小鼠心肌缺血后适应模型的建立及其相关影响因素

目的建立小鼠心肌缺血后适应模型,评价其对缺血再灌注损伤的保护作用以及相关的影响因素。方法120只成年雄性KM小鼠随机接受缺血后适应、缺血再灌注以及假手术3种不同的处理方式。后适应组采用开胸手术结扎左冠状动脉缺血45 min,建立急性心肌梗死模型,在完全再灌注早期给予反复短暂再通/闭塞的后适应。采用Evans blue和TTC染色的方法确定缺血心肌面积以及梗死心肌面积,并测定血清磷酸肌酸激酶(CK)、丙二醛(MDA)及超氧化物歧化酶(SOD)以及血流动力学指标。结果缺血后适应1组、后适应2组以及预适应 后适应组梗死心肌的重量百分比显著小于缺血-再灌注组,P<0.05;后适应2组以及预适应 后适应组与后适应1组比较差异无显著性;延迟后适应组梗死心肌的重量百分比与缺血-再灌注组比较差异无显著性,P>0.05。同时缺血后适应可以明显地降低缺血再灌注血清CK、MDA的浓度,提高SOD的水平以及改善血流动力学的恶化。结论在恢复冠脉血流的早期施行缺血后适应可以有效地减少小鼠再灌注心肌损伤。     

中介素后处理对大鼠肾脏缺血再灌注损伤中肿瘤坏死因子-ɑ与白细胞介素-6表达的影响

目的探讨中介素(IMD)后处理对大鼠肾脏缺血再灌注损伤(IRI)中肿瘤坏死因子(TNF)-ɑ与白细胞介素(IL)-6表达的影响。方法将24只健康雄性SD(Sprasue-Dawley)大鼠随机分为正常对照组、IRI组、0.9%氯化钠注射液组、IMD后处理组。动物切除右肾后,饲养1周后制作肾脏IRI模型(夹闭左肾动脉45 min),24 h后留取肾组织与血清,0.9%氯化钠注射液组于再灌注前5 min 0.9%氯化钠注射液1 ml腹腔注射,IMD后处理组于再灌注前5 min IMD(2 nmol/kg)溶于1ml生理盐水后腹腔注射。过碘酸雪夫(PAS)染色观察肾组织的病理变化,半定量分析肾脏病理损伤;全自动生化仪常规检测血清尿素氮和肌酐;酶联免疫吸附试验(ELISIA)法检测血清IL-6和TNF-ɑ的表达。结果 PAS染色结果显示,IRI组肾小管和间质病理损伤显著重于正常对照组,病理评分为(0.32±0.12),(6.87±0.72)(P<0.05),IMD后处理组病理损伤(病理评分为4.33±0.81)则显著轻于IRI组,表现为细胞坏死,刷状缘脱落及管型减少(P<0.05)。与正常对照组相比,IRI组尿素氮和肌酐均显著增高(P<0.05);与IRI组相比,IMD后处理组尿素氮和肌酐显著下降(P<0.05)。与正常对照组相比,IRI组IL-6与TNF-ɑ的表达显著增高(P<0.05);而与IRI组相比,IMD后处理组IL-6与TNF-ɑ的表达则显著降低(P<0.05)。IRI组与0.9%氯化钠注射液组以上指标相比差异均无统计学意义(P>0.05)。结论 IMD后处理可减轻肾脏缺血再灌注损伤,其机制至少与抑制炎症因子IL-6与TNF-ɑ生成有关。     

中介素后处理对肾脏缺血再灌注损伤后微血管密度及缺氧诱导因子的影响

目的 探讨中介素（IMD）后处理对大鼠肾脏缺血再灌注损伤（IRI）后损伤肾组织微血管密度（MVD）及缺氧诱导因子（HIF-1α）表达的影响.方法 正常雄性SD大鼠24只随机分为：假手术组（Sham组）、IRI组、IRI＋IMD组和IRI＋0.9％氯化钠注射液（NS）组.大鼠右肾切除1周后左肾制作肾脏IRI模型.各组分别于恢复血流后的24 h留取血液及肾组织标本.检测血清的尿素氮和肌酐水平;过碘酸雪夫（PAS）染色观察肾组织的病理变化;CD31免疫组织化学标记MVD计数;实时荧光定量-聚合酶链反应（RT-PCR）法检测HIF-1α mRNA的表达.结果 IRI、IRI＋NS、IRI＋IMD组的尿素氮、肌酐水平和肾小管及间质病理损伤均明显高于Sham组（均P＜0.01）;IRI＋IMD组的尿素氮、肌酐水平以及肾小管和间质病理损伤也较IRI和IRI＋NS组显著减低或减轻（均P＜0.01）.与Sham组相比,IRI及IRI＋NS组肾间质的MVD增多;IRI＋IMD组肾间质的MVD较IRI及IRI＋NS组增多更为明显（均P＜0.01）.RT-PCR结果显示：IRI及IRI＋NS组HIF-1α mRNA表达较Sham组增多;IRI＋IMD组HIF-1α mRNA表达较IRI及IRI＋NS组显著增多（均P＜0.01）.结论 中介素作为后处理药物能够在肾脏IRI后增加缺血组织的MVD并上调血管生成因子HIF-1α的表达,从而参与损伤肾组织的血管新生.     

PI3K/Akt信号转导通路在乌司他丁后处理减轻CPB下心脏瓣膜置换术患者心肌细胞凋亡中的作用

目的 探讨磷脂酰肌醇3-激酶(PI3K)/丝氨酸-苏氨酸蛋白激酶(Akt)信号转导通路在乌司他丁后处理减轻CPB下心脏瓣膜置换术患者心肌细胞凋亡中的作用.方法 择期CPB下心脏瓣膜置换术患者40例,性别不限,年龄21 ～ 59岁,心功能和ASA分级Ⅱ或Ⅲ级.采用随机数字表法,将患者分为2组(n=20):生理盐水对照组(C组)和乌司他丁后处理组(U组).U组于主动脉开放前5min经主动脉根部灌注乌司他丁4000～5000 U·kg-1 ·min-1(剂量1万U/kg);C组给予等容量生理盐水.于升动脉开放后45 min时取右心耳心肌组织,检测Akt、磷酸化Akt(p-Akt)与细胞色素c、caspase9、Bcl-2、Bax表达和细胞凋亡情况,并计算Bcl-2与Bax表达的比值(Bcl-2/Bax)和凋亡指数(AI).结果 与C组比较,U组心肌组织p-Akt与Bcl-2表达上调,心肌组织细胞色素c、caspase-9及Bax表达下调,Bcl-2/Bax升高,AI降低(P＜0.05).结论 乌司他丁后处理通过激活PI3K/Akt信号转导通路减轻CPB下心脏瓣膜置换术患者心肌细胞凋亡.     

米诺环素后处理通过抑制PARP过度活化减轻心肌缺血/再灌注损伤

目的:探讨米诺环素后处理能否通过抑制多腺苷二磷酸核糖聚合酶1(PARP-1)过度活化减轻大鼠心肌缺血/再灌注(I/R)损伤。方法:结扎大鼠冠状动脉左前降支45 min,再灌注2 h,建立心肌I/R模型。将90只雄性Wistar大鼠随机分为假手术(sham)组,I/R组,低、高剂量米诺环素组及PARP抑制剂3-氨基苯甲酰胺(3-AB)组。氯化三苯基四氮唑(TTC)和伊文思蓝双染法检测心肌梗死范围,HE染色观察心肌组织形态学改变,TUNEL法评估心肌细胞凋亡程度,酶联免疫吸附法测定血清肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)含量,Western blot法检测再灌注心肌及外周血白细胞内PARP-1活化产物多腺苷二磷酸核糖(PAR)的表达。结果:与sham组比较,心肌、外周血白细胞内PAR表达及血清TNF-α、IL-1β含量明显升高。与I/R组比较,米诺环素低、高剂量及3-AB后处理组均能显著减少梗死范围及心肌细胞凋亡程度,同时明显降低心肌、外周血白细胞内PAR表达及血清TNF-α、IL-1β含量。米诺环素高剂量组与3-AB组比较无显著差异。结论:米诺环素后处理可能通过抑制心肌及外周血白细胞PARP过度活化减轻大鼠心肌I/R损伤。     

Postconditioning the Isolated Working Rat Heart

PURPOSE: Despite the attention focussed on postconditioning (postC; brief cycles of reperfusion/ischaemia at the onset of reperfusion, conferring cardioprotection against reperfusion injury), an infarct sparing effect for postC in the isolated working heart model has not been reported. The purpose of this study was to develop a cardioprotective postC protocol in this model. METHODS: Hearts from male Wistar rats (210-350 g) were perfused either retrogradely (Langendorff) or in the working mode. For functional studies 30 or 35 min global ischaemia (GI) and 20 or 25 min GI were applied in the Langendorff and working heart models respectively. Infarct size was measured after 35 min regional ischaemia (RI) in both models. In the latter studies hearts were subdivided into low (36.5 degrees C) and high (37 degrees C) temperature groups (during both ischaemia and initial reperfusion). In all groups hearts were either freely reperfused (nonPostC) or postconditioned (postC) by 6 x 10 s ischaemia/reperfusion cycles. RESULTS: In both perfusion modes postC only elicited an infarct sparing effect after a slight elevation in temperature to 37 degrees C (Langendorff: L-nonPostC = 47.99 +/- 3.31% vs. L-postC = 27.81 +/- 2.49%, p < 0.0001; and work = W-nonPostC: 35.81 +/- 3.67% vs. W-postC = 17.74 +/- 2.72%, p < 0.001). However, only in the Langendorff group could postC conserve post-ischaemic function, while no significant recoveries were seen in the working hearts. CONCLUSION: We demonstrated an infarct sparing effect for postC in the working heart model, which unlike the Langendorff model, was not associated with functional preservation. The infarct sparing effect of postC in both models was however extremely sensitive to even slight fluctuations in temperature.     

低氧诱导因子-1α在后处理心肌保护效应中的作用

目的 探讨后处理减轻心肌缺血/再灌注损伤的效应与低氧诱导因子-1α（HIF-1α）的关系.方法 建立大鼠心肌缺血/再灌注及后处理模型,以心肌梗死面积、乳酸脱氢酶（LDH）活性来反映心肌的损伤程度,运用Western blot、real time-PCR等方法检测心肌组织中HIF-1α的表达情况,以探讨其是否参与了后处理的心肌保护效应.结果 后处理缩小了缺血/再灌注所致的心梗面积,降低了血清LDH活性,同时上调了心肌组织中HIF-1α的表达.预先给予HIF-1α脯氨酸羟化酶抑制剂DMOG使HIF-1α表达上调后,后处理减轻心肌损伤的效应进一步增强.结论 后处理减轻缺血/再灌注所致心肌损伤的效应可能与其上调心肌组织中HIF-1α的蛋白表达有关.     

The effects of short-interval postconditioning in preventing testicular ischemia-reperfusion injury in rats

Aim

Testicular torsion can lead to testicular damage. During reperfusion, tissue damage is more severe. The aim of this study was to investigate the protective effect of short-interval postconditioning and determine the optimal time of reperfusion for postconditioning.

Materials and Methods

Thirty-five adult male rats were divided into 5 subgroups: Sh (sham operated), TD (torsion + detorsion), PC5 (torsion + postconditioning 5 seconds), PC10 (torsion + postconditioning-10 seconds), PC20 (torsion + postconditioning 20 seconds). Torsion was created by rotating the left testis counterclockwise 1080° and the testis fixed to the scrotum with 3 sutures. Torsion was maintained for 4 hours. The testicular artery was visualized, and before detorsion of the testis, an atraumatic vessel clamp was applied to prevent reperfusion in all study groups. Then, detorsion of the testis was performed. In the TD group, the clamp was released just after detorsion; in the PC5 group, the clamp was released for 5 seconds and closed for 10 seconds (10 times); in the PC10 group, the clamp was released for 10 seconds and closed for 10 seconds (10 times); and in the PC20 group, the clamp was released for 20 seconds and closed for 10 seconds (10 times). Then, all testes were reperfused for a 1-hour period in all study groups. After this period, the rats were sacrificed, and the left testes were removed and evaluated histopathologically and biochemically. The Mann-Whitney U test was used for statistical analyses.

Results

Tissue malondialdehyde levels were 79.3 ± 10.6, 231.7 ± 102.3, 71.3 ± 12.6, 73.8 ± 13.7, and 124.3 ± 48.0 nmol/g tissue in the Sh, TD, PC5, PC10, and PC20 groups, respectively. Tissue malondialdehyde levels were significantly lower in the PC5 and PC10 groups (P < .05) compared to the other groups. However, mean histopathologic grade was lower in all postconditioning groups compared to the control group, but the difference was significant only in the PC5 group (P < .05).

Conclusion

We conclude that short-interval postconditioning can reduce reperfusion injury in ischemic tissue and the optimal mode of short-interval postconditioning is 5 seconds × 10 times. This technique seems easily applicable, and a similar technique may be used during testicular surgery.     

七氟醚预处理联合后处理对大鼠心肌缺血再灌注时血栓素A2和前列腺素I2的影响

目的 评价七氟醚预处理联合后处理对大鼠心肌缺血再灌注时血栓素A2和前列腺素I2的影响.方法 健康雄性Wistar大鼠50只,体重250～280 g,采用随机数字表法,将大鼠随机分为5组(n=10):假手术组(S组)、缺血再灌注组(I/R组)、七氟醚预处理组(Spr组)、七氟醚后处理组(Spo组)和七氟醚预处理联合七氟醚后处理组(Spr+po组).I/R组、Spr组、Spo组和Spr+po组采用结扎左冠状动脉前降支30 min时进行再灌注的方法制备心肌缺血再灌注模型,S组仅在左冠状动脉前降支下穿线.Spr组进行七氟醚预处理:于缺血前30 min吸入2.5%七氟醚15 min,洗脱15 min;Spo组进行七氟醚后处理:再灌注前1 min开始吸入2.5%七氟醚,持续5 min;Spr+po组进行七氟醚预处理和后处理.再灌注2 h时取动脉血样,测定血MB型磷酸肌酸激酶同工酶(CK-MB)、乳酸脱氢酶(LDH)、心肌肌钙蛋白I(cTnI)、血栓素B2(TXB2)、6-酮-前列腺素F1α(6-keto-PGF1α)的水平和血小板最大聚集率,并计算TXB2与6-keto-PGF1α的比值(TXB2/6-keto-PGF1α).取心肌组织,电镜下观察病理学结果,进行线粒体损伤评分,并测定线粒体的比表面和面数密度.结果 与S组比较,I/R组血CK-MB、LDH、cTnI、TXB2、6-keto-PGF1α的水平、TXB2/6-keto-PGF1α血小板最大聚集率及线粒体损伤评分升高,线粒体的比表面和面数密度降低(P<0.05或0.01);与I/R组比较,Spr组和Spo组血CK-MB、LDH、cTnI的水平、TXB2/6-keto-PGF1α和线粒体损伤评分降低,血6-keto-PGF1α浓度、线粒体的比表面和面数密度升高(P<0.05或0.01);与Spr组和Spo组比较,Spr+po组血CK-MB、LDH、cTnI、TXB2的水平、TXB2/6-keto-PGF1α血小板最大聚集率和线粒体损伤评分降低,血6-keto-PGF1α浓度、线粒体的比表面和面数密度升高(P<0.05).Spr+po组心肌损伤程度轻于Spr组和Spo组.结论 与七氟醚预处理或后处理比较,两种方法联合应用可抑制血栓素A2的释放和促进前列腺素I2的释放,从而进一步减轻了大鼠心肌缺血再灌注损伤.

Abstract：

Objective To investigate the effect of sevoflurane preconditioning-postconditioning on thromboxane A2 and prostaglandin I2 during myocardial ischemia-reperfusion (I/R) in rats. Methods Fifty healthy male Wistar rats weighing 250-280 g were randomly divided into 5 groups (n = 10 each) : sham operation group (group S) , I/R group, sevoflurane preconditioning group (group Spr), sevoflurane postconditioning group (group Spo)and combination of sevoflurane preconditioning and postconditioning group (group Spr + po). Myocardial I/R was produced by occlusion of anterior descending branch of left coronary artery for 30 min followed by 2 h reperfusion in anesthetized rats. In group S the anterior descending branch was only exposed but not ligated. Group Spr received 15 min inhalation of 2.5 % sevoflurane and 15 min wash-out 30 min before ischemia. Group Spo received 5 min inhalation of 2.5% sevoflurane 1 min before reperfusion. Arterial blood samples were taken at 2 h of reperfusion for determination of the levels of MB isoenzyme of creatine kinase (CK-MB) , lactate dehydrogenase (LDH) , cardiac troponin I (cTnI), thromboxane B2(TXB2), and 6-keto-prostaglandin (6-keto-PGF1α) and platelet maximum aggregation rate. TXB2/6-keto-PGF1α ratio was calculated. The myocardial tissues were taken for microscopic examination. Mitochondria] injury was assessed by using Flameng score and stereology (Specific surface, δ and Numerical density on area, NA) .Results Compared with group S, the levels of CK-MB, LDH, cTnI, TXE2 and 6-ketoPGF1α, TXB2/6-keto-PGF1α ratio, platelet maximum aggregation rate and Flameng score were significantly increased, while δ and NA were significantly decreased in group I/R (P < 0.05 or 0.01) . The levels of CK-MB,LDH and cTnI, TXB2/6-keto-PGF1α ratio and Flameng score were significantly lower, and 6-keto-PGF1α level, δand NA were significantly higher in Spr and Spo groups than in group I/R ( P < 0.05 or 0.01) . The levels of CKMB, LDH, cTnI and TXB2 , TXB2/6-keto-PGF1α ratio, platelet maximum aggregation rate and Flameng score were significantly lower and 6-keto-PGF1α level,δ and NA were significantly higher in group Spr + po than in Spr and Spo groups(P < 0.05). Conclusion Sevoflurane preconditioning-postconditioning can reduce myocardial I/R injury through inhibiting the release of thromboxane A2 and promoting the release of prostaglandin I2 in rats.