Bradykinin limits infarction when administered as an adjunct to reperfusion in mouse heart: the role of PI3K,Akt and eNOS

Attenuation of reperfusion injury by growth factors has recently been linked to recruitment of phosphatidylinositol-3 kinase (PI3K) and protein kinase B (Akt), a pathway also linked to the phosphorylation of eNOS by bradykinin. We, therefore, hypothesised that bradykinin would limit infarct size when given as an adjunct to reperfusion. Using an isolated perfused mouse heart model of ischaemia/reperfusion injury, we show that 100 nmol/l bradykinin, administered upon reperfusion, attenuates infarct size (32 +/- 2% to 22 +/- 2%, P < 0.01). This protection was abrogated by concomitant administration of the PI3K inhibitor, wortmannin (100 nmol/l), whereas wortmannin alone had no impact upon infarct size (31 +/- 3% and 30 +/- 1%, respectively). In eNOS knockout hearts, bradykinin was not seen to be protective (31 +/- 2% versus 32 +/- 2%), yet knockout hearts could be rescued with the nitric oxide donor, S-nitroso-N-acetyl penicillamine (SNAP) (1 micromol/l) (17 +/- 4%, P < 0.01). Using western blot analysis, we show that bradykinin administration results in rapid, robust phosphorylation of both Akt and eNOS, greater than that seen in control hearts upon reperfusion (Akt/eNOS phosphorylation: 68 +/- 7/122 +/- 29 AU versus 32 +/- 5/47 +/- 10 AU respectively, P < 0.01). This pattern of Akt phosphorylation was mimicked in the absence of eNOS, whereas Akt phosphorylation was inhibited by wortmannin. Exogenous nitric oxide administration had no impact upon Akt phosphorylation. Therefore, we demonstrate that exogenous bradykinin, administered at reperfusion, limits infarct size with concomitant rapid phosphorylation of Akt and eNOS, and that this protection is dependent upon the presence of eNOS. These results may open new avenues for research into clinical limitation of reperfusion injury following acute myocardial infarction.     

PI3K/AKT信号通路与心肌缺血再灌注损伤

PI3K/AKT通路是重要的抗细胞凋亡/促增殖信号通路,在细胞的生长、存活、增殖、迁移及代谢等方面有重要作用,并且越来越多的研究表明其在心肌缺血再灌注损伤的病理生理过程中也发挥重要作用,本文就此作一综述。     

Bradykinin prevents reperfusion injury by targeting mitochondrial permeability transition pore through glycogen synthase kinase 3beta

Although bradykinin has been demonstrated to protect the heart at reperfusion, the detailed cellular and molecular mechanisms that mediate the protection remain elusive. Here we aimed to determine whether bradykinin protects the heart at reperfusion by modulating the mitochondrial permeability transition pore (mPTP) opening through glycogen synthase kinase 3beta (GSK-3beta). Bradykinin given at reperfusion reduced infarct size in isolated rat hearts subjected to 30 min regional ischemia followed by 2 h of reperfusion. The infarct-limiting effect of bradykinin was reversed by atractyloside, an opener of the mPTP, suggesting that bradykinin may protect the heart at reperfusion by modulating the mPTP opening. In support of this observation, bradykinin prevented the collapse of mitochondrial membrane potential (DeltaPsi(m)), an index of the mPTP opening. Bradykinin increased GSK-3beta phosphorylation at reperfusion, and the selective inhibitor of GSK-3beta SB216763 reduced infarct size and prevented the loss of DeltaPsi(m) by mimicking the effect of bradykinin. The effect of bradykinin on GSK-3beta phosphorylation was blocked by wortmannin and LY294002, and bradykinin increased Akt phosphorylation at reperfusion. Further experiments showed that the MEK inhibitor PD98059 prevented the effect of bradykinin on GSK-3beta. However, the mTOR/p70s6K pathway inhibitor rapamycin did not alter bradykinin-induced GSK-3beta phosphorylation and bradykinin failed to alter phosphorylation of either mTOR or p70s6K at reperfusion. Taken together, these data suggest that bradykinin protects the heart at reperfusion by modulating the mPTP opening through inhibition of GSK-3beta. The PI3-kinase/Akt pathway and ERK, but not the mTOR/p70s6K pathway account for the suppression of GSK-3beta by bradykinin.     

NECA at reperfusion limits infarction and inhibits formation of the mitochondrial permeability transition pore by activating p70S6 kinase

The A₁/A₂ adenosine agonist 5′-(N-ethylcarboxamido) adenosine (NECA) limits infarction when administered at reperfusion. The present study investigated whether p70S6 kinase is involved in this anti-infarct effect. Adult rat ventricular myocytes were isolated and incubated in tetramethylrhodamine ethyl ester (TMRE, 100 nM), which causes cells to fluoresce in proportion to their mitochondrial membrane potential. A reduction in TMRE fluorescence serves as an indicator of collapse of the mitochondrial transmembrane potential. Cells were subjected to H₂O₂ (200 μM), which like ischemia induces loss of mitochondrial membrane potential. Fluorescence was measured every 3 min and to facilitate quantification membrane potential was arbitrarily considered as collapsed when fluorescence reached less than 60% of the starting value. Adding NECA (1 mM) to the cells prolonged the time to fluorescence loss (48.0 ± 3.2 min in the NECA group versus 29.5 ± 2.2 min in untreated cells, P < 0.001) and the mTOR/p70S6 kinase inhibitor rapamycin (5 nM) abolished this protection (31.3 ± 3.4 min). Since cyclosporine A offered similar protection, mitochondrial permeability transition pore formation is a likely cause of the H₂O₂-induced loss of potential. The direct GSK-3β inhibitor SB216763 (3 μM) also prolonged the time to fluorescence loss (49.2 ± 2.1 min, P < 0.001 versus control), and its protection could not be blocked by rapamycin (42.2 ± 2.3 min, P < 0.001 versus control). NECA treatment (100 nM) of intact isolated rabbit hearts at reperfusion after 30 min of regional ischemia decreased infarct size from 33.0 ± 3.8% of the risk zone in control hearts to 11.8 ± 2.0% (P < 0.001), and rapamycin blocked this NECA-induced protection (38.3 ± 3.7%). A comparable protective effect was seen for SB216763 (1 μM) with infarct size reduction to 13.5 ± 2.3% (P < 0.001). NECA treatment (200 nM) of intact rabbit hearts at reperfusion also resulted in phosphorylation of p70S6 kinase more than that seen in untreated hearts. This NECA-induced phosphorylation was blocked by rapamycin. These experiments reveal a critical role for p70S6 kinase in the signaling pathway of NECA’s cardioprotection at reperfusion. Returned for 1st revision: 3 November 2005 1st revision received: 3 February 2006 Returned for 2nd revision: 23 February 2006 2nd revision received: 1 March 2006     

Protein kinase C protects preconditioned rabbit hearts by increasing sensitivity of adenosine A2b-dependent signaling during early reperfusion

Although protein kinase C (PKC) plays a key role in ischemic preconditioning (IPC), the actual mechanism of that protection is unknown. We recently found that protection from IPC requires activation of adenosine receptors during early reperfusion. We, therefore, hypothesized that PKC might act to increase the heart's sensitivity to adenosine. IPC limited infarct size in isolated rabbit hearts subjected to 30-min regional ischemia/2-h reperfusion and IPC's protection was blocked by the PKC inhibitor chelerythrine given during early reperfusion revealing involvement of PKC at reperfusion. Similarly chelerythrine infused in the early reperfusion period blocked the increased phosphorylation of the protective kinases Akt and ERK1/2 observed after IPC. Infusing phorbol 12-myristate 13-acetate (PMA), a PKC activator, during early reperfusion mimicked IPC's protection. As expected, the protection triggered by PMA at reperfusion was blocked by chelerythrine, but surprisingly it was also blocked by MRS1754, an adenosine A(2b) receptor-selective antagonist, suggesting that PKC was somehow facilitating signaling from the A(2b) receptors. NECA [5'-(N-ethylcarboxamido) adenosine], a potent but not selective A(2b) receptor agonist, increased phosphorylation of Akt and ERK1/2 in a dose-dependent manner. Pretreating hearts with PMA or brief preconditioning ischemia had no effect on phosphorylation of Akt or ERK1/2 per se but markedly lowered the threshold for NECA to induce their phosphorylation. BAY 60-6583, a highly selective A(2b) agonist, also caused phosphorylation of ERK1/2 and Akt. MRS1754 prevented phosphorylation induced by BAY 60-6583. BAY 60-6583 limited infarct size when given to ischemic hearts at reperfusion. These results suggest that activation of cardiac A(2b) receptors at reperfusion is protective, but because of the very low affinity of the receptors endogenous cardiac adenosine is unable to trigger their signaling. We propose that the key protective event in IPC occurs when PKC increases the heart's sensitivity to adenosine so that endogenous adenosine can activate A(2b)-dependent signaling.     

Sildenafil and vardenafil but not nitroglycerin limit myocardial infarction through opening of mitochondrial K(ATP) channels when administered at reperfusion following ischemia in rabbits

Phosphodiesterase-5 (PDE-5) inhibitors including sildenafil and vardenafil induce powerful preconditioning-like cardioprotective effect against ischemia/reperfusion injury through opening of mitochondrial K(ATP) channels in the heart. The goal of these studies was to demonstrate the protective effect of sildenafil and vardenafil on reperfusion injury and to compare it with the antianginal vasodilator nitroglycerin (NTG). In addition, we determined the role of mitochondrial K(ATP) channels in protection. Adult male New Zealand white rabbits were anesthetized and subjected to ischemia by 30 min of coronary artery occlusion followed by 3 h of reperfusion. Seven groups were studied. 1-Controls; 2-Sildenafil (total dose: 0.71 mg/kg; i.v.) infused for 65 min starting 5 min before reperfusion; 3-Sildenafil+5-hydroxydecanoate (5-HD, blocker of mitochondrial K(ATP) channel, total dose: 5 mg/kg) administered as 2 bolus injections; 4-Vardenafil (total dose: 0.014 mg/kg; iv) administered as in group 2; 5-Vardenafil+5-HD administered as in group 3; 6-5-HD administered as two bolus injections and 7-Nitroglycerin (NTG, total dose: 2 microg kg(-1) min(-1)) administered as in group 2. Infarct size was reduced in sildenafil (19.19+/-1.3%) as well as vardenafil (17.0+/-2.0%) treated groups as compared to controls (33.8+/-1.7%). However, NTG failed to confer similar cardioprotection (31.5+/-0.8%). 5-HD blocked the cardioprotective effects of sildenafil and vardenafil as shown by an increase in infarct size (34.0+/-1.1% and 28.3+/-1.9%, respectively). Both sildenafil and vardenafil protect the ischemic myocardium against reperfusion injury through a mechanism dependent on mitochondrial K(ATP) channel opening.     

PI3K/Akt信号通路与肝纤维化

PI3K/AKT信号通路可以通过调控基因表达,从而在细胞的存活、分化、生长、运动和凋亡等多种生理和病理过程中起到重要作用。尤其在肝纤维化的进展中,此信号通路发挥了重要的调节作用。本文将对目前有关PI3K/AKT信号通路在参与肝纤维化形成中,如何调控细胞外基质的降解、影响HSC的活化及调节肝窦毛细血管化等作用机制作一综述。这些资料不仅可以揭示相关疾病条件下,多个细胞与信号因子之间复杂的相互作用机制,而且能够突出通过阻断PI3K/AKT信号通路可以保护和治疗肝纤维化这一潜在的临床意义。     

Phosphoproteomic characterization of DNA damage response in melanoma cells following MEK/PI3K dual inhibition

Targeted therapeutics that block signal transduction through the RAS–RAF–MEK and PI3K–AKT–mTOR pathways offer significant promise for the treatment of human malignancies. Dual inhibition of MAP/ERK kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) with the potent and selective small-molecule inhibitors GDC-0973 and GDC-0941 has been shown to trigger tumor cell death in preclinical models. Here we have used phosphomotif antibodies and mass spectrometry (MS) to investigate the effects of MEK/PI3K dual inhibition during the period immediately preceding cell death. Upon treatment, melanoma cell lines responded by dramatically increasing phosphorylation on proteins containing a canonical DNA damage-response (DDR) motif, as defined by a phosphorylated serine or threonine residue adjacent to glutamine, [s/t]Q. In total, >2,000 [s/t]Q phosphorylation sites on >850 proteins were identified by LC-MS/MS, including an extensive network of DDR proteins. Linear mixed-effects modeling revealed 101 proteins in which [s/t]Q phosphorylation was altered significantly in response to GDC-0973/GDC-0941. Among the most dramatic changes, we observed rapid and sustained phosphorylation of sites within the ABCDE cluster of DNA-dependent protein kinase. Preincubation of cells with the inhibitors of the DDR kinases DNA-dependent protein kinase or ataxia-telangiectasia mutated enhanced GDC-0973/GDC-0941–mediated cell death. Network analysis revealed specific enrichment of proteins involved in RNA metabolism along with canonical DDR proteins and suggested a prominent role for this pathway in the response to MEK/PI3K dual inhibition.Dysregulation of the RAS–RAF–MAP/MEK and PI3K–AKT–mTOR pathways represents a common theme in human cancer. The importance of these interconnected pathways is highlighted by the frequency of mutational activation of pathway members including RAS, RAF, and PI3K, as well as inactivation of the inhibitory phosphatase and tensin homolog (PTEN) (1, 2). Targeted therapies that block signaling through the RAS–RAF–MEK pathway, including specific inhibitors of oncogenic forms of BRAF (e.g., BRAF-V600E, which is observed in ∼50% of melanomas) and of the downstream effector MEK have shown clinical efficacy in melanoma and other tumor types (3, 4). Likewise, suppression of cell-survival signaling through inhibition of PI3K has been shown to kill cancer cells (5, 6). Although inhibition of either pathway individually can elicit measureable responses, feedback through signal-transduction networks often limits the effectiveness of single-agent therapies. Mounting evidence suggests that dual inhibition of the RAS–RAF–MEK and PI3K–AKT–mTOR pathways will demonstrate improved efficacy over single-agent therapies.At the heart of the RAS–RAF–MEK and PI3K–AKT–mTOR pathways is a web of phosphorelay networks in which individual phosphorylation sites serve as nodes. Many key nodes reside on protein kinases, where phosphorylation individually and in aggregate modulates the amplitude and specificity of downstream signaling. Our understanding of these networks has been shaped by studies using phosphospecific antibodies against these individual, site-specific phosphorylation events including ERK1/2 at Thr202/Tyr204 (7, 8) and AKT at Thr308 (9, 10). Although this strategy has proven successful, the generation of sensitive phosphospecific reagents capable of reading out signal unambiguously remains a challenge. Likewise, multiply phosphorylated sequences or those occurring adjacent to other posttranslational modifications can confound data interpretation. A key limitation is that phosphospecific antibodies are intended to interrogate only a single node in a signal-transduction network, so that even when multiplexed they provide only a narrow portal through which to view the dynamic system.Mass spectrometry (MS) proteomics provides a platform to dissect signaling networks in breadth and depth. Although theoretically the phosphorylated peptides can be profiled directly from digested cell lysates, interrogating signal-transduction networks requires enrichment of modified peptides from the cellular milieu. One approach involves immunoaffinity enrichment (IAE) with antibodies recognizing classes of phosphopeptides, such as phosphotyrosine (11). IAE methods also have been reported for assaying phosphorylation in the AKT (12) and DNA damage-response (DDR) signaling networks (13, 14), using antibodies that recognize phosphorylation within a local sequence context. For substrates of AKT family kinases, phosphorylation occurs within an RXRXX[s/t] sequence, where X represents any amino acid and [s/t] refers to the phosphorylated serine or threonine (12). For substrates of ataxia-telangiectasia mutated (ATM) and other DDR kinases, phosphorylation occurs on serine or threonine adjacent to glutamine, [s/t]Q (15). In contrast to phosphospecific antibodies directed against a single sequence, motif-specific antibodies recognize a degenerate motif and permit enrichment of an array of peptides. Likewise, these reagents provide a readout for screening conditions by immunoblot before phosphopeptide enrichment and LC-MS/MS.In the current study, we noticed that MEK/PI3K dual inhibition in melanoma lines resulted in a marked DDR. Using motif-directed IAE and MS proteomics, we investigated the signaling elicited by small-molecule inhibitors of MEK and PI3K currently in clinical development to establish a molecular understanding of this response.     

Involvement of ERK1/2, p38 and PI3K in megakaryocytic differentiation of K562 cells

Megakaryocytic differentiation of myelogenous leukemia cell lines induced by a number of chemical compounds mimics, in part, the physiological process that takes place in the bone marrow in response to a variety of stimuli. We have investigated the involvement of mitogen‐activated protein kinases (MAPKs) [extracellular signal‐regulated protein kinase (ERK1/2) and p38] and phosphoinositide 3‐kinase (PI3K) signaling pathways in the differentiated phenotypes of K562 cells promoted by phorbol 12‐myristate 13‐acetate, staurosporine (STA), and the p38 MAPK inhibitor SB202190. In our experimental conditions, only STA‐treated cells showed the phenotype of mature megakaryocytes (MKs) including GPIbα expression, DNA endoreduplication, and formation of platelet‐like structures. We provide evidence supporting that basal activity, but not sustained activation, of ERK1/2 is required for expression of MK surface markers. Moreover, ERK1/2 signaling is not involved in cell endomitosis. The PI3K pathway exerts dual regulatory effects on K562 cell differentiation: it is intimately connected with ERK1/2 cascade to stimulate expression of surface markers and it is also necessary, but not sufficient, for polyploidization. Finally, apoptosis and megakaryocytic differentiation exhibit different sensitivity to p38 down‐regulation: it is required for expression of early specific markers but is not involved in cell apoptosis. The present work with K562 cells provides new insights into the molecular mechanisms regulating MK differentiation. The results indicate that a precise orchestration of signals, including ERK1/2 and p38 MAPKs as well as PI3K pathway, is necessary for acquisition of features of mature MKs.     

HB-EGF enhances restitution after intestinal ischemia/reperfusion via PI3K/Akt and MEK/ERK1/2 activation

BACKGROUND & AIMS: Early recovery of intestinal function after injury occurs by restitution, a complex process with a poorly understood molecular basis. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent chemotactic factor that is induced during ischemia/reperfusion in vivo and intestinal wounding in vitro. The role of HB-EGF in intestinal restitution and the underlying intracellular signaling pathways involved were investigated. METHODS: Adult rats were subjected to intestinal ischemia, with histologic and biochemical damage assessed during the first 3 hours of reperfusion. The effect of recombinant HB-EGF (rHB-EGF) on structural and functional recovery of the intestine by restitution was evaluated in vivo. Scrape wounding of intestinal epithelial cell monolayers was used to elucidate the mechanisms of intrinsic and rHB-EGF-induced restitution. RESULTS: Early structural recovery occurred within 3 hours of reperfusion and was attributed to restitution rather than proliferation. HB-EGF treatment significantly improved structural recovery and accelerated functional recovery of the gut barrier. In vivo restitution was preceded by activation of Akt and extracellular signal-regulated kinase (ERK) 1/2, which were accelerated and enhanced by HB-EGF treatment. Blocking of ErbB-1, phosphatidylinositol 3-kinase (PI3K)/Akt, or mitogen-activated protein kinase/ERK kinase (MEK)/ERK activity resulted in significant reduction in intrinsic and HB-EGF-induced restitution in vitro. Endogenous HB-EGF was shown to play an essential role in wound-induced ErbB-1 and ERK1/2 activation and in intrinsic restitution. CONCLUSIONS: Endogenous HB-EGF, ErbB-1, PI3K/Akt, and MEK/ERK are involved in intrinsic restitution. rHB-EGF enhances restitution in vivo and in vitro in a PI3K/Akt- and MEK/ERK1/2-dependent fashion.     

红细胞生成素对大鼠心肌缺血-再灌注后抗氧化酶及NO等的影响

目的：探讨红细胞生成素（erythropoietin，EPO）对大鼠心肌缺血-再灌注损伤心肌组织中超氧化物岐化酶（SOD）、丙二醛（MDA）、谷胱甘肽过氧化物酶（GSH-px）、过氧化氢酶（CAT）、一氧化氮（NO）及一氧化氮合酶（NOS）表达的影响。方法：以左冠脉穿线结扎法制备心肌缺血再灌注模型，造模前24h开始给药。在大鼠心肌缺血30min再灌注24h后分别检测心肌组织的MDA，GSHpx，SOD，CAT，NO及NOS。结果：EPO干预组的SOD活力有明显增高，MDA含量明显下降（P均〈0．05），GSHpx及CAT含量均显著提高（P〈0．05）。同时，EPO的预处理也降低了NO和NOS的含量（P〈0．01）。结论：对于大鼠心肌缺血再灌注损伤，EPO干预可以提高多种抗氧化酶活性，同时对NO产生增多有一定的抑制作用。     

杨梅素通过磷脂酰肌醇3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白信号通路诱导MTB感染巨噬细胞发生自噬的研究

目的对杨梅素通过磷脂酰肌醇3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/Akt/mTOR)信号通路诱导MTB感染的巨噬细胞发生自噬进行研究,从而探讨杨梅素抗结核作用的机理。方法用CCK8法检测杨梅素对细胞增殖的影响,确定安全的用药范围;以H37Ra菌株感染的小鼠巨噬细胞Raw 264.7为模型组,并设空白组和药物处理组。按感染复数(MOI,即细菌:细胞=10:1)加入模型组、药物处理组,共孵育4 h后,磷酸盐缓冲液(PBS)洗3次以弃掉未进入胞内的MTB。药物处理组分别用不同浓度(12.5、25、50、100μmol/L)的杨梅素作用24 h,Western blot法检测自噬相关蛋白即"微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)和p62"表达水平的变化,并以此筛选出杨梅素促进自噬的最佳作用浓度;100μmol/L杨梅素作用于感染细胞72 h后,0.1%聚乙二醇辛基苯基醚(Triton X-100)冰上裂解细胞10 min,菌落形成单位(CFU)法检测巨噬细胞胞内荷菌量;杨梅素作用感染细胞不同时间(30、60、180 min)后Western blot测定PI3K/Akt/mTO...     

吸入一氧化氮对犬肺血栓栓塞症再灌注后血气及血流动力学的影响

目的 建立血栓栓塞1周的犬肺栓塞(PTE)模型,观察吸入20 ppm一氧化氮(NO)对取栓再灌注后血气及血流动力学的影响并探讨其可能的机制.方法 对该PTE犬模型行取栓术,观察再灌注0、2、4、6 h后生命体征、血气变化,通过漂浮导管监测肺血流动力学的变化,再灌注6 h后肺泡灌洗液(BALF)中的白蛋白含量(g/L)、肺组织湿干重比值(W/D)、肺泡腔多核中性粒细胞(PMN)数.结果再灌注2 h后与再灌注前比较(下同)平均肺动脉压(MPAP)升高[(3.20±0.53)kPa对(2.27±0.67)kPa,F=63,P=0.02],再灌注6 h后,心率增快最明显[(175±8)次/min对(155±5)次/min,F=38.72,P=0.01],氧合指数下降(41.70±8.04对54.71±3.78,F=48.36,P=0.03);再灌注后2 h,吸入NO组与再灌注组(下同)比较MPAP降低((2.53±0.40)kPa对(3.20±0.53)kPa,F=55,P=0.04],4 h,氧合指数有升高趋势(49.17±7.37对39.71±7.31,F=2.36,P=0.11),再灌注后2、4、6 h,吸入NO组肺血管阻力较再灌注组也有减小趋势(F=1.49,P=0.26),6 h后吸入NO组肺泡腔PMN数低于再灌注组[(19±6)个/10 HPF对(31±11)个/10 HPF,F=98,P=0.01]. 结论 血栓栓塞1周的PTE犬模型再灌注后导致了肺再灌注损伤,再灌注不同时间点对肺组织损伤程度不一.吸入20 ppm NO可降低PTE再灌注损伤导致的升高的肺动脉压,减少PMN向肺组织的迁移而减轻再灌注损伤,可能减轻肺微血管渗漏.     

PI3K/AKT信号通路与辐射抵抗机制的研究概况

辐射抵抗的三个主要机制为内在辐射敏感性、肿瘤细胞增殖和乏氧。磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)信号通路是肿瘤细胞的存活通路之一,该通路的激活与辐射抵抗密切相关。该文就PI3K/AKT信号通路与辐射抵抗机制作一综述。     

参麦注射液对兔心肌缺血再灌注内皮功能的影响

目的 :观察兔心肌缺血再灌注 (I/R)损伤中内皮功能改变 ,参麦注射液 (SMI)对其影响及作用机制。方法 :检测假手术对照组、心肌I/R模型组及心肌I/R加SMI治疗组 ,不同时期血中一氧化氮代谢产物 (NOP)和内皮素 (ET)含量及心肌组织NOP、ET、总超氧化物歧化酶 (T SOD)和丙二醛 (MDA)含量 ,并电镜观察心肌超微结构。结果 :I/R模型组与假手术对照组比较缺血 4 0min、再灌注 4 0min血清NOP明显降低 ,血浆ET显著增高 ;再灌注 4 0min后心肌组织NOP、T SOD明显降低 ,ET、MDA明显增高 ,心肌超微结构发生异常改变。而I/R加SMI治疗组与I/R模型组比较上述改变减轻。结论 :心肌I/R导致血管内皮功能紊乱 ;SMI能够改善I/R时的内皮功能 ,减轻心肌I/R损伤     

Hydrophilic bile salt ursodeoxycholic acid protects myocardium against reperfusion injury in a PI3K/Akt dependent pathway

The opening of mitochondrial permeability transition pore (PTP) during reperfusion injury of heart has been well demonstrated and thus controlling PTP would attenuate the myocardial damage and cell death. Ursodeoxycholic acid (UDCA) is a hydrophilic bile salt and has been shown to prevent apoptosis in hepatocytes by inhibiting the opening of PTP. Here we demonstrate the role of UDCA in preventing the reperfusion injury of heart through its ability to inhibit PTP. Wistar rats underwent 30 min left coronary artery occlusion (LCA) followed by 180 min reperfusion after treatment with 40 mg/kg per iv infusion of UDCA over 30 min before LCA occlusion. Other groups of rats were treated with PTP agonist atractyloside(5 mg/kg) or PI3 kinase inhibitor wortmannin (16 ug/kg) before UDCA treatment. UDCA treatment prior to LCA occlusion, activated phosphorylation of Akt and Bad. Phosphorylating Bad prevented its translocation in to mitochondria, there by preventing the down regulation of Bcl-2 expression and PTP opening. This was confirmed by reduced cytochrome C release from intramitochondrial space in to the cytosol and hence reduced cell death either by apoptosis (4.8 vs 11.8%, P<0.001, UDCA treated against control group) or necrosis (reduced MI area in UDCA treated group (22.1%) compared to control group(46.4%), P<0.001). In contrast, inhibition of Akt activation with PI3K inhibitor wortmannin or opening the PTP with atractyloside abolished, UDCA mediated cytoprotective effects. Studies on primary culture cardiomyocytes also confirmed our in vivo results of UDCA on cell survival. These results altogether demonstrate that UDCA protect the heart against reperfusion injury by inhibiting the PTP in a PI3K/Akt dependent pathway.     

Adenosine A2A and A2B receptors work in concert to induce a strong protection against reperfusion injury in rat hearts

We aimed to test if stimulation of both adenosine A_2A and A_2B receptors is required to produce an effective cardioprotection against reperfusion injury. Isolated rat hearts were subjected to 30-min regional ischemia followed by 2 h of reperfusion. The adenosine A₁/A₂ receptor agonist 5′-(N-ethylcarboxamido) adenosine (NECA) given at reperfusion reduced infarct size, an effect that was reversed by both the adenosine A_2A antagonist SCH58261 and the A_2B antagonist MRS1706. The A_2B agonist BAY 60-6583 but not the selective A_2A agonist CGS21680 reduced infarct size. Interestingly, a combination of BAY 60-6583 and CGS21680 further reduced infarct size. These results suggest that both A_2A and A_2B receptors are involved in NECA's anti-infarct effect at reperfusion. NECA attenuated mitochondrial swelling upon reperfusion and this was blocked by both SCH58261 and MRS1706, indicating that activation of A₂ receptors with NECA can modulate reperfusion-induced mitochondrial permeability transition pore (mPTP) opening. In support, NECA also prevented oxidant-induced loss of mitochondrial membrane potential (ΔΨ_m) and matrix Ca²⁺ overload in cardiomyocytes via both the A₂ receptors. In addition, NECA increased mitochondrial glycogen synthase kinase-3β (GSK-3β) phosphorylation upon reperfusion and this was again blocked by SCH58261 and MRS1706. In conclusion, A_2A and A_2B receptors work in concert to prevent reperfusion injury in rat hearts treated with NECA. NECA may protect the heart by modulating the mPTP opening through inactivating mitochondrial GSK-3β. A simultaneous stimulation of A_2A and A_2B receptors at reperfusion is required to produce a strong cardioprotection against reperfusion injury.     

L-nitro-arginine inhibits increase in endothelin binding sites induced by ischemia and reperfusion

We have demonstrated that ischemia and reperfusion promoted augmented contractile response to endothelin-1 (ET) in coronary arteries in the presence of polymorphonuclear leukocytes (PMN). It has been also reported that ischemia and reperfusion increase ET binding sites in cardiac membrane in isolated rat heart perfused by blood cell-free system. To determine the role of PMN and L-arginine to nitric oxide (NO) pathway in these phenomena, isolated perfused rabbit hearts were subjected to 30 min of global ischemia followed by 30 min of reflow in the absence or presence of PMN and 10(-5)M of L-nitro-arginine (LNA). PMN was prepared with Percoll density gradients from peritoneal exudate elicited by glycogen. PMN activated with 10(-6)M of phorbol myristate acetate or their supernatant were infused into the coronary perfusion circuit after 5 min of reflow. LNA was added to perfusate also after reflow. The effect of superoxide dismutase (SOD: 50 IU/ml) was also determined. After the end of protocols, membrane fraction was isolated from the hearts for (125)I-ET-1 binding assay. ET-1 binding (Bmax) showed a significant increase by ischemia and reperfusion (P<0.01 vs control). That was markedly augmented with addition of activated PMN or their supernatant (both P<0.01), but abolished either by LNA or SOD (P<0.01 and P<0.05, respectively). These results indicate that increase in ET-receptor by ischemia and reperfusion is mediated by free radicals generated via L-arginine to NO pathway.     

Remote postconditioning by humoral factors in effluent from ischemic preconditioned rat hearts is mediated via PI3K/Akt-dependent cell-survival signaling at reperfusion

Short non-lethal ischemic episodes administered to hearts prior to (ischemic preconditioning, IPC) or directly after (ischemic postconditioning, IPost) ischemic events facilitate myocardial protection. Transferring coronary effluent collected during IPC treatment to un-preconditioned recipient hearts protects from lethal ischemic insults. We propose that coronary IPC effluent contains hydrophobic cytoprotective mediators acting via PI3K/Akt-dependent pro-survival signaling at ischemic reperfusion. Ex vivo rat hearts were subjected to 30 min of regional ischemia and 120 min of reperfusion. IPC effluent administered for 10 min prior to index ischemia attenuated infarct size by ≥55% versus control hearts (P < 0.05). Effluent administration for 10 min at immediate reperfusion (reperfusion therapy) or as a mimetic of pharmacological postconditioning (remote postconditioning, RIPost) significantly reduced infarct size compared to control (P < 0.05). The IPC effluent significantly increased Akt phosphorylation in un-preconditioned hearts when administered before ischemia or at reperfusion, while pharmacological inhibition of PI3K/Akt-signaling at reperfusion completely abrogated the cardioprotection offered by effluent administration. Fractionation of coronary IPC effluent revealed that cytoprotective humoral mediator(s) released during the conditioning phase were of hydrophobic nature as all hydrophobic fractions with molecules under 30 kDa significantly reduced infarct size versus the control and hydrophilic fraction-treated hearts (P < 0.05). The total hydrophobic effluent fraction significantly reduced infarct size independently of temporal administration (before ischemia, at reperfusion or as remote postconditioning). In conclusion, the IPC effluent retains strong cardioprotective properties, containing hydrophobic mediator(s) < 30 kDa offering cytoprotection via PI3K/Akt-dependent signaling at ischemic reperfusion.     

AKT/PI3K信号通路介导的干细胞动员在抗心梗后心律失常中的作用

目的 探讨心肌梗死（MI）后丝氨酸/苏氨酸/磷脂酰肌醇-3激酶（AKT/PI3K）信号通路介导的心脏干细胞（CSCs）动员对抗心梗后心律失常的影响.方法 将大鼠随机分为5组,每组20只：对照组（CON组）、结扎组（LI组）、结扎＋注射组（LI＋LY组）、缺血预处理＋结扎组（IP＋LI组）、缺血预处理＋结扎＋注射组（IP＋LI＋LY组）.采用结扎LAD的方法建立心梗模型,采用流式细胞分析、ELISA法等检测各组AKT/PI3K信号通路相关指标的表达情况及心律失常的发生情况.结果 LI＋LY组炎症反应显著高于其他各组,IP＋LI组显著低于IP＋LI＋LY组、LI＋LY组、LI组（P＜0.05）;IP＋LI组比LI组CD34＋、Oct4＋细胞的数量多（P＜0.05）;与CON组相比,IP＋LI组SDF-1表达显著增加,LI＋LY组及LI组表达明显减少（P＜0.05）;与LI组比较,IP＋LI组心律失常的发生率明显降低,QTc间期也明显缩短（P＜0.05）.结论 通过激活内源性Akt/PI3K信号通路,可有效促进内源性心脏干细胞动员及归巢,使心肌细胞再生,从而改善心脏功能,降低心肌缺血后室性心律失常的发生.