后适应在兔心肌缺血再灌注损伤中的实验研究

目的：观察缺血后适应对兔心肌缺血再灌注损伤（IRI）的保护作用。方法将30只新西兰大白兔随机分为缺血-再灌注组（IR组）,缺血后适应组,后适应加5-羟基癸酸盐（5-HD）组,延迟后适应组及假手术组5组,每组6只。采用硫代巴比妥酸比色法检测5组大白兔心肌组织丙二醛含量,依文思兰加氯化四氮唑兰双重染色法及称重法测定心肌梗死范围大小。结果丙二醛含量缺血后适应组显著低于IR组和延迟后适应组（均P＜0.01）,与后适应加5-HD组相近,但显著高于假手术组（P＜0.05）。心肌梗死面积在IR组、后适应加5-HD组、延迟后适应组之差异无统计学意义（P＞0.05）,但大于缺血后适应组,差异均有显著统计学意义（均P＜0.01）。结论缺血后适应在兔心肌IRI模型中有明显的心肌保护作用;特异性线粒体ATP敏感钾通道抑制剂5-HD可完全取消后适应的心肌保护作用;延迟5min后实行后适应操作其保护作用完全消失。     

Postconditioning of the small intestine: which is the most effective algorithm in a rat model?

Background

Mesenteric ischemia is a serious clinical condition requiring immediate surgical intervention. The unavoidable ischemic-reperfusion (IR) injury may be ameliorated using the appropriate postconditioning protocol. The aim of the present study was to investigate the optimal postconditioning algorithm in a rat model of intestinal ischemic–reperfusion injury.

Materials and methods

Male Wistar rats were randomized into five groups (n = 10), one sham-operated, one IR, and three postconditioned groups, each with different protocols. The animals were subjected to 60 min of mesenteric ischemia, followed by 60 min of reperfusion. Postconditioning was applied at the onset of reperfusion using three different algorithms. Arterial pressure and mucosal microcirculation were monitored throughout the experiment. Mesenteric pH was determined at the early phase of reperfusion. Blood and tissue samples were taken at the end of reperfusion for histologic evaluation, serum lactate dehydrogenase, serum creatine kinase, serum tumor necrosis factor-α, serum interleukin-6, detailed mucosal antioxidant, and scavenger capacity assays.

Results

The shorter and intermediate length cycles of postconditioning enhanced mucosal microcirculation and redox state and significantly delayed the normalization of mesenteric pH. Furthermore, milder histopathologic lesions and lower concentrations of serum necroenzymes and proinflammatory cytokines were detected compared with the IR group. The protective effect of postconditioning using longer cycles could only be seen in a tendentious manner.

Conclusions

In a rat model of intestinal ischemia–reperfusion, the shorter and intermediate length cycles of postconditioning proved to be more effective than the use of longer cycles.     

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.     

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。     

Nrf2-ARE通路在缺血后处理和吡那地尔后处理减轻大鼠离体心脏缺血再灌注损伤中的作用

目的 探讨核转录因子NF-E2相关因子2(Nrf2)-抗氧化反应元件(ARE)通路在缺血后处理和吡那地尔后处理减轻大鼠离体心脏缺血再灌注损伤中的作用.方法 健康雄性SD大鼠,体重200～250 g,4～5月龄,采用Langendorff灌注装置建立离体心脏灌注模型,取模型制备成功的心脏56个,采用随机数字表法,将其随机分为7组(n=8):正常对照组(C组)、缺血再灌注组(I/R组)、缺血后处理组(IP组)和不同浓度的吡那地尔后处理组(PP1组、PP2组、PP3组、PP4组).平衡灌注20min后,C组继续灌注100 min;I/R组停止灌注40 min,复灌60min;IP组停止灌注40 min,于再灌注开始给予6次间隔灌注10 s停止灌注10 s,复灌58 min;PP1组、PP2组、PP3组、PP4组停止灌注40 min,于再灌注开始时灌注含5、10、30、50 μmol/L吡那地尔的K-H液5min,继续灌注55 min.于平衡灌注末及复灌结束时测定左室发展压(LVDP)和左室舒张期末压(LVEDP);于复灌结束时取左心室心肌,分别采用RT-PCR法和Western blot法检测心肌Nrf2、醌氧化还原酶1(NQO1)、超氧化物歧化酶1(SOD1)及血红素加氧酶1(HO-1)的mRNA及蛋白表达水平.结果 与C组比较,其余各组Nrf2、NQO1、SOD1及HO-1的mRNA和蛋白表达均上调,I/R组、PP1组和PP2组复灌结束时LVDP降低,LVEDP升高(P＜0.05).与I/R组比较,IP组、PP3组和PP4组Nrf2、NQO1、SODI及HO-1的mRNA和蛋白表达均上调,IP组、PP1组、PP2组、PP3组和PP4组复灌结束时LVDP升高,LVEDP降低(P＜0.05).结论 缺血后处理和吡那地尔后处理可通过激活Nrf2-ARE通路减轻大鼠心肌缺血再灌注损伤.     

舒芬太尼后处理对大鼠心肌缺血再灌注时细胞凋亡的影响

目的 探讨舒芬太尼后处理对大鼠缺血再灌注时心肌细胞凋亡的影响.方法 健康雄性SD大鼠36只,体重250 ～ 280 g,采用随机数字表法,将大鼠随机分为3组(n=12):假手术组(S组)、缺血再灌注组(I/R组)和舒芬太尼后处理组(SP组).采用结扎左冠状动脉30 min,再灌注120min的方法制备心肌缺血再灌注模型.S组只挂线不结扎左冠状动脉;SP组于再灌注即刻静脉注射舒芬太尼3.0 μg/kg.于缺血再灌注期间记录HR和MAP.于再灌注120 min时,处死大鼠,取心脏,测定心肌梗死面积,采用RT-PCR测定心肌Bax mRNA和Bcl-2 mRNA的表达,采用TUNEL法检测心肌凋亡细胞,计算凋亡指数.结果 三大鼠各时点HR比较差异无统计学意义(P＞0.05);与S组比较,I/R组心肌缺血再灌注期间MAP降低(P＜0.05),SP组差异无统计学意义(P＞0.05),I/R组和SP组凋亡指数和Bax mRNA表达水平升高,I/R组Bcl-2 mRNA表达水平降低,SP组Bcl-2 mRNA表达水平升高(P＜0.05);与I/R组比较,SP组心肌梗死面积、凋亡指数和Bax mRNA表达水平降低,Bcl-2 n.RNA表达水平升高(P＜0.05).结论 舒芬太尼后处理可能通过上调Bcl-2表达,下调Bax表达,抑制细胞凋亡,从而减轻大鼠心肌缺血再灌注损伤.     

三七总皂苷后处理减轻兔失血性休克复苏期心肌损伤

目的 探讨三七总皂苷（PNS）后处理对兔失血性休克复苏期心肌损伤的影响和机制。

方法 选择雄性新西兰兔30只，11周龄，体质量2.5～3.5 kg。随机分为五组：假手术组（S组）、失血性休克复苏组（H组）、PNS后处理组（P组）、18-β甘草酸（18-AGA）+PNS后处理组（AP组）、18-AGA组（A组），每组6只。S组行气管插管，右侧颈总动脉和右侧股静脉置管，不做放血处理。H组、P组、AP组和A组行气管插管后，制备失血性休克复苏模型。P组于造模成功后45 min给予PNS 100 mg/kg，AP组于造模前腹腔注射18-AGA 75 mg/kg，于造模成功后45 min给予PNS 100 mg/kg，A组造模前腹腔注射18-AGA 75 mg/kg。于复苏1 h时取兔心肌组织，采用Western blot法检测线粒体Cx43含量，采用TUNEL法检测心肌细胞凋亡率，通过透射电镜观察心肌线粒体超微结构，进行线粒体超微结构评分。

结果 与S组比较，H组、AP组、A组心肌线粒体Cx43相对含量明显降低（P＜0.05），H组、P组、AP组、A组心肌细胞凋亡率明显升高，线粒体超微结构评分明显升高（P＜0.05）。与H组比较，P组心肌线粒体Cx43相对含量明显升高，心肌细胞凋亡率明显降低（P＜0.05），AP组、A组心肌线粒体Cx43相对含量明显降低（P＜0.05），P组和AP组粒体超微结构评分明显降低（P＜0.05）。与P组比较，AP组、A组心肌线粒体Cx43相对含量明显降低，心肌细胞凋亡率明显升高，线粒体超微结构评分明显升高（P＜0.05）。S组心肌线粒体膜完整光滑，H组线粒体嵴断裂不清，线粒体完整性丧失，P组线粒体稍肿胀但结构相对完整，AP组线粒体肿胀明显，A组线粒体嵴断裂，线粒体完整性丧失。

结论 三七总皂苷后处理可通过上调线粒体Cx43表达减轻兔失血性休克复苏期心肌损伤。     

盐酸法舒地尔后适应对大鼠心肌缺血/再灌注损伤的影响及其机制

目的 通过主动脉根部模拟冠状动脉内给药,观察盐酸法舒地尔后适应对大鼠急性心肌缺血/再灌注损伤的保护作用,并探讨其机制.方法 60只SD大鼠随机分为假手术组、缺血再灌注组、PI3K抑制剂组、盐酸法舒地尔组、盐酸法舒地尔+ PI3K抑制剂组,每组12只.各组于再灌注180 min后处死大鼠,分别测定心功能参数、血浆心肌酶、细胞凋亡指数和心肌梗死范围.结果 盐酸法舒地尔组心肌细胞凋亡指数、心肌梗死范围与缺血再灌注组比较明显减少(P＜0.01)；给予PI3K抑制剂,盐酸法舒地尔的后适应效应消失,即盐酸法舒地尔+PI3K抑制剂组心肌细胞凋亡指数、心肌梗死范围与盐酸法舒地尔组比较明显增加(P＜0.01).结论 盐酸法舒地尔后适应的机制可能与激活PI3K-Akt传导通路有关.