PI3K/Akt信号通路在七氟醚后处理减轻大鼠局灶性脑缺血再灌注损伤中的作用

目的 评价磷脂酰肌醇3激酶/蛋白质丝氨酸苏氨酸激酶(PI3K/Akt)信号通路在七氟醚后处理减轻大鼠局灶性脑缺血再灌注损伤中的作用.方法 雄性SD大鼠64只,体重300～350 g,随机分为4组(n=16):局灶性脑缺血再灌注组(IR组)、七氟醚后处理组(SP组)、七氟醚后处理+Wortmannin组(SW组)和Wortmannin组(W组).采用电凝法阻断左侧大脑中动脉,夹闭双侧颈总动脉60 min恢复灌注的方法制备大鼠局灶性脑缺血再灌注模型.再灌注即刻SP组和SW组吸入2.5%七氟醚60 min,夹闭双侧颈总动脉30 min时SW组和W组股静脉输注Wortmannin 0.6 mg/kg.于再灌注24、48、72 h时行神经功能评分,最后1次评分后处死大鼠取脑,测定脑梗死体积,采用Western blot法检测左侧脑组织磷酸化Akt(p-Akt)和磷酸化Bad(p-Bad)的表达水平.结果 与IR组相比,SP组和SW组各时点神经功能评分升高,再灌注72 h时脑梗死体积比降低,脑组织p-Akt和p-Bad表达上调(P＜0.05),W组上述指标差异无统计学意义(P＞0.05);与SP组相比,SW组各时点神经功能评分降低,再灌注72 h时脑梗死体积比升高,脑组织p-Akt和p-Bad表达下调(P＜0.05).结论 PI3K/Akt信号通路激活后p-Bad表达上调可能参与了七氟醚后处理减轻大鼠局灶性脑缺血再灌注损伤的过程.     

Biological stress response terminology: Integrating the concepts of adaptive response and preconditioning stress within a hormetic dose-response framework

Many biological subdisciplines that regularly assess dose-response relationships have identified an evolutionarily conserved process in which a low dose of a stressful stimulus activates an adaptive response that increases the resistance of the cell or organism to a moderate to severe level of stress. Due to a lack of frequent interaction among scientists in these many areas, there has emerged a broad range of terms that describe such dose-response relationships. This situation has become problematic because the different terms describe a family of similar biological responses (e.g., adaptive response, preconditioning, hormesis), adversely affecting interdisciplinary communication, and possibly even obscuring generalizable features and central biological concepts. With support from scientists in a broad range of disciplines, this article offers a set of recommendations we believe can achieve greater conceptual harmony in dose-response terminology, as well as better understanding and communication across the broad spectrum of biological disciplines.     

缺血后处理和CGS-21680后处理对兔心肌缺血再灌注后细胞凋亡的影响

目的:观察缺血后处理和选择性腺苷A2a受体激动剂CGS-21680后处理对兔缺血再灌注损伤心肌的影响,并探讨其作用机制。方法:健康新西兰大耳白兔36只,随机分成三组:缺血再灌注组(I/R组)、缺血后处理组(IPO组)、CGS-21680后处理组,每组12只。建立兔心肌I/R模型,于再灌注末颈静脉取血测定血清肌酸激酶同工酶(CK-MB);采用伊文思蓝和NBT染色后计算各组兔心肌梗死范围;原位末端标记(TUNEL)法检测各组兔心肌细胞凋亡;实时荧光定量RT-PCR检测各组心肌组织半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)mRNA和Bcl-2mR-NA的表达。结果:(1)与I/R组比较,IPO组和CGS-21680后处理组CK-MB水平显著下降,心肌梗死范围分别减少34.5%、31.2%。(2)TUNEL法结果:IPO组和CGS-21680组的细胞凋亡指数(AI)分别为9.57±1.34%和11.31±0.97%,与IR组(18.32±1.75%)相比明显降低并有显著差异(P0.01)。(3)荧光定量RT-PCR结果:与IR组比较,IPO组和CGS-21680组的促凋亡基因Caspase-3mRNA的表达明显下调,抗凋亡基因Bcl-2mRNA的表达显著上调,P均0.05。结论:缺血后处理和CGS-21680后处理可减少心肌梗死范围,减少细胞凋亡,保护I/R损伤心肌,其机制可能与下调促凋亡基因Caspase-3和上调抗凋亡基因Bcl-2的表达有关。     

缺血后适应对老年冠心病患者缺血再灌注肢体血管内皮功能的影响

目的 观察缺血再灌注不同时刻给予缺血后适应,对老年冠心病患者血管内皮功能的保护作用.方法 选取老年冠心病患者54例,随机分为3组,缺血再灌注组、后适应组及延迟后适应组.建立肢体缺血再灌注模型,在再灌注不同时刻(1 min内及1 min后)分别给予后适应组及延迟后适应组缺血后适应干预,通过超声检测肱动脉血流介导的舒张功能(FMD),观察缺血再灌注前后反应性充血血管内径的变化.结果 缺血再灌注组再灌注后FMD明显减小(P＜0.05),再灌注1 min内给予缺血后适应有明显的血管内皮功能保护作用,与缺血再灌注组比较,后适应组FMD明显提高(6.70±2.36 vs 3.05±0.91,P＜0.05),而延迟后适应组则失去了内皮功能保护作用(3.17±1.04 vs 3.05±0.91,P＞0.05).结论 再灌注1 min内给予缺血后适应可以改善血管内皮功能,但延迟后适应的保护作用消失.     

大鼠体内给药途径的探索

目的设计一种新的给药途径,以探讨药物发挥作用的关键时刻以及最佳时刻,并通过在体心肌缺血再灌注模型进行验证。方法新的给药途径为:左心室-主动脉-冠状动脉途径给药,从右侧颈总动脉插管,经动脉瓣到左心室,显示特征性心室波待稳定后,经该管直接给予溶有还原型谷胱甘肽(120mg/kg)的生理盐水1ml至心室,于1min内注入,与常规右侧股静脉给药途径作对比,采用经典大鼠在体缺血再灌注模型验证新法给药途径的优点。结果分别经左心室—主动脉—冠状动脉途径和静脉途径给药,主动脉血浆药物浓度均在1min时达峰值;相同剂量GSH(120mg/kg)经左心室—主动脉—冠状动脉途径给药后主动脉血浆药物浓度[(810±100)μmol/L]明显高于静脉途径给药[(351±50)μmol/L]。在再灌注早期经左心室—主动脉—冠状动脉途径注射GSH(120mg/kg)干预和静脉注射GSH(120mg/kg)干预,与缺血再灌注对照组相比,心梗面积均显著减小[分别为(23±4)%、(39±2)%和(52±1)%],差异有统计学意义(P<0.05),但经左心室—主动脉—冠状动脉途径减小得更明显,差异有统计学意义(P<0.05)。肌酸激酶、丙二醛含量在再灌注末与静脉途径相比[(53±1)U/ml,(3.6±0.4)μmol/L],经左心室—主动脉—冠状动脉途径减少更明显[(47±3)U/ml,(3.2±1)μmol/L]。结论与经静脉给药相比,经左心室—主动脉—冠状动脉途径注药,可显著提高注射期间主动脉和冠状动脉血浆中的药物浓度,是一条可供药物干预的新途径,并且可以更好地保护心肌缺血再灌注损伤。     

Postconditioning A new link in nature’s armor against myocardial ischemia–reperfusion injury

Reperfusion injury is a complex process involving several cell types (endothelial cells, neutrophils, and cardiomyocytes), soluble proinflammatory mediators, oxidants, ionic and metabolic dyshomeostasis, and cellular and molecular signals. These participants in the pathobiology of reperfusion injury are not mutually exclusive. Some of these events take place during the very early moments of reperfusion, while others, seemingly triggered in part by the early events, are activated within a later timeframe. Postconditioning is a series of brief mechanical interruptions of reperfusion following a specific prescribed algorithm applied at the very onset of reperfusion. This algorithm lasts only from 1 to 3 minutes depending on species. Although associated with re–occlusion of the coronary artery or re–imposition of hypoxia in cell culture, the reference to ischemia has been dropped. Postconditioning has been observed to reduce infarct size and apoptosis as the “end games” in myocardial therapeutics; salvage of infarct size was similar to that achieved by the gold standard of protection, ischemic preconditioning. The cardioprotection was also associated with a reduction in: endothelial cell activation and dysfunction, tissue superoxide anion generation, neutrophil activation and accumulation in reperfused myocardium, microvascular injury, tissue edema, intracellular and mitochondrial calcium accumulation. Postconditioning sets in motion triggers and signals that are functionally related to reduced cell death. Adenosine has been implicated in the cardioprotection of postconditioning, as has e–NOS, nitric oxide and guanylyl cyclase, opening of K_ATP channels and closing of the mitochondrial permeability transition pore. Cardioprotection by postconditioning has also been associated with the activation of intracellular survival pathways such as ERK1/2 and PI3 kinase – Akt pathways. Other pathways have yet to be identified. Although many of the pathways involved in postconditioning have also been identified in ischemic preconditioning, some may not be involved in preconditioning (ERK1/2). The timing of action of these pathways and other mediators of protection in postconditioning differs from that of preconditioning. In contrast to preconditioning, which requires a foreknowledge of the ischemic event, postconditioning can be applied at the onset of reperfusion at the point of clinical service, i.e. angioplasty, cardiac surgery, transplantation.     

线粒体通透性转换孔与缺血后处理

新近提出的缺血后处理具有重要的潜在临床应用价值.缺血后处理对缺血-再灌注损伤具有显著的抗损伤保护作用.线粒体通透性转换孔的开放抑制被证实可能在缺血后处理中起关键作用.     

处理时间窗对七氟醚后处理新生大鼠缺氧/缺血脑损伤长期保护作用的影响

目的 对七氟醚后处理新生大鼠缺氧/缺血(hypoxic/ischemic,H/I)脑损伤模型长期保护作用的有效处理时间窗进行研究探讨. 方法 新生7dSD大鼠240只,按随机数字表法分为假手术组(Sham组)、H/I组、0h后处理组(P0组)、3h后处理组(P3组)、6h后处理组(P6组)、12h后处理组(P12组)(每组40只).各组行左侧颈总动脉结扎术(Sham组不结扎),吸入8％O2+92％N2混合气体处理2h,制备大鼠H/I脑损伤模型.制模后,P0组、P3组、P6组、P12组分别于0、3、6、12 h吸入含2％七氟醚的湿化混合气体30 min.于H/I处理后7d,测定死亡率、大鼠体重;取脑称重,测定左右脑质量比;左右脑切片尼氏染色,观察海马CA1区皮质神经元存活情况,计数神经元,计算左右侧正常神经元密度比值.于H/I处理后21～28 d行悬吊实验检测运动能力,29～34 d用Morris水迷宫实验检测大鼠学习、记忆能力. 结果 各组死亡率差异无统计学意义(P＞0.05).与Sham组比较,其余各组左右脑质量比降低(P＜0.05),神经元计数减少(P＜0.05),逃避潜伏期延长、平台穿越次数减少.与H/I组比较,P0组、P3组、P6组左右脑质量比降低减少(P＜0.05),神经元计数改善(P＜0.05),逃避潜伏期明显缩短、平台穿越次数增加(P＜0.05);P12组与H/I组比较,组间差异无统计学意义(P＞0.05).各组悬吊时间差异无统计学意义(P＞0.05). 结论 在七氟醚后处理大鼠脑H/I模型的保护作用上,6h以内是后处理保护作用最为有效的时间窗;这种保护作用具有可持续性,可改善由于H/I脑损伤而带来的学习、记忆功能障碍.     

缺血后适应调控树鼩脑缺血海马CA1区Akt（pS473）和Akt（pT308）过磷酸化的抗凋亡机制

观察树鼩脑缺血海马神经元Akt（pS473）和Akt（pT308）磷酸化改变对CA1区细胞凋亡的影响,探讨缺血后适应（PC）抑制细胞凋亡的可能机制。方法 用光化学诱导法诱导树鼩脑缺血并建立缺血PC模型;用免疫组织化学TACS原位凋亡检测试剂盒检测皮层及海马CA1区神经元凋亡数量,用免疫组织化学法检测Akt（pS473）和Akt（pT308）表达的空间分布,用ELISA法检测海马CA1区神经元Akt（pS473）和Akt（pT308）磷酸化水平;用电子显微镜观察其神经元超微结构改变。 结果 脑缺血后神经元皱缩,核消失以缺血24h为著。脑缺血后4 h、24 h及72 h皮层及海马CA1区神经元TUNEL阳性细胞数明显增加（P<0.01）,海马CA1区神经元Akt (pS473) 和Akt (pT308)的表达及磷酸化水平明显升高,以脑缺血4 h的改变最明显,分别为(152.3±3.5) units/mg、(130.8±2.6) units/mg和(149.5±4.7)units/mg和 (42.35±2.49) units/mg、(19.23±1.41) units/mg和(23.38±1.32) units/mg (P<0.01）。缺血PC组神经元损伤明显减轻,皮层及海马CA1区TUNEL阳性细胞明显减少（P<0.01）,且与海马神经元Akt (pT308)活化水平呈平行改变(P<0.05）。结论 树鼩脑缺血海马CA1区细胞凋亡与Akt(pS473) 和Akt (pT308)过磷酸化的信号机制有关,缺血PC抑制海马神经元Akt(pS473) 和Akt(pT308)双磷酸化可能具有抗凋亡作用。