应激蛋白、自身防御与心肌

应激蛋白或热休克蛋白是细胞自身保护的一种内在本能。热休克蛋白是细胞或组织暴露在不足以致命的高热中合成的应激蛋白并对随后的高热应激反应产生耐受或抵抗,这类诱导的应激蛋白从细菌到人类的各种生物都存在,且诱导因素还包括低血糖、反应性氧(过氧化氢和超氧阴离子)和缺血等。动物和人类心肌均存在预处理的心肌保护现象,尽管70KDa热休克蛋白(HSP 70)族中的应激蛋白基因表达可在缺血鼠心脏中选择性诱导,但应激蛋白对预处理心肌是否有保护作用仍有争论,一项支持应激蛋白在预处理心肌中作用的研究表明:心肌缺血前使动物经过高热(诱导热休克蛋白效应)对心肌缺血和再灌注损伤有一定的保护作用,然而仅     

热休克蛋白70心肌保护的研究进展

热休克蛋白是细胞在应激条件下产生的一类高度保守的蛋白质,通过稳定细胞内变性的蛋白质;减轻细胞内的离子紊乱;保护血管内皮细胞的功能;干扰应激所启动的细胞凋亡程序等方面来发挥心肌缺血/再灌注损伤的保护作用。现对热休克蛋白及其心肌缺血/再灌注损伤的保护作用及机制进行综述。     

左旋卡尼汀对兔心肌缺血再灌注损伤的保护

目的探讨左旋卡尼汀在心肌缺血再灌注损伤状态下对心肌的保护作用。方法制备兔缺血再灌注模型,实验分为空白对照组、盐水对照组和左旋卡尼汀组,左旋卡尼汀组心肌缺血30 min后给予左旋卡尼汀。观察各组缺血再灌注过程中心电图的动态改变,以及再灌注结束后动脉血游离脂肪酸、超氧化物歧化酶、丙二醛、肌酸激酶的含量和组织中Na -K -ATP酶和Ca2 -Mg2 -ATP酶活性;用Western blot法检测结扎点以下5 mm处左心室全层心肌热休克蛋白70的含量。结果盐水对照组和左旋卡尼汀组均造成明显的心电图动态改变,与盐水对照组比较,左旋卡尼汀组心电图ST段出现有效改善;左旋卡尼汀组分别与盐水对照组和空白对照组相比,游离脂肪酸和丙二醛含量均显著减少(P<0.05);Na -K -ATP酶、Ca2 -Mg2 -ATP酶活性及超氧化物歧化酶的含量显著增多(P<0.05),肌酸激酶含量有下降趋势(P>0.05);心肌热休克蛋白70含量显著增多(P<0.05)。结论左旋卡尼汀可能诱导产生热休克蛋白70,并对心肌缺血再灌注损伤有保护作用。     

心肌晚期预适应及其研究进展

晚期预适应是一种由多种应激因素诱发，对心肌缺血再灌注损伤起保护作用的适应性反应。其发生机制与热休克蛋白、抗氧化酶、蛋白激酶Ｃ及氧自由基、腺苷等生物活性物质有关。因而深入了解心肌缺血的病理生理过程和寻找新的防治方法具有重要意义。     

热应激对心肌缺血耐受性影响的机制

心肌缺血后常会引起一系列生化改变 ,这些改变将影响心肌细胞的生存 ,临床上采用一些方法恢复心肌供血 ,但这些措施未必能使心肌细胞功能恢复 ,原因是恢复供血引发再灌注损伤。热应激为一简单而行之有效的应激方法 ,给心肌短暂热应激能起到保护心肌作用。本文就热应激耐受机制作一概述。     

热休克蛋白对短暂心肌缺血所致蛋白质聚集的影响

为了观察短暂心肌缺血后心肌中蛋白质聚集物的产生，探讨热休克蛋白70及αB-晶状体蛋白对心肌中蛋白质聚集物的影响，采用雄性Wistar大鼠制备在体心缺血—再灌注损伤模型。通过乙醇磷钨酸电镜观察蛋白质聚集物产生，采用免疫电镜观察热休克蛋白70及αB-晶状体蛋白对蛋白质聚集物的影响。结果发现：①缺血15min再灌注30min时，心肌细胞中开始出现形态不规则的蛋白质聚集物，聚集物主要分布在核周、线粒体周围及其两极。再灌注4h，蛋白质聚集物达到高峰，24h后逐渐减少，72h基本恢复正常。②大鼠经热休克预处理及缺血预适应后，15min缺血及4h或24h再灌注所致的心肌蛋白质聚集物的产生明显减少，恢复速度加快，再灌注24h已基本恢复正常。③通过免疫电镜观察，在假手术对照组，心肌中热休克蛋白70表达水平很低。大鼠经热休克预处理后，心肌中热休克蛋白70表达增多，经缺血15min再灌注4h后，热休克蛋白70与心肌中蛋白质聚集物共分布。在假手术对照组心肌中，αB-晶状体蛋白有一定量的组成型表达，并均匀分布于胞浆之中。经热休克预处理、缺血预适应后缺血15min再灌注4h心肌中，αB-晶状体蛋白向肌丝移位，主要位于z线两侧的明带，且与蛋白质聚集物共分布。本研究首次揭示了缺血—再灌注损伤时心肌细胞中蛋白质聚集物的形成、空间分布及其动态变化规律；证实了热休克预处理及心肌缺血预适应可明显减轻缺血—再灌注所致心肌蛋白质聚集。     

金纳多对心肌保护作用机制的实验研究

目的:观察金纳多注射液对兔心肌缺血再灌注后热休克蛋白70的诱导作用及其心肌保护作用的机制。方法:取成功建立心肌缺血30 min再灌注模型的新西兰大白兔30只,根据灌注液的不同随机分为两组:①盐水组(n=15),根据再灌注时间的不同平均(n=5)分为再灌注1 h、3 h和5 h 3种情况;②金纳多组(n=15),根据再灌注时间的不同平均(n=5)分为再灌注1 h、3 h和5 h 3种情况。另取5只为正常对照组(无心肌缺血)。采用western blot蛋白印迹技术检测各组心肌热休克蛋白70的含量,并进行半定量分析;检测各组血清总超氧化物歧化酶、丙二醛及肌酸激酶变化。结果:①心肌组织热休克蛋白70含量:缺血区和非缺血区在正常对照组、盐水组、金纳多组心肌热休克蛋白70均分别显示极少表达、轻度或明显表达及过度表达。缺血区和非缺血区各组间比较均有极显著性差异(P<0．01),各组的缺血区与非缺血区比较(除了盐水组再灌注5 h外)均有显著性差异(P<0．05～0．01)。②血清总超氧化物歧化酶含量:与正常对照组比较,再灌注不同时间的盐水组和金纳多组血清总超氧化物歧化酶含量均降低,且有显著性差异(P<0．05- 0．01);与同一再灌注时间盐水组比较,金纳多组均增高,有极显著性差异(P<0．01)。③血清丙二醛含量:与正常对照组比较,再灌注1 h和3 h时,盐水组和金纳多组血清丙二醛均增高,且有显著性差异(P<0．01或P<0．05),再灌注5 h基本恢复至正常对照组水平。且金纳多组较盐水组明显降低有显著差异(P<0．05或P<0．01)。④血清肌酸激酶含量;与正常对照组比较,盐水组再灌注1 h、3 h、5 h和金纳多组再灌注3 h、5 h血清肌酸激酶含量增高,有极显著性差异(P<0．01);且再灌注不同时间的金纳多组较同一再灌注时间盐水组降低,有显著性差异(P<0．05或P<0．01)。结论:心肌缺血再灌注3小时后热休克蛋白70开始明显表达,金纳多注射液可以诱导热休克蛋白70及早且大量表达,对心肌具有保护作用;金纳多的抗氧化作用可能与热休克蛋白70的表达有关。     

AMPK与心肌缺血-再灌注损伤

心肌缺血-再灌注损伤是急性心肌梗死后溶栓疗法、经皮冠状动脉介入治疗、冠状动脉旁路移植术后心肌损伤的重要机制之一。单磷酸腺苷激活的蛋白激酶(AMPK)不仅是细胞内能量代谢的重要调控因子,而且能通过抗氧化应激、内质网应激、抑制凋亡、调节自噬、抗炎、参与缺血预处理或缺血后处理等心肌保护效应减轻心肌缺血-再灌注损伤。因此,AMPK对防治心肌缺血-再灌注损伤有潜在的临床意义。     

黄芪预处理大鼠心肌保护效应的实验研究

目的本文研究黄芪预处理对大鼠心肌的保护效应以及心肌保护效应与热休克蛋白之间的关系.方法用在体大鼠复制缺血再灌注损伤模型,将大鼠随机分成对照组、缺血再灌组、黄芪组三组,观察心肌组织学、SOD酶的活性、MDA含量变化、心肌梗死范围和HSP70蛋白的表达的变化.结果黄芪预处理组明显减少梗死面积和减少MDA的产生,保护细胞中SOD酶活性和超微结构,HSP70表达水平明显高于对照组及缺血再灌注组.结论黄芪处理可减少缺血再灌注心肌损伤,并能诱导心肌细胞HSP70的表达.     

局部热应激预处理对缺血/再灌注损伤的心脏保护作用

目的探讨热应激预处理诱导产生的热休克蛋白70（HSP70）对缺血/再灌注损伤心脏的保护作用的机制。方法应用冠脉结扎法制备心脏缺血/再灌注损伤模型及热应激预处理模型。将实验大鼠随机分为热应激预处理组（HP+IR组）与非预处理组（IR组）,对比观察两组动物心脏缺血/再灌注后0、4、8、12、24h时心脏HSP70的表达一氧化氮合酶、（NOS）活力及大鼠血清门冬氨酸转氨酶（AST）,丙氨酸转氨酶（ALT）和乳酸脱氢酶（LDH）的活性与心脏病理组织学改变。结果热应激预处理组各时间点心脏HSP70的表达均比非预处理组同一时间点高,而NOS活力及血清AST、ALT、LDH的酶活性较非预处理组低,病理损伤也比非预处理组减轻。结论热应激预处理诱导产生的HSP70可能通过抑制NO的产生,从而降低氧自由基对心脏的损害,起到保护心脏的作用。     

谷氨酰胺对离体大鼠心脏缺血再灌注损伤的影响

目的研究谷氨酰胺对心肌缺血再灌注损伤时心功能和心肌酶的影响。方法30只sD大鼠取心脏建立Langendorff灌注模型,随机分为三组：（1）对照组（A组,正常灌注90min）;（2）缺血再灌注组（B组,全心缺血30min,复灌60min）;（3）谷氨酰胺组（c组,取心脏前3小时静脉注射谷氨酰胺0．75g/kg,全心缺血30min,复灌60min）。记录三组心脏的血流动力学水平,测定冠脉流出液肌钙蛋白I水平,测定复灌后心肌的热休克蛋白70水平。结果实与缺血再灌注组相比,谷氨酰胺组的左室发展压（LVDP）、左室内压上升/下降速率（±dp/dt）和心率明显增加,左室舒张末期压力（LVEDP）和肌钙蛋白I水平显著降低（P均〈0．05）,谷氨酰胺组热休克蛋白70表达水平显著增加（P〈0．05）。结论谷氨酰胺能改善缺血再灌注损伤大鼠心脏的血流动力学,减轻心肌损伤,这些作用可能与其诱导热休克蛋白70表达相关。     

Impact of hyperthermic preconditioning on postischemic hepatic microcirculatory disturbances in an isolated perfusion model of the rat liver

Sublethal hyperthermia and the following recovery from this heat exposure, referred to as hyperthermic preconditioning, elicits a transient state of tolerance to oxidative insults through an intracellular protective response: stress response. The impact of hyperthermic preconditioning on hepatic microcirculatory disturbance, which is one of the determinants of ischemia/reperfusion-induced injury of the liver, was investigated by using intravital fluorescence microscopy. Thirty minutes of ischemia and a subsequent 120 minutes of reperfusion was induced in an in situ isolated perfusion model of Sprague-Dawley rats. Heat stress was given by whole-body hyperthermia, and a subsequent recovery was allowed for 18 or 48 hours, respectively. Postischemic decrease in sinusoidal perfusion rate and sinusoidal diameter, leukocyte stagnation in sinusoids, and leukocyte adhesion in postsinusoidal venules were significantly attenuated in both hyperthermia-pretreated groups. A recovery of bile production, a reduction of liver enzyme release, and an attenuation of tissue edema and histological damage were also observed. A marked expression of heat shock protein (HSP) 70 and heme oxygenase (HO-1)/HSP32 was correlatively observed in the liver tissue coincident with the induction of these protective effects. Hyperthermic preconditioning provides a continuous long-term and constant inhibitory effect (up to 48 hours after heat exposure) on postischemic injury of the liver through the attenuation of microcirculatory disturbances. These beneficial effects might be associated with a concomitant increase in HSP70 and HO-1/HSP32 expression.     

Mechanisms of the beneficial actions of ischemic preconditioning on subcellular remodeling in ischemic-reperfused heart

Cardiac function is compromised by oxidative stress which occurs upon exposing the heart to ischemia reperfusion (I/R) for a prolonged period. The reactive oxygen species (ROS) that are generated during I/R incur extensive damage to the myocardium and result in subcellular organelle remodeling. The cardiac nucleus, glycocalyx, myofilaments, sarcoplasmic reticulum, sarcolemma, and mitochondria are affected by ROS during I/R injury. On the other hand, brief periods of ischemia followed by reperfusion, or ischemic preconditioning (IPC), have been shown to be cardioprotective against oxidative stress by attenuating the cellular damage and alterations of subcellular organelles caused by subsequent I/R injury. Endogenous defense mechanisms, such as antioxidant enzymes and heat shock proteins, are activated by IPC and thus prevent damage caused by oxidative stress. Although these cardioprotective effects of IPC against I/R injury are considered to be a consequence of changes in the redox state of cardiomyocytes, IPC is considered to promote the production of NO which may protect subcellular organelles from the deleterious actions of oxidative stress. The article is intended to focus on the I/R-induced oxidative damage to subcellular organelles and to highlight the cardioprotective effects of IPC. In addition, the actions of various endogenous cardioprotective interventions are discussed to illustrate that changes in the redox state due to IPC are cardioprotective against I/R injury to the heart.     

Effect of Ischemia-Reperfusion on Heat Shock Protein 70 and 90 Gene Expression in Rat Liver: Relation to Nutritional Status

Heat shock proteins are intracellular proteinsassociated with a generalized response of cells tostress. The purpose of this study was to assess RNAlevels of heat shock protein 70 and 90 in fed or fasted rat livers during ischemia-reperfusion.Northern blot analysis of heat shock proteins wasperformed. Adenosine triphosphate and glutathione wereassessed. In baseline conditions, livers of fasted ratsshowed a twofold increase in mRNA for both heat shockproteins and 38% and 43% reductions in adenosinetriphosphate and glutathione, respectively, whencompared with organs from fed rats. After ischemia,livers of fasted rats presented a twofold decrease inheat shock protein mRNA, while no changes were observedin livers of fed rats; reduced glutathione and adenosinetriphosphate decreased 55% and 50% in fasted livers and 25% and 20% in fed organs,respectively. After 120 min of reperfusion, heat shockprotein mRNA rose threefold in fasted livers, while aslight decrease was observed in the fed group; reduced glutathione and adenosine triphosphate returnedto 65% and 70% of baseline values in fasted livers and85% and 90% in fed organs, respectively. In conclusion,the nutritional status affects heat shock protein expression determined by reperfusion. Thereduced antioxidant status leading to increasedoxidative stress could be the mechanism underlying thephenomenon.     

基质金属蛋白酶-9及其抑制物TIMP-1表达与醋柳黄酮治疗大鼠缺血-再灌注损伤心肌的关系研究

目的：观察基质金属蛋白酶（MMP）-9及其抑制物TIMP-1表达与醋柳黄酮治疗大鼠缺血-再灌注损伤心肌的关系研究,探讨醋柳黄酮对心肌缺血再灌注损伤的保护机制。方法：将50只大鼠随机分组：模型组、氯沙坦组、醋杉P黄酮大小剂量组及假手术组。结扎左冠状动脉前降支,30min后松开制作心肌缺血再灌注模型,免疫组织化学检测再灌注心肌组织MMP-9及其抑制物TIMP-1表达。结果：免疫组化结果示模型组较假手术组MMP-9、TIMP-1表达增高;醋柳黄酮大小剂量组较模型组MMP-9表达减少,TIMP-1表达增多。且有统计学意义。结论：醋柳黄酮对再灌注损伤心肌有保护作用,其作用机制与抑制心肌中MMP-9表达,促进TIMP-1表达有关。     

热休克蛋白70对过氧化氢所致乳鼠心肌细胞核仁损伤的保护作用

为探讨氧化应激时体外培养的新生Wistar大鼠心肌细胞核仁损伤以及热休克蛋白70对损伤核仁的保护作用。用0.5mmol/L过氧化氢处理原代培养的心肌细胞0,30,60min，采用甲苯胺兰染色核仁及电镜技术观察核仁结构的改变；并通过热休克预处理及反义技术诱导或阻断热休克蛋白70的表达，观察其对核仁损伤的保护作用。结果发现，光镜下过氧化氢损伤组心肌细胞核仁染色颗粒数增多，电镜下核仁结构松散，核仁组份分离。热休克预处理导致心肌细胞中热休克蛋白70表达明显增加，并使过氧化氢缺致心肌细胞核仁损伤明显减轻，免疫组织化学显示过氧化氢可引起热休克蛋白70从胞浆到胞核，再到核仁的移位，热休克蛋白70反义寡核苷酸很大程度上能阻断热休克预处理对心肌细胞核仁损伤的保护作用。结果提示，过氧化氢可导致体外培养的新生大鼠心肌细胞核仁结构损伤，热休克蛋白70高表达及其向核仁的移位对上述损伤具有明显保护作用。     

Importance of small heat shock protein 20 (hsp20) C-terminal extension in cardioprotection

Recent studies show that overexpression of small heat shock protein 20 (Hsp20) in mouse hearts reduces infarct size and improves cardiac performance. However, it is not known whether Hsp20 exerts its protective action through improved calcium handling or chaperone activity. The C-terminal extensions of small heat shock proteins, such as alphaB-crystallin and Hsp25, are implicated in chaperoning activity. Through adenovirus mediated overexpression of Hsp20 with C-terminal extension substitution, we delineated the mechanism of protection. Neonatal and adult rat cardiomyocytes overexpressing either the full-length Hsp20 or Hsp20 with a C-terminal extension substitution were subjected to simulated ischemia for 14-16 h followed by reperfusion 6-8 h. Overexpressing Hsp20 with a C-terminus extension substitution did not protect against simulated ischemia/reperfusion in either adult (98+/-8.8% LDH release of control) or neonatal cardiomyocytes (103+/-1.8% CK release of control) as measured by creatine kinase (CK) and lactate dehydrogenase (LDH) cell viability assays (n=4, P<0.05). However, this Hsp20 C-terminal substitution mutant increased calcium transients 33+/-11% and cell contraction amplitude 60+/-15% as quantified through epifluorescence microscopy (n=16 to 34 cells per heart from 4 to 5 hearts, P<0.05). In contrast, overexpression of the full-length Hsp20 protected cultured adult (53+/-8.5% LDH release of control) and neonatal rat (57+/-8.3% CK release of control) cardiomyocytes from simulated ischemia/reperfusion injury. This overexpression also increased calcium transients 30+/-10% and cell contraction amplitude 50+/-10%. These novel data suggest that the C-terminal extension of Hsp20 is essential for cardioprotection. Hsp20 renders this protection through its C-terminal extension protein domain, while this part of the protein is not involved in the Hsp20 ability to increase both calcium transients and cell contraction.     

热休克蛋白27在脑缺血耐受中的作用

热休克蛋白27( heat shock protein 27,HSP27)是小分子热休克蛋白家族成员之一,其分子伴侣功能以及与细胞死亡通路的相互作用介导了应激状态下的细胞存活.脑缺血时,缺血灶以及远隔区域HSP27表达量及其磷酸化水平变化与神经元对缺血的耐受性有关.然而,HSP27在脑缺血耐受中的神经保护机制至今尚不完...     

Distinct roles of autophagy in the heart during ischemia and reperfusion: roles of AMP-activated protein kinase and Beclin 1 in mediating autophagy

Autophagy is an intracellular bulk degradation process for proteins and organelles. In the heart, autophagy is stimulated by myocardial ischemia. However, the causative role of autophagy in the survival of cardiac myocytes and the underlying signaling mechanisms are poorly understood. Glucose deprivation (GD), which mimics myocardial ischemia, induces autophagy in cultured cardiac myocytes. Survival of cardiac myocytes was decreased by 3-methyladenine, an inhibitor of autophagy, suggesting that autophagy is protective against GD in cardiac myocytes. GD-induced autophagy coincided with activation of AMP-activated protein kinase (AMPK) and inactivation of mTOR (mammalian target of rapamycin). Inhibition of AMPK by adenine 9-beta-d-arabinofuranoside or dominant negative AMPK significantly reduced GD-induced autophagy, whereas stimulation of autophagy by rapamycin failed to cause an additive effect on GD-induced autophagy, suggesting that activation of AMPK and inhibition of mTOR mediate GD-induced autophagy. Autophagy was also induced by ischemia and further enhanced by reperfusion in the mouse heart, in vivo. Autophagy resulting from ischemia was accompanied by activation of AMPK and was inhibited by dominant negative AMPK. In contrast, autophagy during reperfusion was accompanied by upregulation of Beclin 1 but not by activation of AMPK. Induction of autophagy and cardiac injury during the reperfusion phase was significantly attenuated in beclin 1(+/-) mice. These results suggest that, in the heart, ischemia stimulates autophagy through an AMPK-dependent mechanism, whereas ischemia/reperfusion stimulates autophagy through a Beclin 1-dependent but AMPK-independent mechanism. Furthermore, autophagy plays distinct roles during ischemia and reperfusion: autophagy may be protective during ischemia, whereas it may be detrimental during reperfusion.