蒺藜总皂苷对大鼠缺血再灌注损伤心肌ICAM-1表达及中性粒细胞浸润的影响

目的观察蒺藜总皂苷对缺血再灌注损伤心肌炎症因子ICAM-1表达及中性粒细胞浸润的影响。方法可逆性冠脉左前降支结扎缺血45min再灌注2h复制MI／R模型,SD雄性大鼠32只,随机分成假手术组、模型组、GSTT大剂量组、GSTT小剂量组,每组8只。HE染色观察心肌中性粒细胞浸润,测定心肌MPO活性,免疫组化测定心肌组织ICAM-1的表达。结果HE染色镜下观察模型组心肌中性粒细胞浸润严重,而GSTT大、小剂量组则较轻;GSTT大、小剂量组MPO活性显著降低（P〈0．01或0．05）;缺血45min再灌注2h心肌组织ICAM-1无明显表达。结论GSTT对MI／R炎症损害有保护作用,可降低IVIPO活性,减轻中性粒细胞浸润。     

白介素8和兔心肌缺血／再灌注损伤研究

目的 探讨白介素8（rhIL-8)参与兔心肌缺血／再灌注损伤的机制,为减轻再灌注损伤探索新的治疗途径。方法 结扎兔冠状动脉左前降支（left anterior descending coronary artery,LAD)造成缺血1小时,再灌注3.5小时,实验分两组：缺血／再灌注组（MI／R,n=8)和假结扎组（Sham Mi/R,n=8)。结果 MI／R组发生严重的心肌损伤,包括受累心肌髓过氧化物酶（myeloperoxidase,MPO)活性增大和血清肌酸磷酸激酶－MB同工酶（CPK－MB）、异构前列腺素（epi-PGF2a)。水平增高（均P＜0．01）。血清IL－8浓度逐渐升高,免疫组化示受损心肌区血管内皮基底膜呈IL－8阳性染色。结论 血管内皮细胞释放的IL－8是吸引中性粒细胞浸润于缺血区心肌,造成缺血／再灌注损伤的因素之一。     

抗肿瘤坏死因子-α单克隆抗体对心肌缺血再灌注损伤的保护作用及与核因子KB活化的关系

目的研究抗肿瘤坏死因子-α单克隆抗体(Anti-TNF-αmAb)对心肌缺血再灌注损伤的保护作用及与心肌核因子-kB(NF-kB)活化之间的关系。方法新西兰兔152只分为①缺血-再灌注组;②抗肿瘤坏死因子-α单克隆抗体预处理组;③假手术对照组。用酶联免疫吸附实验测定心肌组织中TNF-α的表达,凝胶电泳迁移率分析检测NF-kB的活性,髓过氧化酶法定量测定心肌组织中浸润的中性粒细胞。结果心肌缺血-再灌注使NF-kB迅速活化;心肌TNF-α的表达也明显增高,其与中性粒细胞浸润升高有相关性(r=0.764,P<0.05);Anti-TNF-α-mAb能抑制NF-kB的活化、减少TNF-α的表达及中性粒细胞浸润。结论心肌缺血-再灌注能刺激心肌NF-kB的活化,活化的NF-kB启动TNF-α的表达而参与缺血-再灌注损伤的发生过程。抗肿瘤坏死因子-α单克隆体对心肌缺血再灌注损伤的保护作用不仅在于直接减少TNF-α,而且可以抑制TNF-α对心肌NF-kB的活化的反馈激活作用。     

白介素-1β预处理对心肌细胞粘附分子表达的影响

为观察IL－1β预处理后心肌细胞粘着分子（CAMs)表达的变化及其与延迟保护（DP）作用的关系，在大鼠心肌缺血再灌注（I／R）模型上设假手术对照组（C组）、生理盐水组（NS组）和IL－1β预处理组（ILPC组），采用原位杂交、免疫组化及酶法检测低剂量IL－1β注入大鼠后即刻、12h和24h心肌细胞间粘附分子－1（ICAM-1)mRNA及其蛋白质表达和白细胞功能相关抗原－1（CD11a)蛋白质表达的变化，以及中性粒细胞（PMNs)的浸润数，并测定心肌梗死面积。结果发现，与C组比较，NS组和ILPC组各时相点ICAM－1 mRNA及其蛋白质表达和CD11a蛋白质表达以及PMNs浸润数均显著增加（P＜0．05或0．01），但ILPC组后24h I/R末，上述指标的增幅明显减少（P＜0．05），且心肌梗死面积显著下降（P＜0．05）。即刻和12h,ILPC组与NS组比较差异无显著性意义（P＞0．05）。提示，低剂量IL－1β预处理可使心肌延迟期CAMs表达减少而产生延迟保护作用。     

中性粒细胞激活在心肌缺血再灌注损伤中的作用

由于心肌缺血再灌注损伤（ischemic／reperfusioninjury,I／R）机制的复杂性,使其成为临床上经常面临和较难防治的问题。在其发生发展的众多机制中,中性粒细胞（PMN）活化、心肌浸润及脱颗粒可能是最终的环节或途径。大量研究表明中性粒细胞能通过释放细胞毒性物质如蛋白水解酶、氧自由基、脂类介质、各种炎性介质及阻塞毛细血管等引起缺血再灌注时的心肌损伤。认识这一细胞机制无疑有助于研究并找到防治心肌缺血再灌注损伤的方法。本文就近年来中性粒细胞激活在心肌缺血再灌注损伤中作用机制的及其防治的相关研究综述如下。     

大鼠缺血再灌注后小动脉ICAM-1与NOS表达关系的实验研究

目的：观察大鼠缺血再灌注后细胞间黏附分子（ICAM-1）和一氧化氮合酶（NOS）表达及其相互关系。方法：将大鼠分为对照组、缺血45min组和缺血再灌注1、3、6、12、24h组,建立失血性休克模型并给予治疗,用光镜和电镜观察小动脉形态学改变,并用免疫组化法检测ICAM-1、NOS在小动脉内皮和平滑肌细胞表达的阳性细胞数。结果：（1）缺血45min后NOS表达量首先增加,再灌后1h达到高峰,3h表达减弱。与对照组比较,差异均有统计学意义（P〈0．05）;（2）缺血再灌后1h组ICAM-1表达量增加,再灌后3h达到高峰,并持续至24h,与对照组比较,差异均有统计学意义（P〈0．05）。结论：ICAM-1介导了小动脉缺血再灌注损伤,NOS对小动脉缺血再灌注损伤具有保护作用,NOS对ICAM-1有抑制作用。     

大鼠心肌缺血再灌注损伤及中性粒细胞浸润与一氧化氮心肌保护作用的研究

目的 中性粒细胞(Neu)心肌浸润是心肌缺血-再灌注(I/R)损伤的重要机制之一。本实验通过在SD大鼠中建立心肌I/R模型,研究I/R过程中心肌损伤与中性粒细胞浸润的关系,并通过左型精氨酸(L-Arg)干预,探讨一氧化氮(NO)对中性粒细胞浸润的影响及其在心肌I/R损伤中的保护作用。方法 实验分对照(CON)组、L-Arg处理组。每组又分缺血前、缺血40 min、再灌 1h、再灌 3h和再灌6 h 5个时间点,分别测定各组各时间点血清肌酸磷酸激酶(CPK)及心肌匀浆丙二醛(MDA)值,并观察心肌组织病理学的改变。结果 (1)CPK及心肌匀浆MDA测定结果:再灌注后各时相血清CPK及肌匀浆MDA含量较缺血前明显上升,再灌注3h后达到高峰。L-Arg组中再灌注后各时相点血清CPK及肌匀浆MDA明显低于CON组(P<0.05)。(2)H-E染色结果:缺血前心肌细胞完整,边界清楚,心肌横纹清晰。CON组:再灌后1h出现Neu心肌浸润,再灌3 h Neu心肌浸润更明显,累及心肌全层,伴有局灶心肌坏死;再灌6 h后Neu浸润尤甚,大片心肌坏死,并可见小血管内Neu聚集现象。L-Arg组:再灌注后Neu心肌浸润明显减少,再灌6 h心肌坏死灶较CON组明显缩小。结论 (1)心肌I/R过程中有大量Neu心肌浸润,与心肌损伤密切。(2)L-Arg能抑制Neu心肌浸润,具有心肌保护作用。     

镁盐对犬心肌缺血-再灌注损伤的保护作用

建立整体犬急性心肌缺血-再灌注模型，在心肌缺血后再灌注前静脉滴注硫酸镁（MgSO4），以观察镁盐对再灌注区心肌组织超氧化物歧化酶（SOD）活性和脂质过氧化反应代谢产物——丙二醛（MDA）含量的影响，着重分析与血清肌酸磷酸激酶（CPK）水平和心肌超微结构病变的关系。结果表明，镁盐组再灌注区心肌组织SOD活性（1.19±0．21U·（mgProt）－1）较再灌组（0.31±0．06U·（mgPro）－1）明显升高（P＜0．01），而MDA含量明显降低（P＜0．01），同时伴有血清CPK水平下降（P＜0．01），超微结构损伤亦较再灌组轻。结果提示，镁益对缺血后再灌注心肌具有明显的保护作用。     

异丙酚对大鼠离体心脏缺血/再灌注损伤时心肌c-fos基因表达的影响

目的观察异丙酚对离体大鼠全心缺血再灌注损伤心肌c-fos基因表达的影响,探讨其对缺血再灌注损伤心肌保护作用机制。方法Wistar大鼠36只,随机分为对照组和异丙酚组,每组又分为缺血前、缺血30min和复灌30min3个亚组。通过离体心肌灌注模型,采用免疫组化及RT-PCR观察心肌c-fos蛋白及c-fo smRNA表达水平的变化。结果与缺血前相比,缺血30min及再灌注30min亚组的c-fos蛋白的光密度值及c-fos mRNA表达水平均增加（P〈0．01）;与对照组相比,异丙酚各亚组的c-fos蛋白光密度值及c-fos mRNA表达水平均降低（P〈0．01）。结论c-fos基因参与了心肌缺血再灌注损伤的基因调节。异丙酚可减轻心肌c-fos基因的表达,并可避免或减轻心肌缺血再灌注损伤。     

丙泊酚对糖尿病大鼠肢体缺血再灌注心肌组织内TNF—α和NF—KB的影响

目的探讨细胞因子、核转录因子在糖尿病大鼠肢体缺血再灌注后心肌组织损伤中的作用机制及丙泊酚的保护作用机制。方法将SD大鼠随机分为正常大鼠（非糖尿病大鼠）和糖尿病大鼠两系列,用链脲佐菌素（STZ）＋高脂高糖饮食法建立糖尿病大鼠模型。正常大鼠随机分为：①对照组（Ic组）;②肢体缺血再灌注组（ILIR组）;③肢体缺血再灌注＋丙泊酚组（I LIR＋Pro组）;糖尿病大鼠分为：①对照组（ⅡC组）;②肢体缺血再灌注组（ⅡLIR组）;③丙泊酚组（ⅡPro组）;④肢体缺血再灌注＋丙泊酚组（ⅡLIR＋Pro组）,每组8只一采用免疫组织化学法对TNF-α【和NF—KB进行定性、半定量检测。用放射免疫法对TNF-α进行定量检测。结果糖尿病大鼠系列与正常大鼠系列相比,对应的各组NF—KB和TNF-α的表达均升高。两系列中肢体LIR后心肌组织的NF—KB和TNF-α的表达均升高。用丙泊酚后两系列的NF—KB和TNF-α的表达均下降。结论（1）肢体缺血再灌注可引起正常和糖尿病大鼠心肌组织损伤;②糖尿病能加重肢体LIR引起的心肌损伤;③丙泊酚能降低正常和糖尿病大鼠肢体缺血再灌注后心肌组织中NF—KB和TNF-α的表达,对肢体缺血再灌注诱发心肌组织损害有保护作用。     

Influence of clinically used antioxidants on radiation-induced expression of intercellular cell adhesion molecule-1 on HUVEC

Purpose: The effects of antioxidants (N-acetyl-l-cysteine [NAC] and pyrrolidine dithiocarbamate [PDTC]) on radiation-induced ICAM-1 expression on human umbilical vein endothelial cells (HUVEC) were investigated. Materials and methods : The expression of ICAM-1 on HUVEC was determined by flow cytometry up to 72h after X-irradiation. Functional competence of induced ICAM-1 was assessed by adhesion experiments with human polymorphonuclear neutrophils on irradiated HUVEC. Results: Preincubation of cells with both or either NAC and PDTC was unable to reduce radiation-induced ICAM-1 expression on HUVEC. In fact, by themselves, these antioxidants induced a significant increase of ICAM-1 expression, which in comparison with a radiation dose of 7Gy after 24h was nine times higher for PDTC, and more than double for NAC. Treatment with NAC clearly restrained TNF-alpha-induced ICAM expression on HUVEC, while preincubation of cells with PDTC showed synergistic effects. Conclusions: The role of reactive oxygen intermediates in signal transduction pathways leading to ICAM-1 expression should be investigated further. Furthermore, antioxidants may exert a proinflammatory role, as revealed by the induction of ICAM-1 expression on endothelial cells. The inhibition of TNF-alpha-induced ICAM-1 expression by NAC might have clinical implications because this substance is used as a radioprotector in radiotherapy.     

Influence of clinically used antioxidants on radiation-induced expression of intercellular cell adhesion molecule-1 on HUVEC.

PURPOSE: The effects of antioxidants (N-acetyl-L-cysteine [NAC] and pyrrolidine dithiocarbamate [PDTC]) on radiation-induced ICAM-1 expression on human umbilical vein endothelial cells (HUVEC) were investigated. MATERIALS AND METHODS: The expression of ICAM-1 on HUVEC was determined by flow cytometry up to 72 h after X-irradiation. Functional competence of induced ICAM-1 was assessed by adhesion experiments with human polymorphonuclear neutrophils on irradiated HUVEC. RESULTS: Preincubation of cells with both or either NAC and PDTC was unable to reduce radiation-induced ICAM-1 expression on HUVEC. In fact, by themselves, these antioxidants induced a significant increase of ICAM-1 expression, which in comparison with a radiation dose of 7 Gy after 24h was nine times higher for PDTC, and more than double for NAC. Treatment with NAC clearly restrained TNF-alpha-induced ICAM expression on HUVEC, while preincubation of cells with PDTC showed synergistic effects. CONCLUSIONS: The role of reactive oxygen intermediates in signal transduction pathways leading to ICAM-1 expression should be investigated further. Furthermore, antioxidants may exert a pro-inflammatory role, as revealed by the induction of ICAM-1 expression on endothelial cells. The inhibition of TNF-alpha-induced ICAM-1 expression by NAC might have clinical implications because this substance is used as a radioprotector in radiotherapy.     

心肌毛细血管六胺银染色法及缺血预适应对心肌毛细血管的影响

探讨显示心肌毛细血管的方法及用该法观察缺血预适应（ischemic preconditioning,IP)对毛细血管的影响,制备套扎前降支 脉犬模型,缺血再灌注（ischemia-reperfusion,IR)组予缺血1h,再灌注2h;IP组在缺血与再灌注之前,予缺血和再灌注各5min反复4次, 实验终点取缺血心肌进行六胺银染色显示毛细血管,并定量比较,结果六胺银法染色显示心肌毛细管清晰;IP组缺血心肌毛细血管数、周长和面积密度较IR一增加。认为六胺银法显示心肌毛细血管可靠实用;IP减轻缺血后毛细血管的破坏,提示具有毛细血管保护效应。     

大鼠缺血性心肌损伤后早期抑制核因子κB对心功能的影响

目的研究大鼠缺血性心肌损伤后早期抑制核因子κB（NF-κB）对心功能的影响,并探讨其作用机制。方法应用大剂量异丙肾上腺素（ISO）建立大鼠缺血性心肌损伤模型,造模后24 h存活的大鼠随机分为实验组和治疗组,并设立对照组。治疗组给予腹腔注射吡咯烷二硫代氨基甲酸盐（PDTC）100 mg/kg体重,1次/d,连续26 d。检查心肌病理形态学、心肌NF-κB激活情况、心肌毛细血管密度、心肌单核细胞趋化蛋白-1（MCP-1）mRNA表达情况及血清MCP-1水平,并在实验第14、28 d进行超声心动图检查,观察心脏结构及心功能变化情况。结果实验组与治疗组心肌坏死面积无明显差异;PDTC抑制缺血性心肌损伤后心肌NF-κB的激活,下调心肌MCP-1 mRNA的表达及血清MCP-1水平,并导致肉芽组织替代受损心肌组织延迟,心肌坏死边缘区毛细血管密度减低。实验第28 d,治疗组心功能降低较实验组更为明显。结论大鼠缺血性心肌损伤后早期抑制NF-κB,可能对心功能造成不良影响,其机制可能与抑制NF-κB的激活,进而下调MCP-1的表达、影响心肌修复有关。     

维生素E对大鼠心肌缺血再灌注损伤的影响

目的观察大鼠心肌缺血再灌注（MIR）时ICAM-1和自由基代谢的变化及维生素E对其的影响,探讨大鼠MIR损伤的可能机制。方法大鼠30只,分为假手术对照组（sham）、MIR组和维生素E＋MIR组,免疫组织化学法检测心肌ICAM-1蛋白表达。检测血清、心肌组织SOD、MDA和GSH-PX及心肌线粒体Na＋-K＋-ATP酶、Mg＋＋-ATP酶、Ca＋＋-ATP酶活性等自由基代谢指标。结果与sham组相比,MIR组心肌线粒体Na＋-K＋-ATP酶、Mg＋＋-ATP酶、Ca＋＋-ATP酶活性明显下降;用维生素E4周后心肌线粒体Na＋-K＋-ATP酶、Ca＋＋-ATP酶活性明显升高。与sham组相比,MIR组血清、心肌MDA明显升高,血清、心肌SOD和GSH-PX明显降低;用维生素E4周后心肌MDA低于MIR组,血清、心肌SOD和GSH-PX水平高于MIR组。与sham组相比,MIR组心肌ICAM-1蛋白质表达明显升高;用维生素E后心肌ICAM-1蛋白质表达低于MIR组。结论MIR时有大量ICAM-1蛋白表达,与心肌损伤关系密切。维生素E可通过减少ICAM-1蛋白表达水平,起心肌保护作用。在MIR时自由基产生增多,维生素E能加速自由基的清除,增强机体的抗氧化能力,从而保护心肌组织免受氧化损伤。     

High-resolution imaging with (99m)Tc-glucarate for assessing myocardial injury in rat heart models exposed to different durations of ischemia with reperfusion.

(99m)Tc-Glucarate ((99m)Tc-GLA) is a novel infarct-avid imaging agent. The aim of this study was to evaluate the role of (99m)Tc-GLA for assessing the severity of myocardial ischemia-reperfusion injury in rat heart models exposed to varied durations of left coronary artery (LCA) occlusion with reperfusion using a high-resolution SPECT system, FASTSPECT. We also wanted to clarify whether a rapid sequence of 3-dimensional imaging with FASTSPECT can quantify uptake and washout kinetics of cardiovascular imaging agents in small-animal heart models. METHODS: The ischemic-reperfused rat heart models were created by ligating the LCA for 30 min (IR30, n = 12) or 90 min (IR90, n = 6) (IR = ischemia-reperfusion) and releasing the ligature for 30 min. Dynamic images were acquired over a 2-h period after (99m)Tc-GLA was intravenously injected. The ischemic area at risk (IAR) was determined by Evans blue staining. Necrosis was assessed with triphenyltetrazolium chloride (TTC) staining and a transmission electron microscope (TEM). RESULTS: The infarct size of the left ventricle (% IAR) on TTC staining was smaller in IR30 (49.2 +/- 4.3) than in IR90 (73.4 +/- 4.7, P < 0.05), which exhibited evidence of more severe irreversible injury than the IR30 heart on TEM. FASTSPECT images demonstrated hot spot accumulations of (99m)Tc-GLA in all hearts. The washout of (99m)Tc-GLA from the ischemic-reperfused area in IR90 was significantly slower than that in IR30. The ratio of the hot spot to normal myocardial activity was 4.1 +/- 0.3 in IR30 and 7.1 +/- 1.1 in IR90 (P < 0.05). The hot spot size (% IAR) (58.4 +/- 2.7 in IR30 vs. 75.9 +/- 2.7 in IR90, P < 0.05) related significantly to the infarct size. CONCLUSION: The severity of myocardial injury induced by ischemia-reperfusion can be assessed by FASTSPECT imaging with (99m)Tc-GLA. The results suggest that (99m)Tc-GLA will be clinically useful in detecting and quantifying acute necrotic myocardium. The FASTSPECT imaging with the rat heart models provides a solution-specific approach with high-resolution and fast dynamic acquisition for kinetic studies of new myocardial imaging agents.     

预运动训练对脑梗死大鼠脑内谷氨酸含量及其受体mRNA表达的影响

目的:探讨预运动训练对大鼠脑梗死后缺血侧纹状体处谷氨酸(Glu)水平及其代谢型受体(mGluR1)mRNA表达的影响,探讨预运动训练对缺血性脑梗死的保护机制。方法:将Sprague-Dawley大鼠随机分为假手术组、缺血组和缺血前运动组。运动方案采用电动跑台,30mim/d,20m/min,5d/week,共2周。采用微透析技术收集各组大鼠缺血前、缺血期间(40,80和120min)和再灌注后(40,80,120,160,200和240min)的脑细胞外液,用于测定兴奋性氨基酸Glu含量的变化。同时运用RT-PCR技术半定量分析缺血80min和再灌注240min时纹状体脑组织内的mGluR1 mRNA表达水平。结果:缺血组Glu水平在缺血40、80、120min和再灌注40、120、160min时间点与缺血前比较有显著性差异(P<0.05,P<0.01)。运动组各时间点的Glu水平普遍显著低于缺血组(P<0.05,P<0.01),而缺血时各时间点显著高于假手术组(P<0.05,P<0.01)。缺血80min和再灌注240min时缺血组和运动组mGluR1 mRNA表达均显著高于假手术组,运动组显著低于缺血组(P<0.05)。结论:预运动训练对随后发生的脑梗死纹状体内重要的兴奋性氨基酸递质Glu的过度释放有一定程度的抑制作用,这种抑制作用可能与mGluR1 mRNA的下调有某种联系。     

大鼠局灶脑缺血和(或)再灌流模型中IL-1β的表达及其与白细胞浸润的关系

目的　探讨白细胞介素 1(IL 1)在脑缺血和 (或 )再灌流中的动态变化及其与白细胞浸润的关系。方法　采用大鼠大脑中动脉梗塞模型 ,分别用免疫组织化学方法检测IL 1β、酶组化方法检测髓过氧化物酶 (MPO)的动态变化 ,用图象分析进行定量分析。结果　显示缺血 3h及再灌流 0 .5hIL 1β表达增高 (P <0 .0 5 )并逐渐增高 ,2 4h达高峰 (P <0 .0 1) ,在持续缺血 12 0h仍有意义 ,而再灌流 12 0hIL 1β恢复至缺血前水平 (P >0 .0 5 )。缺血组和缺血再灌流组IL 1β与MPO水平呈非常显著正相关 (P <0 .0 1)。结论　提示缺血脑区IL 1β是由局部神经元、星形细胞和血管内皮细胞合成 ,其介导的缺血再灌流损伤与白细胞浸润有密切关系 ,说明在脑缺血和 (或 )再灌流损伤中 ,高浓度的IL 1β是一个重要因素。     

Evaluation of fatty acid metabolism in hearts after ischemia-reperfusion injury using a dual-isotope autoradiographic approach and tissue assay for metabolites of tracer.

We investigated whether changes in myocardial uptake of fatty acid tracer after reperfusion following transient myocardial ischemia were closely related to alterations in intracellular fatty acid oxidation. METHODS: Using a fatty acid tracer of (131)I- and (125)I-labeled 15-(p-iodophenyl)-9-methylpentadecanoic acid (9MPA), the myocardial uptake and metabolites were determined by dual-tracer autoradiography and thin-layer chromatography in rats 3 or 14 d after reperfusion following 5 or 15 min of ischemia induced by coronary artery ligation. RESULTS: 9MPA metabolites processed via beta-oxidation were lower in the ischemic region (IR) than in non-IR 3 d after 5 min of ischemia, despite no reduction of tracer uptake in IR. Oxidation of 9MPA was recovered 14 d after 15 min of ischemia in association with normalization of tracer uptake in IR, whereas both uptake and oxidation of 9MPA were markedly impaired 3 d after 15 min of ischemia, accompanied by slow clearance of myocardial tracer. CONCLUSION: Normal uptake of fatty acid tracer early after reperfusion does not always imply preserved intracellular fatty acid oxidation. However, reduction of tracer uptake might reflect impaired fatty acid oxidation.