ERK1/2、JNK、p38MAPK蛋白表达均显著升高(p-ERK1/2:1.13±0.38比0.48±0.34;p-JNK:1.40±0.15比0.36±0.15;p-p38MAPK:0.47±0.15比0.21±0.17,均P＜0.05);与模型组比较,EP各处理组在不同时间点均能显著抑制ERK1/2、JNK、p38MAPK的活化(均P＜0.05). 结论 EP能有效防治小鼠I/R肾脏损伤,可能与其调控炎症相关因子及MAPK信号转导的表达有关.     

缺血预适应在大鼠肢体缺血再灌注后胃黏膜损伤中的作用

背景:肢体缺血再灌注作为应激原而引起胃黏膜损伤,导致应激性溃疡的发生。目的:观察肢体缺血再灌注对胃黏膜的损伤,了解肢体缺血再灌注对胃黏膜损伤的作用及其部分机制,以及短暂多次肢体缺血在胃黏膜损伤发生中的作用。设计:随机分组设计、对照动物实验。单位:华北煤炭医学院病理生理学教研室。材料:实验于2002-01/06在华北煤炭医学院病理生理学实验室完成。选择健康成年的雄性Wistar大鼠54只随机数字表法分为3组,每组18只。缺血再灌注组:按Rosenthal方法复制模型,乙醚浅麻醉下以橡皮带环绕结扎大鼠双后肢根部,阻断血流4h后松解,恢复血流灌注4h后自腹主动脉放血处死。缺血预适应组:如上法预先阻断双后肢血流5min,然后恢复血流灌注5min,反复4次,其后操作同缺血再灌注组。对照组:操作同缺血再灌注组,但松弛结扎双后肢,不阻断血流。方法:取各组胃黏膜制作切片于光学显微镜和电子显微镜下进行观察,按Guth标准测定各组胃黏膜损伤指数,在721型分光光度计上于650nm波长比色,计算胃结合黏液量,同时测定胃黏膜血流量、胃黏液中磷脂、氨基己糖的含量、血浆和胃组织一氧化氮含量及胃黏膜一氧化氮合酶活性。主要观察指标:胃黏膜损伤指数、胃结合黏液量、胃黏膜血流量、胃黏液中磷脂、氨基己糖的含量、血浆和胃组织一氧化氮含量及胃黏膜一氧化氮合酶活性。结果:纳入动物54只,均进入结果分析。①缺血再灌注组大鼠胃黏膜损伤严重,光镜下可见黏膜腺体水肿、充血,糜烂、解体,在黏膜基底部及黏膜下层可见炎细胞浸润,即溃疡形成。缺血预适应组胃黏膜较完整,损伤程度较缺血再灌注组轻;电镜下缺血再灌注组胃壁细胞、主细胞细胞器结构不完整,遭到破坏。同样缺血预适应组各类细胞损伤轻于缺血再灌注组[损伤指数分别为18.00±10.71,34.00±15.01,P<0.01]。②缺血再灌注组和缺血预适应组大鼠胃黏膜血流量及胃结合黏液量、胃黏液中磷脂、氨基己糖的含量均明显低于对照组[分别为(2.12±0.56),(10.84±2.56),(25.52±2.97)mL/(kg·h);(2.01±0.91),(2.79±0.73),(3.99±0.87)mg;(7.68±1.95),(9.74±1.04),(11.98±1.98)mg/g;(3.83±1.18),(5.42±0.47),(5.76±1.21)mg/g,P<0.05,0.01]。缺血预适应组胃黏膜血流量及胃结合黏液量、胃黏液中磷脂、氨基己糖的含量均高于缺血再灌注组。③缺血再灌注组和缺血预适应组血浆与胃黏膜组织一氧化氮含量及一氧化氮合酶活性显著高于对照组[分别为(250.0±5.6),(270.0±11.3),(210.0±7.4)μmol/L;(9.34±0.67),(11.34±1.00),(7.50±0.67)μkat/g,P<0.01],缺血预适应组血浆与胃黏膜组织一氧化氮含量和胃黏膜的一氧化氮合酶活性又显著高于缺血再灌注组。结论:肢体缺血再灌注作为应激原可导致胃黏膜损伤,引起应激性溃疡;缺血预适应可减轻肢体缺血再灌注后的胃黏膜损伤。     

瘦素预处理和缺血预处理在小鼠心肌缺血/再灌注损伤中的心肌保护机制

目的 观察瘦素预处理和缺血预处理(IPC)对心肌缺血/再灌注损伤(MIRI)模型小鼠的心肌保护作用,探讨瘦素参与的机制.方法 将36只C57BL/6小鼠按随机数字表法分为5组:①假手术组(12只);②短暂缺血/再灌注(I/R)组(6只):缺血3 min后再灌注5 min,进行3个短暂的循环;⑨标准I/R组(6只):缺血30 min后再灌注120 min,④IPC组(6只):行3个短暂I/R循环后再进行标准I/R操作;⑤瘦素预处理组(6只):缺血前30min腹腔注射50μg/kg小鼠重组瘦素.假手术组和短暂I/R组分别于再灌注后0(RP刻)、5、30、120 min取血,动态监测血清瘦素水平;其余各组于再灌注后120 min处死小鼠,观察心肌梗死面积、心肌瘦素水平、髓过氧化物酶(MPO)活性,以及血清瘦素、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)水平.结果 短暂I/R处理后5 min血清瘦素水平即较假手术组同期显著升高[(6.24±2.34)μg/L比(1.35±0.45)μg/L],30 min达峰值[(12.36±1.33)μg/L],之后逐渐下降,与假手术组水平同期比较差异无统计学意义[(1.96±1.33)μg/L比(1.16±0.25)μg/L,P＞0.05].与标准I/R组比较,瘦素预处理组与IPC组均可显著缩小梗死面积[(11.50±2.86)%、(9.00±1.90)%比(37.00±2.53)%],降低心肌瘦素[(8.36±3.42)μg/g、(6.71±2.03)μg/g比(15.51±3.92)μg/g]、MPO((17.10±3.95)μg/g、(13.33±2.88)μg/g比(30.83±4.06)μg/g]以及血清瘦素[(15.03±1.87)μ/L、(11.85±0.72)μg/L比(29.55±2.31)μg/L]、TNF-α[(35.10±10.12)ng/L、(27.04±5.18)ng/L比(81.34±14.20)ng/L]、IL-6[(167.39±72.83)ng/L、(149.13±37.69)ng/L比(477.30±29.09)ng/L]水平(均P＜0.01).结论 低剂量瘦素预处理可模仿经典的IPC以减轻MIRI,瘦素参与IPC的心肌保护机制可能与减轻炎症反应有关.     

预刺激小脑顶核对缺血再灌注大鼠脑蛋白激酶C同工酶表达的影响

背景预先电刺激小脑顶核(fastigial nucleus,FN)具有明确的缺血脑保护作用,但其机制尚不十分清楚.研究缺血诱导的蛋白激酶C(proteinkinase C,PKC)同工酶γ,δ异常表达,可使人们从新的角度去认识和探索预先电刺激FN缺血脑保护作用的机制.目的观察预刺激脑缺血大鼠小脑顶核不同时相PKC同工酶γ,δ蛋白表达.设计随机对照实验.地点和对象实验地点重庆医科大学神经病学研究所.Wistar雄性大鼠48只,随机将动物分为单纯缺血再灌注组(I/R),假手术组(I/R'),刺激小脑齿状核(dentate nucleus,DN)I/R组(I/RDN),刺激小脑FNI/R组(I/PFN);其中I/PDN,I/RFN又分别分为3组缺血前1,4,7 d刺激.每组动物为6只.干预采用线栓法大鼠大脑中动脉栓塞再灌注模型,缺血时间均为1.5 h再灌注24 h;于缺血前1,4,7 d分别刺激小脑顶核、齿状核1 h.主要观察指标以尾状核冠状切面作为观察对象,应用免疫组织化学方法观察对照组、假手术组、刺激小脑顶核组和齿状核组PKCγ,δ的表达情况.结果缺血前1,4,7d刺激小脑齿状核各组、单纯缺血再灌注组、假手术组PKC γ,δ阳性细胞数比较无显著性差异(P＞0.05),而缺血前1,4,7 d预刺激小脑顶核能明显抑制PKC γ,δ蛋白的表达(t=2.372～6.632,P＜0.05).结论缺血性脑损害能诱导PKC γ,δ蛋白表达上调,预先电刺激小脑顶核的缺血脑保护作用可能与其下调PKC γ,δ蛋白表达有关.     

缺血预处理对缺血骨骼肌收缩功能的影响

背景缺血预处理能有效提高骨骼肌缺血耐受性,减轻骨骼肌缺血再灌注期间的坏死范围,但缺血预处理对骨骼肌收缩功能的影响文献报道不多.目的探讨缺血预处理对骨骼肌缺血再灌注期间收缩功能的影响.设计以实验动物为研究对象的随机对照研究.单位华中科技大学同济医学院及解放军第二五二医院.材料实验地点为解放军第二五二医院中心实验室.选用健康雄性SD大鼠14只.方法采用大鼠后肢缺血再灌注模型,将14只大鼠随机分为对照组和实验组.对照组持续缺血4 h,再灌注1 h;实验组缺血5 min,再灌注5 min,重复3次后,持续缺血4 h再灌注1 h.测定缺血再灌注期间腓肠肌收缩功能变化及再灌注1 h后,血清磷酸激酶(CK),丙二醛和腓肠肌99锝m亚甲基二磷酸钠(99TcmMDP)吸收量变化.主要观察指标缺血预处理对腓肠肌收缩力及对血清CK,丙二醛及99TcmMDP吸收量的影响.结果实验组腓肠肌收缩力在缺血4h时为(14.32±5.05)g,再灌注1 h时为(25.71±7.58)g,对照组分别为(0,4 73±2.05)g,两者相比差异有显著性意义(P＜0.05),实验组血清CK为(104.85±9.84)nkat/L,丙二醛为(3988.60±455.92)nmol/L,99TcmMDP吸收量为(56.0±8.1)mBq/g·mir,对照组CK为(136.36±14.50)nkat/L,丙二醛为(6 542.90±536.72)nmol/L,99TcmMDP吸收量为(97.3±5.8)mBq/g·min,两者相比差异有显著性意义(P＜0.05,P＜0.01).结论缺血预处理能有效改善缺血再灌注期间骨骼肌的收缩力,减轻骨骼肌坏死程度.因此,缺血预处理对缺血骨骼肌的收缩功能具有保护作用.     

钙离子、蛋白激酶C与大鼠心肌预适应相关性研究

目的 :观察钙离子、蛋白激酶C在大鼠心肌预适应保护机制中的作用。方法 :64只雄性SD大鼠随机分为 8组 ,A组正常灌流 2 0min缺血 40min再灌 3 0min(I/R) ;B组短暂缺血复灌 +I/R ;C组短暂缺血复灌 +L- 型钙通道阻滞剂 +I/R ;D组短暂缺血复灌 +PKC阻滞剂 +I/R ;E组钙通道激动剂 +I/R ;F组钙通道激动剂 +PKC阻滞剂 +I/R ;G组L- 型钙通道阻滞剂 +I/R ;H组PKC阻滞剂 +I/R。结果 :实验中观察到B、E组与A、E、C组相比 ,各项指标均提示心肌保护作用较好。结论 :短暂钙离子内流增加通过蛋白激酶C诱导出心肌内源性保护作用。     

局灶性脑缺血预处理对大鼠肿瘤坏死因子表达影响的实验研究

目的观察局灶性缺血预处理对大鼠肿瘤坏死因子（TNF—a）的表达影响，探讨TNF—a与缺血耐受的关系及其在内源性保护机制中的作用。方法利用线栓法建立局灶性脑缺血耐受的动物模型。选用30只SD大鼠．随机将30只大鼠分为实验组：预缺血＋缺血（IP＋MCAO）；假手术组（SS＋MCAO）：假手术代替IP，余同实验组：对照组（SS＋SS），每组10只。评价指标包括神经功能缺损评分、光镜下组织病理改变及免疫组织化学染色和图像分析比较各组TNF-a的表达变化。结果局灶性IP能够明显改善3d后的大鼠的神经功能评分，减轻组织学的损伤，下调了TNF—a的表达，IOD值实验组（8109．53±571．21）对比假手术组（10704．72±584．01），差异有统计学意义（F=233．59，P〈0．05）。结论局灶性缺血预处理对随后的脑梗死有明显的保护作用．能够诱导缺血耐受的产生，其可能的机制是通过下调肿瘤坏死因子的表达。     

Potentiation of a survival signal in the ischemic heart by resveratrol through p38 mitogen-activated protein kinase/mitogen- and stress-activated protein kinase 1/cAMP response element-binding protein signaling

Resveratrol (3,4',5-trihydroxy-trans-stilbene), a naturally occurring polyphenolic compound found abundantly in grape skins and red wines, has been found to pharmacologically precondition the heart against ischemia reperfusion injury through the potentiation of a survival signal involving cAMP response element-binding protein-dependent phosphatidylinositol 3-kinase-Akt-BclII pathway. The present study was designed to determine whether, similar to ischemic preconditioning, resveratrol uses mitogen-activated protein kinases (MAPKs) as upstream signaling targets. The isolated rat hearts were preperfused for 15 min with Krebs-Henseleit bicarbonate buffer in the absence (control) or presence of extracellular signal-regulated kinase (ERK) 1/2 inhibitor 2'-amino-3'-methoxyflavone (PD98059), p38 MAPK inhibitor 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole (SB-202190), mitogen- and stress-activated protein kinase 1 (MSK-1) inhibitor N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89), protein kinase A inhibitor (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3fg: 3',2',1'-kl]-pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ester (KT5720), resveratrol only, resveratrol plus PD98059, resveratrol plus SB-202190, resveratrol plus H89, or resveratrol plus KT5720. Consistent with previous reports, resveratrol provided cardioprotection as evidenced by its ability to improve postischemic ventricular function, reduction of myocardial infarct size, and cardiomyocyte apoptosis. The cardioprotection afforded by resveratrol was partially abolished with PD98059 or SB-202190, suggesting that ERK1/2 and p38 MAPK play roles in resveratrol-mediated preconditioning. An MSK-1 inhibitor, H89, abolished resveratrol-mediated preconditioning, indicating MSK-1 to be the downstream target molecule for both ERK1/2 and p38 MAPK. KT5720 had no effect on resveratrol-mediated cardioprotection. Corroborating these results, Western blot analysis revealed phosphorylation of ERK1/2, p38 MAPK, MAPK-activated protein (MAPKAP) kinase 2, and MSK-1 with resveratrol and inhibition of phosphorylation with corresponding inhibitors. These results showed for the first time that resveratrol triggers an MAPK signaling pathway involving ERK1/2 and p38 MAPK, the former using MSK-1 as the downstream target and the latter, using both MAPKAP kinase 2 and MSK-1 as downstream targets.     

Possible involvement of PKC-δ in the abrogated cardioprotective potential of ischemic preconditioning in hyperhomocysteinemic rat hearts

The present study has been designed to investigate the possible role of protein kinase C-delta (PKC-δ) in hyperhomocysteinemia-induced attenuation of cardioprotective potential of ischemic preconditioning (IPC). Rats were administered l-methionine (1.7 g/kg/day, p.o.) for 4 weeks to produce hyperhomocysteinemia. Isolated Langendorff perfused normal and hyperhomocysteinemic rat hearts were subjected to global ischemia for 30 min followed by reperfusion for 120 min. Myocardial infarct size was assessed macroscopically using triphenyltetrazolium chloride (TTC) staining. Coronary effluent was analyzed for lactate dehydrogenase (LDH) and creatine kinase (CK) release to assess the degree of cardiac injury. Moreover, the oxidative stress in heart was assessed by measuring lipid peroxidation and superoxide anion generation. The ischemia-reperfusion (I/R) was noted to produce myocardial injury as assessed in terms of increase in myocardial infarct size, LDH and CK in coronary effluent and oxidative stress in normal and hyperhomocysteinemic rat hearts. In addition, the hyperhomocysteinemic rat hearts showed enhanced I/R-induced myocardial injury with high degree of oxidative stress as compared with normal rat hearts subjected to I/R. Four episodes of IPC (5 min each) afforded cardioprotection against I/R-induced myocardial injury in normal rat hearts as assessed in terms of reduction in myocardial infarct size, LDH, CK and oxidative stress. On the other hand, IPC mediated myocardial protection against I/R-injury was abolished in hyperhomocysteinemic rat hearts. Treatment with rottlerin (10 μM), a selective inhibitor of PKC-δ did not affect the cardioprotective effects of IPC in normal rat hearts; but its treatment significantly restored the cardioprotective potentials of IPC in hyperhomocysteinemic rat hearts. The high degree of oxidative stress produced in hyperhomocysteinemic rat hearts during reperfusion may activate PKC-δ, which may be implicated in the observed paradoxically abrogated cardioprotective potentials of IPC in hyperhomocysteinemic rat hearts.     

Remote hindlimb preconditioning and hepatoprotection: NO-table strides against liver ischaemia/reperfusion injury

Hepatic IR (ischaemia/reperfusion) injury is an important clinical problem complicating liver surgery and transplantation. IPC (ischaemic preconditioning) is a strategy whereby brief episodes of IR in an organ can induce an adaptive response to protect against subsequent prolonged IR injury. However, trauma to vessels supplying the target organ is unavoidable using the technique of direct IPC. One amenable strategy would be to apply the protective preconditioning stimulus to an organ distant or remote from the target organ of interest, a technique known as RIPC (remote IPC). In the present issue of Clinical Science, Abu-Amara and co-workers utilize hindlimb RIPC as a novel therapeutic strategy against liver IR injury and investigate the mechanistic contribution of NO to hepatoprotection by administering C-PTIO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt], an NO scavenger. Their experiments set the stage for more definitive studies to demonstrate a discernible benefit for the utility of RIPC in liver surgery and transplantation.     

Ischemic preconditioning protects post-ischemic oxidative damage to mitochondria in rat liver

This study examined the effect of ischemic preconditioning (IPC) in protecting against a hepatic ischemia/reperfusion (I/R) injury, with particular focus on mitochondrial damage. Rat liver was preconditioned by 10 min of ischemia and 10 min of reperfusion. Immediately after IPC, liver was subjected to 90 min of sustained ischemia followed by 5 h of reperfusion. The hepatic I/R increased serum aminotransferase activity and mitochondrial lipid peroxidation 5 h after reperfusion. IPC attenuated these increases. Whereas the mitochondrial glutathione content and glutamate dehydrogenase activities were lower in the I/R group, these decreases were attenuated by IPC. During IPC, the tissue peroxide levels increased after 10 min of ischemia and were normalized after 10 min of reperfusion. In association with the IPC-derived transient increase in the peroxide levels, the significant production of peroxides observed at 10 min of reperfusion after 90 min of ischemia was attenuated. Furthermore, whereas the mitochondria isolated from rat liver after 5 h of reperfusion were rapidly swollen, the swelling rate was attenuated in the mitochondria from rat liver subjected to IPC before the sustained ischemia. The hepatic ATP and adenosine levels were 38% and 46% lower during the reperfusion, respectively. These decreases were attenuated by IPC. Thus, these results suggest that IPC protects the mitochondria against the deleterious effects of I/R, and this protection is associated with the reduced oxidative stress.     

单磷酰脂A和缺血预处理对大鼠缺血/再灌注心肌细胞凋亡 Bcl-2 Bax蛋白表达的影响

目的 研究单磷酰脂A预处理(MLA-P)和缺血预处理(IP)对大鼠缺血/再灌注(I/R)心肌细胞凋亡及相关基因 Bcl-2和Bax蛋白表达的影响。方法 复制I/R损伤模型,采用末端标记技术(TUNEL)检测心肌细胞凋亡;应用免疫组化SABC法检测Bcl-2和Bax蛋白表达。结果I/R组凋亡细胞较多,MLA-P组及IP组凋亡细胞明显少于I/R组(P<0.01)。Bcl-2和Bax蛋白表达在I/R组均较多,MLA-P组及IP组Bcl-2表达明显高于I/R组(P<0.01),而Bax蛋白表达明显低于I/R组(P<0.01)。MLA-P组与IP组各指标均无显著性差异(P>0.05)。结论 MLA-P可抑制I/R诱发的心肌细胞凋亡,Bcl-2和Bax蛋白表达在心肌凋亡的发生中起重要作用。MLA-P与IP两者对心肌细胞凋亡及相关基因表达影响的作用相近。     

缺血预处理保护大鼠移植胰及与细胞凋亡的相关性

背景:胰腺移植过程中,缺血再灌注损伤会导致许多并发症,直接威胁着供胰和受体本身的存活,严重时可导致移植失败。缺血预处理可使靶器官在随后的缺血中得到保护,目前成为器官移植研究的热点之一。目的:观察缺血预处理对大鼠移植胰缺血再灌注损伤的早期保护作用及其与细胞凋亡的相关性。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、麻醉科。材料:雄性SD大鼠70只(250～320g),3～6个月。方法:实验于2001-09/2004-04在西京医院胃肠外科实验室完成。正常大鼠6只为对照组,糖尿病大鼠模型成功24只,采用随机数字法分为缺血再灌注组6只和缺血预处理1次组6只(缺血5min再灌注5min1次)、缺血预处理2次组6只(缺血5min再灌注5min2次)和缺血预处理3次组6只(缺血5min再灌注5min3次),缺血再灌注组和缺血预处理组均行单纯胰腺移植,24只SD大鼠为供体。主要观察指标:①各组大鼠再灌注前、后血糖;再灌注后2h血清中肿瘤坏死因子α和一氧化氮的含量、移植胰组织中超氧化物歧化酶,髓过氧化物酶和丙二醛含量。②用原位末端标记法观察移植胰组织细胞凋亡情况;WesternBlot法检测移植胰组织Bax和Bcl-2蛋白表达情况。结果:①各组大鼠再灌注前、后血糖变化:缺血再灌注组和缺血预处理各组较再灌注前血糖下降;缺血预处理2次组血糖显著低于缺血再灌注组、缺血预处理1次组和缺血预处理3次组(P<0.05)。②各组大鼠再灌注后2h血清中肿瘤坏死因子α含量:缺血预处理各组较缺血再灌注组血清中肿瘤坏死因子α含量低;缺血预处理2次组较缺血预处理1次组和缺血预处理3次组肿瘤坏死因子α含量低(P<0.05)。③各组大鼠再灌注后血清一氧化氮的含量:缺血预处理各组较缺血再灌注组血清中一氧化氮含量高;缺血预处理2次组较缺血预处理1次组和缺血预处理3次组一氧化氮含量高(P<0.05)。④各组大鼠再灌注后移植胰组织中超氧化物歧化酶含量:缺血预处理各组较缺血再灌注组超氧化物歧化酶活性高;缺血预处理2次组较缺血预处理1次组和缺血预处理3次组超氧化物歧化酶活性高(P<0.05)。⑤各组大鼠再灌注后移植胰组织中丙二醛和髓过氧化物酶含量:缺血预处理各组丙二醛含量和髓过氧化物酶活性较缺血再灌注组低;缺血预处理2次组较缺血预处理1次组和缺血预处理3次组低。⑥各组大鼠移植胰组织细胞凋亡情况:再灌注后缺血预处理各组较缺血再灌注组移植胰组织中凋亡指数值低;缺血预处理2次组显著低于较缺血预处理1次组和缺血预处理3次组(P<0.05)。⑦各组大鼠移植胰组织Bax和Bcl-2蛋白表达情况:再灌注后缺血再灌注组胰组织Bax蛋白高表达,Bcl-2蛋白低表达,缺血预处理各组再灌注后移植胰组织Bax蛋白低表达,Bcl-2蛋白高表达,而缺血预处理2次组Bcl-2蛋白表达最高,Bax蛋白表达最低。结论:缺血预处理对大鼠移植胰的缺血再灌注损伤具有早期保护作用,可能与提高超氧化物歧化酶的活性、增加内源性一氧化氮的合成、下调肿瘤坏死因子α和减轻多形核白细胞黏附与聚集有关;缺血预处理可以减少移植胰缺血再灌注后的细胞凋亡,可能与减轻多形核白细胞黏附与聚集、减少氧自由基、上调Bcl-2蛋白和下调Bax蛋白有关;缺血5min再灌注5min2次是最佳的大鼠移植胰缺血预处理诱导办法。     

小胶质细胞耗竭消除了白质缺血预处理的作用

缺血预处理(ischemic preconditioning,IPC)是通过短暂、无害的缺血性暴露增强对随后发生的缺血障碍耐受性的现象。IPC的机制主要在灰质约占大脑85%的啮齿动物卒中模型中进行研究。人类脑白质占脑容量的50%,是卒中损伤的关键组成部分。我们使用小鼠视神经开发了一种新的中枢神经系统白质IPC模型,并确定了相关的免疫信号通路。我们验证了小胶质细胞对于白质IPC是必需的这一假说。首先用集落刺激因子1受体抑制剂PLX5622处理以耗尽小胶质细胞。视神经在体内暴露于短暂性缺血,72 h后急性分离,并进行氧-葡萄糖剥夺(OGD)以模拟缺血性损伤。通过记录复合动作电位(CAPs)和使用定量体视学的显微镜来评估轴突功能和结构的恢复。结果显示小胶质细胞耗竭消除了IPC介导的保护作用。在对照小鼠中,与非预处理视神经相比,预处理视神经的CAP恢复有所改善。然而在PLX5622处理的小鼠中,预处理和非预处理视神经之间的CAP恢复没有差异。小胶质细胞缺失还消除了IPC对OGD后轴突完整性和成熟(APC+)少突胶质细胞存活的保护作用。IPC介导的保护与视网膜损伤无关,表明它是由白质缺血暴露的固有的机械过程引起的。我们得出结论,预处理的小胶质细胞对白质中的IPC至关重要。"预处理的小胶质细胞"表型可能对其他中枢神经系统病变有保护作用,是值得探索的神经治疗领域。     

不同处理方法心肌保护作用的比较研究

目的研究常规预适应、后处理与药物后处理对大鼠离体心脏再灌注损伤保护作用的异同并探讨其机制。方法 SD大鼠共50只,随机分为5组:停灌/再灌组(I/R);预适应组(IPC);后处理组(POS);吗啡后处理组(MOR);预适应加后处理组(IPC+POS),行Langendorff心脏灌流。观测其血流动力学、心肌梗死面积、肌酸激酶(CK)、乳酸脱氢酶(LDH)以及超微结构变化。结果与I/R相比,4个实验组均能明显的改善心功能,减小心梗面积,减少心肌酶释放,减轻超微结构损伤。而MOR的心肌保护作用明显低于IPC、POS和IPC+POS。此三组的心肌保护效果差异有统计学意义(P<0.05)。结论预适应和后处理可能有大部分相同的心肌保护机制。而药物后处理只能激活部分机制而达到部分心肌保护作用。