缺血后处理对抗大鼠离体心脏缺血再灌注损伤及其作用机制

目的： 研究缺血后处理（postconditioning）对抗大鼠离体心脏缺血再灌注损伤及其作用机制。方法：采用SD大鼠心肌缺血/再灌注模型，并于再灌注一开始即给予3次全心停灌30 s，再灌30 s处理作为缺血后预处理。记录心肌收缩功能指标，以Even’s blue-TTC法监测心肌梗死范围，并对心律失常严重程度进行定量分析。结果：缺血后处理组左室峰压（LVSP）、最大左室收缩速率（+dp/dt_max）以及心率明显高于缺血对照组。缺血后处理可明显缩小心肌梗死范围（22.97%±3.96% vs 缺血对照组 44.30%±13.61%，P<0.01）。观察复灌10 min时心律失常评分发现，缺血后处理组明显低于缺血对照组。缺血后处理组和缺血预处理组具有类似的心肌保护作用。5-HD组LVSP和+dp/dt_max低于缺血后处理组，心律失常评分增高，心肌梗死范围扩大。结论: 缺血后处理对大鼠缺血再灌注损伤具有心脏保护作用，其作用机制可能是部分通过激活线粒体ATP依赖性钾离子(mitoK_ATP)通道起作用。     

缺血后处理对离体大鼠心肌线粒体功能的影响

目的：建立离体大鼠心肌缺血/再灌注损伤模型,观察缺血后处理对大鼠心肌线粒体功能的影响,并探讨线粒体ATP敏感性钾通道（mitoK_ATP）在缺血后处理心肌保护中的作用。方法：采用Langendorff装置建立离体大鼠心肌缺血/再灌注损伤模型。将SD大鼠随机分为对照组（C）、模型组（M）、缺血后处理组（IPO）、5-羟癸酸拮抗缺血后处理组（5-HD+IPO）,每组8只。各组均先灌注平衡20 min,C组：续灌70 min;M组：缺血前灌注4 ℃ St.Thomas停跳液（10 mL/kg）,全心缺血40 min,复灌30 min;IPO组：全心缺血40 min,复灌前先开放10 s,缺血10 s,反复6次,时间为2 min,复灌28 min;5-HD+IPO组：缺血后处理前给予含5-羟癸酸（100 μmol/L）的K-H液灌注5 min,余同IPO组,复灌23 min。观察各组平衡末与再灌注末心肌线粒体膜电位、氧自由基及呼吸功能的变化。结果：(1) 各组再灌注末心肌线粒体膜电位较平衡末显著降低,而C组显著高于其它3组,IPO组明显高于5-HD+IPO与M组,5-HD +IPO组高于M组。(2) 各组再灌注末与平衡末比较,心肌线粒体氧自由基含量显著升高,其中M组显著高于其它3组,5-HD +IPO组高于IPO及C组,IPO组高于C组。(3) 各组再灌注末较平衡末线粒体呼吸功能明显受损,且C组优于其它3组,IPO组优于5-HD+IPO与M组,5-HD +IPO组优于M组。结论：(1) 缺血后处理通过维护线粒体膜电位稳定、减少线粒体氧自由基的产生、保护线粒体呼吸链及功能,减轻心肌的再灌注损伤。(2) 5-HD不能完全阻断缺血后处理的心肌保护作用。(3) 缺血后处理的心肌保护效应可通过激活心肌mitoK_ATP实现,同时还有其它因素参与了缺血后处理的心肌保护。     

缺血后处理减轻兔缺血再灌注心肌细胞损伤

目的探讨缺血后处理对在体兔心肌缺血再灌注心肌细胞凋亡和线粒体结构与功能的影响以及可能机制。方法80只兔随机分为假手术组(sham组)、心肌缺血再灌注组(IR组)、缺血预处理组(IP组)、缺血后处理组(PC组)以及缺血后处理加5-羟葵酸(5-HD)干预组(PC+5-HD组)。用TUNEL法检测心肌细胞凋亡,用透射电镜观察心肌细胞的超微结构,用荧光法检测线粒体膜电位,比色法测线粒体Ca2+浓度、丙二醛(MDA)浓度、超氧化物岐化酶(SOD)活性。结果与IR组比较,PC组和IP组兔心肌细胞凋亡减少,心肌及线粒体形态结构改变明显减轻,线粒体跨膜电位、SOD活性明显升高、线粒体Ca2+浓度、MDA浓度均下降(P<0.05),5-HD部分降低上述作用。结论PC可能通过提高线粒体跨膜电位、降低线粒体氧自由基水平、减轻线粒体钙超载而减轻心肌细胞损伤,其机制可能与线粒体功能损伤有关。     

缺血后处理抑制内质网应激PERK通路减轻大鼠离体心脏缺血/再灌注损伤

目的探讨缺血后处理对大鼠离体心脏缺血再灌注损伤的作用及相关机制。方法 36只Wistar大鼠(257-326g),随机分为3组,对照组(Con组),缺血再灌注组(IR组),缺血后处理组(IPo C组)。采用Langendorff灌流装置建立缺血再灌注损伤模型,TTC染色评价梗死面积,Western blot检测凋亡蛋白及内质网应激相关通路蛋白表达。结果 IR组较Con组梗死面积增加,而Bcl-2表达显著降低,GRP78、CHOP、cleaved caspase12、cleaved caspase3、Bax及p-PERK表达水平显著增加。缺血后处理显著缩小梗死面积,并部分逆转相关蛋白表达的变化。结论缺血后处理减轻大鼠离体心脏缺血再灌注损伤,可能与抑制PERK通路介导的ERS相关凋亡相关。     

无创伤双后肢缺血后处理对移植胰腺缺血再灌注损伤的远隔保护

背景：缺血预处理及缺血后处理是近年来提出减轻缺血再灌注损伤有效方法。 目的：探讨无创伤双后肢缺血后处理对移植胰腺缺血再灌注损伤的影响及机制。 方法：18只糖尿病SD大鼠数字表法随机分为3组,对照组仅行开腹术;缺血再灌注组仅行胰腺移植;缺血后处理组,移植前行非创伤性双后肢缺血后处理。 结果与结论：缺血再灌注组血糖和胰腺组织中丙二醛水平均高于缺血后处理组(P < 0.01)、而超氧化物歧化酶活性低于缺血后处理组(P < 0.01);与缺血后处理组比较,缺血再灌注组胰腺组织凋亡指数明显增高(P < 0.01)。结果提示,无创伤双后肢缺血后处理对大鼠移植胰的缺血再灌注损伤具有保护作用,机制可能与可通过减少超氧化物歧化酶失活,从而清除氧自由基以及减少胰腺细胞凋亡等有关。     

缺血后处理对高血脂大鼠缺血/再灌注血清sICAM的影响

目的 研究缺血后处理对高血脂大鼠缺血/再灌注血清sICAM的影响.方法 选择高血脂SD大鼠36只,随机分为3组:假手术组、缺血再灌注组(对照组)和缺血后处理组,每组12只.制备大鼠心肌缺血再灌注模型.结果 ①血清中肌酸激酶活性的测定:再灌注结束后缺血后处理组和缺血再灌注组肌酸激酶活性明显高于假手术组,缺血后处理组明显低于对照组.②血清sICAM含量:缺血后处理组和对照组血清sICAM含量均高于假手术组(P<0.05).缺血后处理组与对照组相比血清sICAM含量降低(P<0.05).结论 缺血后处理可减轻高血脂大鼠缺血再灌注损伤,其机制可能与抑制白细胞的粘附有关.     

二氮嗪后处理对离体大鼠心功能及线粒体心磷脂的影响

目的:建立离体大鼠心肌缺血/再灌注损伤模型,观察二氮嗪(diazoxide,D)后处理对缺血/再灌注损伤离体大鼠心功能及线粒体心磷脂的影响,并探讨ATP敏感性钾通道在二氮嗪后处理心肌保护中的作用。方法:采用Langendorff装置建立离体大鼠心肌缺血/再灌注损伤模型,将SD大鼠随机分为对照组(control)、缺血再灌注模型组(I/R)、二氮嗪后处理组(I/R+D)、5-羟葵酸拮抗二氮嗪后处理组(I/R+5-HD+D),每组8只,均先灌注平衡20 min。Control组:灌注平衡后续灌70 min;I/R组:缺血前灌注4℃ST.Thomas停跳液,全心缺血40 min,再灌30 min;I/R+D组:全心缺血40 min,缺血后给予含二氮嗪(50μmol/L)的K-H液灌注5 min后,再灌25 min;I/R+5-HD+D组:二氮嗪后处理前给予含5-羟葵酸(100μmol/L)的K-H液灌注5 min,再灌20 min。观察各组续(再)灌注末心率、冠脉流出液量、心功能、心肌酶学及心肌线粒体心磷脂的变化。结果:各组续(再)灌注末比较,I/R组较control组及I/R+D组心率减慢、冠脉流出液量降低,心功能明显受损,心肌酶增加,心磷酯含量减少,但与I/R+5-HD+D无明显差异。结论:二氮嗪后处理通过增加线粒体心磷脂含量,减少心肌酶的释放,改善心脏功能,减轻心肌的再灌注损伤,产生心肌保护作用。5-羟葵酸能够完全阻断二氮嗪的心肌保护作用。     

无创性延迟肢体缺血预适应减轻大鼠脑缺血/再灌注损伤

目的 研究无创性延迟肢体缺血预适应(NDLIP)对大鼠脑缺血/再灌注后氧化损伤的保护作用及其作用机制。方法 连续3d,每天1次3个循环大鼠左后肢无创性5min缺血、5min再灌注,建立NDLIP模型。实验分4组：假手术组、缺血再灌（I/R）组、早期脑缺血预适应+I/R（ECIP+I/R）组及NDLIP+I/R组。进行神经行为评分,检测脑梗死范围,测定再灌末脑组织中海马和皮层超氧化物歧化酶(SOD)以及丙二醛(MDA)含量。结果 与I/R组相比,ECIP+I/R组及NDLIP+I/R组缺血1h,再灌24h后评分有非常显著的降低(P<0.01);脑梗死范围显著减小(P<0.01)。与I/R组比较,ECIP+I/R 组和NDLIP+I/R组的海马和皮层的T-SOD和Mn-SOD活力均升高(P<0.01); MDA含量明显减少(P<0.01)。结论 对脑缺血/再灌注后的氧化损伤,NDLIP具有与ECIP程度相当的保护作用。     

缺血预处理联合缺血后处理对肺移植中缺血再灌注肺损伤的影响

背景：研究显示缺血预处理和缺血后处理在缺血再灌注肺损伤中均具有明显的保护作用。 目的：观察缺血预处理联合缺血后处理对肺移植中缺血再灌注损伤的累积保护效应。 方法：将40只SD大鼠随机等分为假手术组、模型组、缺血预处理组、缺血后处理组和联合处理组。后4组建立缺血再灌注肺损伤动物模型,缺血预处理组、缺血后处理组和联合处理组在造模前或/和造模后反复3次阻断开放左侧肺门。 结果与结论：与缺血预处理组和缺血后处理组相比,联合处理组大鼠肺组织的干质量比、丙二醛和髓过氧化物酶降低(P < 0.05),而肺组织中超氧化物歧化酶活性升高(P < 0.05),肺组织病理损伤程度明显减轻;缺血预处理组与缺血后处理组大鼠肺组织超氧化物歧化酶活性、丙二醛水平及髓过氧化物酶活性接近(P > 0.05),且肺组织病理损伤程度基本相似。而且缺血预处理与联合处理组中超氧化物歧化酶、丙二醛和髓过氧化物酶水平呈正相关。提示缺血预处理和缺血后处理联合应用对于减轻中性粒细胞的侵润和激活及氧化反应对于肺组织的损伤有明显的累积保护效应,从而可以更好的减轻肺缺血再灌注损伤程度。     

不同缺血后处理方案对大鼠肾脏缺血再灌注损伤的保护作用

目的 试建立恰当的大鼠肾脏缺血再灌注损伤（IRI）的缺血后处理模型。方法 选8 ～ 12周健康60只SD雄性大鼠,体质量220 ～ 260 g。随机分为6组：对照组（S组）、缺血再灌注组（IR组）及IR缺血后处理A、B、C、D组,每组10只。S组：分离出双侧肾蒂,仅行右肾蒂结扎和左肾蒂游离;IR组：结扎右侧肾蒂,夹闭左侧肾蒂60 min后恢复灌注;缺血后处理A、B、C、D组在肾脏缺血60 min后分别进行6次（开放10 s ＋ 阻断10 s）、5次（开放20 s ＋ 阻断20 s）、3次（开放10 s ＋ 阻断10 s）、3次（开放2 min ＋ 阻断2 min）的循环,然后充分开放灌注。再灌注24 h后监测肾功能,对肾组织结构损伤程度评分。结果 与IR组相比,A、B、C、D组后处理的血尿素氮（BUN）、血肌酐（SCr）及肾小管损伤程度评分均降低（P 〈 0.05）,肾组织损伤明显减轻。处理组中,A、B两组的BUN、SCr及肾小管损伤程度评分相当（P 〉 0.05）,且缺血后处理效果优于C、D两组（P 〈 0.05）。结论： 采用缺血后处理A组方法和B组方法均可以成功制作出缺血后处理模型。     

他克莫司后处理诱导缺血脊髓对再灌注损伤的耐受*

目的探讨他克莫司后处理能否诱导大鼠缺血脊髓对再灌注损伤的耐受。方法成年雄性SD大鼠30只,随机分为假手术（s0）组、缺血再灌注（IR）组和他克莫司后处理（TP）组,每组10只大鼠,采用经股动脉置管球囊扩张制备脊髓缺血模型,SO组仅行置管,IR组在脊髓缺血20分钟后行再灌注,TP组在脊髓缺血20分钟后再灌注,即刻经左颈总动脉一次性注射他克莫司0．5mg／kg。再灌注后7、14天采用Tarlov评分法检测大鼠后肢运动功能,脊髓组织切片HE染色观察病理学改变。结果SO组大鼠各时间点后肢Tarlov评分均为5分,形态学检测显示脊髓组织结构正常;IR组大鼠Tarlov评分明显降低,脊髓组织呈现出坏死、水肿、空腔形成等缺血再灌注损伤表现;TP组大鼠Tarlov评分结果显著优于IR组,脊髓组织病理变化较IR组为轻。结论建立大鼠脊髓缺血再灌注损伤模型,并初步证实他克莫司后处理能诱导缺血脊髓对再灌注损伤的耐受。     

Quercetin postconditioning attenuates myocardial ischemia/reperfusion injury in rats through the PI3K/Akt pathway

Quercetin (Que), a plant-derived flavonoid, has multiple benefical actions on the cardiovascular system. The current study investigated whether Que postconditioning has any protective effects on myocardial ischemia/reperfusion (I/R) injury in vivo and its potential cardioprotective mechanisms. Male Sprague-Dawley rats were randomly allocated to 5 groups (20 animals/group): sham, I/R, Que postconditioning, Que+{"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 [a phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway inhibitor], and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002+I/R. I/R was produced by 30-min coronary occlusion followed by 2-h reperfusion. At the end of reperfusion, myocardial infarct size and biochemical changes were compared. Apoptosis was evaluated by both TUNEL staining and measurement of activated caspase-3 immunoreactivity. The phosphorylation of Akt and protein expression of Bcl-2 and Bax were determined by Western blotting. Que postconditioning significantly reduced infarct size and serum levels of creatine kinase and lactate dehydrogenase compared with the I/R group (all P<0.05). Apoptotic cardiomyocytes and caspase-3 immunoreactivity were also suppressed in the Que postconditioning group compared with the I/R group (both P<0.05). Akt phosphorylation and Bcl-2 expression increased after Que postconditioning, but Bax expression decreased. These effects were inhibited by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. The data indicate that Que postconditioning can induce cardioprotection by activating the PI3K/Akt signaling pathway and modulating the expression of Bcl-2 and Bax proteins.     

Ubiquitin and endogenous antioxidant enzymes participate in neuroprotection of the rabbit spinal cord after ischemia and bradykinin postconditioning

The aim of this study was to investigate neuroprotective effect of bradykinin postconditioning on the rabbit spinal cord after 20 min of ischemia and 3 days of reperfusion. Bradykinin was administered by single i.p. application at 1, 6, 12 or 24 h after ischemia. Assessment of neurological function of hind limbs (Tarlov score) was estimated. Quantitative analysis was evaluated by Fluoro Jade B method, NeuN and ubiquitin immunohistochemistry in anterior horn neurons of the spinal cord. Histomorphologically distribution of ubiquitin and endogenous antioxidant enzymes (SOD1, SOD2, catalase) immunoreaction was described. Bradykinin postconditioning showed decreased number of degenerated neurons, increased number of surviving neurons and increase in number of ubiquitin positive neurons in all bradykinin postconditioned groups versus ischemia/reperfusion group. According to our results bradykinin postconditioning applied 24 h after ischemia significantly decreased (p < 0.001) number of degenerated neurons versus ischemia/reperfusion group. The least effective time window for bradykinin postconditioning was at 12 h after ischemia. Tarlov score was significantly improved (p < 0.05) in groups with bradykinin postconditioning applied 1, 6 or 24 h after ischemia versus ischemia/reperfusion group. Tarlov score in group with bradykinin application 12 h after ischemia was significantly decreased (p < 0.05) versus sham control group. Neuronal immunoreaction of ubiquitin, SOD1, SOD2 and catalase influenced by bradykinin postconditioning was dependent on neuronal survival or degeneration. In conclusion, bradykinin postconditioning showed protective effect on neurons in anterior horns of the rabbit spinal cord and improved motor function of hind limbs.     

Anti-inflammatory Effect of B-Type Natriuretic Peptide Postconditioning During Myocardial Ischemia–Reperfusion: Involvement of PI3K/Akt Signaling Pathway

High mobility group box 1 protein (HMGB1) plays an important role in myocardial ischemia–reperfusion (I/R) injury. B-type natriuretic peptide (BNP) postconditioning has been reported to reduce myocardial I/R injury. The present study investigated whether postconditioning of BNP could reduce myocardial I/R injury by inhibiting HMGB1 expression and the potential mechanisms in rats. The left anterior descending coronary arteries of rats were occluded to induce ischemia for 30 min and reopened to imitate reperfusion for 4 h. The rats were treated with BNP (0.03 μg/kg min, i.v.) 15 min before reperfusion until the end of the procedure, with or without treatment of LY294002 (an inhibitor of phosphoinositide 3-kinase (PI3K), 0.3 mg/kg, i.v.), which was injected 20 min before reperfusion. Lactate dehydrogenase (LDH), creatine kinase (CK), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and infarct size were measured. Phospho-Akt, total Akt, and HMGB1 expression were assessed by immunoblotting. The results showed that treatment of BNP postconditioning could significantly decrease the infarct size and the levels of LDH and CK after 4-h reperfusion (all p?TNF-α and IL-6 (both p?p?p?PI3K/Akt signaling pathway may be involved in the expression of HMGB1 and the protective effect of BNP postconditioning.     

STAT3介导缺血后处理心肌保护作用的研究

目的研究大鼠心肌经历缺血再灌注损伤后心功能指标的变化,探讨缺血后处理是否具有保护作用,并探寻STAT3在其中的作用机制。方法SD大鼠32只,随机分为4组：对照组、缺血再灌注组、缺血后处理组、缺血后处理组＋NSC-74859（STAT3抑制剂）组。建立大鼠离体心脏工作模型,观察心脏在各组条件下心率、LVSP、＋dp／dtmax、-dp／dtmax、冠脉流量的变化及心肌酶谱的改变。结果缺血后处理组与缺血再灌注组相比,复灌期心率,冠脉流出液中CK及LDH的含量明显降低,左室收缩压、左心室压力变化率、冠状动脉流出量明显升高。而抑制STAT3表达后,此保护效应明显减弱。结论缺血后处理有助于减轻缺血再灌注所致心肌损伤,这一作用由STAT3转录因子介导。     

右美托咪定在大鼠脊髓缺血再灌注损伤中的 保护作用及机制研究

目的 探讨右美托咪定对大鼠脊髓缺血/再灌注损伤的保护作用及PI3K/Akt传导通路在其中的作用。方法 30只成年雄性大鼠随机分为假手术组、模型组和右美托咪定治疗组,每组10只。建立大鼠脊髓缺血/再灌注损伤模型,对再灌注损伤后6 h、12 h、24 h、48 h实验大鼠后肢运动功能进行评分,检测缺血脊髓前角组织中P-AKT的表达水平及神经元的凋亡指数。结果 右美托咪定可改善脊髓缺血/再灌注损伤后实验大鼠的后肢运动功能(P＜0.05);提高脊髓前角P-AKT的表达水平(P＜0.05),抑制缺血/再灌注损伤所致的脊髓神经元的凋亡(P＜0.05)。结论 右美托咪定对脊髓缺血/再灌注损伤有一定的保护作用,其机制可能与激活PI3K/Akt传导通路,从而抑制神经元的凋亡有关。     

Nerve Growth Factor Protects the Ischemic Heart via Attenuation of the Endoplasmic Reticulum Stress Induced Apoptosis by Activation of Phosphatidylinositol 3-Kinase

Background: Increased expression of nerve growth factor (NGF) has been found in the myocardium suffered from ischemia and reperfusion (I/R). The pro-survival activity of NGF on ischemic heart has been supposed to be mediated by phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway. Endoplasmic reticulum (ER) stress, which is activated initially as a defensive response to eliminate the accumulated unfolded proteins, has shown a critical involvement in the ischemia induced myocardial apoptosis. This study was aimed to investigate whether NGF induced heart protection against I/R injury includes a mechanism of attenuation of ER stress-induced myocardial apoptosis by activation of PI3K/Akt pathway.Methods: Isolated adult rat hearts were perfused with a Langendörff perfusion system. Hearts in the Sham group were subjected to 225 min of continuous Krebs-Henseleit buffer (KHB) perfusion without ischemia. Hearts in I/R group were perfused with KHB for a 75-min of equilibration period followed by 30 min of global ischemia and 120 min of KHB reperfusion. Hearts in the NGF group accepted 45 min of euilibration perfusion and 30 min of NGF pretreatment (with a final concentration of 100 ng/ml in the KHB) before 30 min of global ischemia and 120 min of reperfusion. Hearts in K252a and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 groups were pretreated with either a TrkA inhibitor, K252a or a phosphatidyl inositol 3-kinase inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 for 30 min before NGF (100 ng/ml) administration. Cardiac hemodynamics were measured from the beginning of the perfusion. Cardiac enzymes and cardiac troponin I (cTnI) were assayed before ischemia and at the end of reperfusion. Myocardial apoptosis rate was measured by TUNEL staining, and expression of glucose-related protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), caspase-12, total- and phospho-(Ser473)-Akt were assessed by Western blot analyses.Results: NGF pretreatment significantly improved the recovery of post-ischemia cardiac hemodynamics. Reduced creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) activity and cTnI levels, as well as decreased myocardial apoptosis ratio were observed in the NGF group. The improvement of NGF on recovery of cardiac function and alleviation of myocardial injury were completely abolished by K252a or {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. GRP78, caspase-12 and CHOP were highly expressed in ischemic myocardium, while NGF significantly inhibited the overexpression of these proteins which were involved in ER stress-induced myocardial apoptosis. NGF pretreatment also induced phosphorylation of Akt. When the activation of PI3K/Akt pathway is blocked by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, the NGF induced suppression of the apoptosis-related proteins expression was reversed.Conclusions: NGF pretreatment may protect the ischemic heart via inhibition of the ER stress-induced apoptosis; this pro-survival effect is mediated by PI3K/Akt pathway.     

Isoflurane postconditioning induces neuroprotection via Akt activation and attenuation of increased mitochondrial membrane permeability

We have shown that isoflurane application at the onset of reperfusion (postconditioning) reduces brain ischemic injury in rats. This study was designed to determine whether this protection involved activation of prosurvival protein kinases and maintenance of normal mitochondrial membrane permeability. Two-month-old male rats were subjected to a 90-min middle cerebral arterial occlusion. They then were exposed or were not exposed to 2% isoflurane for 1 h. Ischemic penumbral cerebral cortex was harvested immediately and separated into the mitochondrial and cytosolic fractions. We showed that the mitochondrial nicotinamide adenine dinucleotide content in the ischemic penumbral cortex was significantly reduced, suggesting an increased mitochondrial membrane permeability. This increase was partly attenuated by isoflurane postconditioning. The mitochondrial adenosine diphosphate content in the penumbral cortex was reduced no matter whether the animals were postconditioned with isoflurane. The mitochondrial adenosine triphosphate concentration was not different among various experimental conditions. The phospho-Akt in the cytosolic and mitochondrial fractions of the ischemic penumbral cortex was higher than that in the control cortex. This increase trended to be higher in animals with isoflurane postconditioning. A similar change pattern was observed in the mitochondrial phospho-glycogen synthase kinase 3β, an Akt substrate that can regulate the mitochondrial membrane permeability. Isoflurane postconditioning reduced oxygen-glucose deprivation-induced injury of rat cortical neuronal cultures and increased phospho-Akt in these cells. The isoflurane postconditioning-induced protection in the neuronal cultures was decreased by the Akt inhibitor LY294002. These results suggest that isoflurane postconditioning effects may be mediated by Akt and involve reduced mitochondrial membrane permeability.     

PI3K/AKT通路在刺五加保护大鼠缺血再灌注心肌细胞损伤中的作用

目的研究刺五加注射液对缺血再灌注损伤心肌细胞的保护作用机制中PI3K/AKT信号通路的作用。方法取Wistar大鼠幼鼠心室肌细胞进行体外培养,缺氧及复氧各3h建立缺血再灌注损伤细胞模型。培养的心肌细胞分成:正常对照组(Co),缺血再灌注组(I/R),I/R+复方刺五加注射液组(Pi)和I/R+复方刺五加注射液+LY294002(Pi+Ly)。采用免疫细胞化学染色法和Western-bloting检测细胞内P-AKT蛋白含量。结果免疫细胞化学染色法及Western-bloting结果均显示,IR组P-AKT蛋白表达水平明显高于正常对照组;实验组(Pi)与其他各组(IR、Pi+Ly)比较,实验组(Pi)P-AKT蛋白表达水平明显升高(P0.05)。结论刺五加注射液减轻心肌缺血再灌注损伤可能与其激活PI3K/AKT信号通路有关。     

磷脂酰肌醇3激酶／蛋白激酶B通路在缺氧血管内皮细胞凋亡中的作用

目的：探讨磷脂酰肌醇3激酶／蛋白激酶B（PI3 K／Akt）通路在缺氧血管内皮细胞凋亡中的作用。方法（1）常规培养人血管内皮细胞株EA．hy926细胞,取部分人血管内皮细胞株EA．hy926按照随机数字表法分为两组：正常对照组：置于气体成分体积分数5％二氧化碳培养箱中常规培养;缺氧组：置于气体成分体积分数1％氧气、5％二氧化碳和94％氮气的三气培养箱中进行缺氧培养。采用蛋白质印迹法检测正常对照组内皮细胞和缺氧处理3、6、24 h的内皮细胞中Akt活化状态（以pAkt／Akt值表示）,流式细胞仪检测细胞凋亡率。（2）另取部分人血管内皮细胞株EA．hy926按照随机数字表法分为4组：正常对照组,缺氧组：培养方法同前,正常对照＋阻断剂组：用含50μmol／L的LY294002（PI3 K／Akt阻断剂）的培养液常规培养内皮细胞;缺氧＋阻断剂组：用含50μmol／L 的LY294002的培养液缺氧处理内皮细胞。均于培养3 h后收集内皮细胞,采用流式细胞仪检测细胞凋亡率。对Akt活化状态与细胞凋亡率行单因素方差分析和LSD-t检验。结果（1）正常对照组细胞和缺氧处理3、6、24 h的内皮细胞pAkt／Akt值分别为0．67、0．79、0．34和0．35;正常内皮细胞和缺氧处理3、6、24 h 的内皮细胞凋亡率分别为（3．11±0．21）％、（4．57±0．85）％、（6．93±0．58）％、（9．96±2．62）％,组间比较差异有统计学意义（F＝8．96,P＝0．030）。与正常对照组细胞比较,缺氧处理3、6 h的内皮细胞凋亡率升高,差异无统计学意义（t＝1．03、2．70,P＝0．360、0．054）;缺氧处理24 h的内皮细胞凋亡率显著升高,差异有统计学意义（t＝4．99,P＝0．008）。（2）正常对照组、正常对照＋阻断剂组、缺氧组、缺氧＋阻断剂组培养3 h后细胞凋亡率为（2．39±0．50）％、（5．77±1．21）％、（3．76±1．05）％